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S E C O N D E D I T I O N

Metabolic
and
Therapeutic
Aspects
of
Amino Acids
in
ClinIcal Nutrition
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S E C O N D E D I T I O N

Metabolic
and
Therapeutic
Aspects
of
Amino Acids
in
ClinIcal Nutrition

Edited by
Luc a. Cynober

CRC PR E S S
Boca Raton London New York Washington, D.C.
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Library of Congress Cataloging-in-Publication Data

Metabolic & therapeutic aspects of amino acids in clinical nutrition / edited by Luc A.
Cynober. --2nd ed.
p. ; cm.
Rev. ed of: Amino acid metabolism and therapy in health and nutritional disease. 1995.
Includes bibliographical references and index.
ISBN 0-8493-1382-1 (alk. paper)
1. Amino acids--Metabolism. 2. Amino acids--Pathophysiology. 3. Amino acids in
human nutrition. I. Title: Metabolic and therapeutic aspects of amino acids in clinical
nutrition. I. Cynober, Luc A. II. Amino acid metabolism and therapy in health and
nutritional disease.
[DNLM: 1. Amino acids--metabolism. 2. Amino Acids--therapeutic use. 3. Amino
Acids--administration & dosage. QU 60 M5867 2003]
QP561.A4615 2003
612.3'98—dc22 2003047263

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Printed on acid-free paper
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Dedication

Prof. Bernard Beaufrère Prof. Peter Reeds

The second edition of this book is dedicated to the memories of


Prof. Bernard Beaufrère and Prof. Peter Reeds.
Both were first-rate scientists, and their contributions to the field
of amino acid and protein metabolism were considerable.
They were life-loving people and are sorely missed
by their many friends across the world.

Luc A. Cynober
Editor
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Preface
When CRC Press urged me a year ago to supervise a second edition of this book, I accepted
for several reasons. First, this book remained the only one devoted exclusively to the
metabolic and therapeutic aspects of amino acids in clinical nutrition. Second, in the 9
years since the first edition, a number of very important advances had been made in the
field. Third, some new topics had emerged with recent work, for example, on amino acids
and aging, and on arginine as a nutraceutical in cardiovascular disease. There were also
interesting new findings concerning taurine and other sulfur amino acids. Additionally, I
had reached the conclusion that the aftereffects of sports were a catabolic disorder and so
deserved a chapter. Finally, technical considerations concerning amino acid measurements,
which were lacking in the first edition, could now be addressed. So a second edition was
in order.
Most of the authors had already contributed to the first edition. They have done a
fine job once more, not only refining their earlier contributions, but also thoroughly
updating them to make this book a most valuable store of information and insight. Some
of the original authors were unable to contribute again for various reasons, but new
authors bring fresh ideas and new viewpoints. I warmly thank all the contributors.
Despite all possible efforts, some topics (AIDS, transplantation, etc.) remain unad-
dressed in this edition. I may be able to correct this in a third edition. Who knows?
Lastly, I am most grateful for the secretarial assistance of Solange Ngon in the prep-
aration of the book. Her perseverance in handling hundreds of e-mails, opening obstinate
attachments, and dealing with the different versions of the manuscripts was decisive for
a successful outcome.

Luc A. Cynober
Paris
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About the Editor


Luc A. Cynober, Ph.D., is head of the Department of Clinical Biochemistry, Hotel-Dieu
Hospital, Paris, and is professor of nutrition and head of the Biological Nutrition Labo-
ratory at the School of Pharmacy, University of Paris, France.
Dr. Cynober obtained his Pharm.D. degree in 1979 from the School of Pharmacy, Paris
XI University. In 1985, he received his Ph.D. degree in biological and pharmaceutical
sciences from the same university.
Dr. Cynober is a member of the European Society of Parenteral and Enteral Nutrition,
the American Society of Parenteral and Enteral Nutrition, the French-Speaking Society of
Clinical Biology, and the French Society of Biochemistry and Molecular Biology, among
others.
He served from 1992 to 2000 as officer in the executive committee of the European
Society of Parenteral and Enteral Nutrition, and since 2001 as chairman of the French-
Speaking Society of Enteral and Parenteral Nutrition. He served (1987–1992) as editor-in-
chief of Nutrition Clinique & Métabolisme. He is presently (since 1998) editor-in-chief of
Current Opinion in Clinical Nutrition and Metabolic Care.
Among other awards, he has received the Pharmacy Academy Award for his Ph.D.
thesis and the International Research Award of the French Society of Clinical Biology.
Dr. Cynober has presented over 70 invited lectures at international and national
meetings and over 150 guest lectures at universities and institutes. He has published more
than 200 research papers. His current major research interests relate to amino acid metab-
olism and therapy in critical illness and aging.
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Contributors List
Naji N. Abumrad Denis Breuillé
Vanderbilt University Medical Center Nestlé Research Center
Nashville, Tennesee Vers-Chez-Les-Blanc, Switzerland

Jorge E. Albina Philip C. Calder


Rhode Island Hospital and Brown Medical University of Southampton
School Southampton, United Kingdom
Providence, Rhode Island
Antonio C.L. Campos
Birgit Alteheld Federal University of Parana
University of Bonn Brazil
Bonn, Germany
Noël Cano
Adrian Barbul School of Pharmacy and Clinique
Sinai Hospital and Johns Hopkins Medical Résidence due Parc
Institutions Marseille, France
Baltimore, Maryland
Marika Collin
Vickie E. Baracos University of Helsinki
University of Alberta Helsinki, Finland
Edmonton, Alberta, Canada
Luc A. Cynobar
Bernard Beaufrére (Deceased) Hôtel-Dieu Hospital
Centre de Recherche en Nutrition Paris, France
Humaine d’Auverfue
Clermont-Ferrand, France Nicole M. Daignault
Emory University School of Medicine
Luc Bertrand Atlanta, Georgia
Institute of Cellular Pathology and
Université Catholique de Louvain S.W.M. Olde Damink
Brussels, Belgium Academic Hospital
Maastricht, The Netherlands
Petra G. Boelens
VU University Medical Center Dominique Darmaun
Amsterdam, The Netherlands Hôtel-Dieu Hospital
Nantes, France
Yves Boirie
Centre de Recherche en Nutrition
Humaine d’Auverfue
Clermont-Ferrand, France
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Patrick David Daniel P. Griffith


Hôtel-Dieu Hospital Emory University School of Medicine
Paris, France Atlanta, Georgia

Jean-Pascal De Bandt George K. Grimble


Université Paris 5 University of Surrey Roehampton
Paris, France London, United Kingdom

Cornelis H.C. Dejong Svend Høime Hansen


Academic Hospital Rigshospitalet
Maastricht, The Netherlands Copenhagen University Hospital
Copenhagen, Denmark
Nicolaas E.P. Deutz Dieter Häussinger
Academic Hospital Heinrich Heine University
Maastricht, The Netherlands Dusseldorf, Germany

Ketan Dhatariya Milan Holecek


Mayo Clinic and Foundation Charles University School of Medicine
Rochester, Minnesota Hradec Králové, Czech Republic

Peter F. Dubbelhuis Louis Hue


Academic Medical Center Institute of Cellular Pathology and
Amsterdam, The Netherlands Université Catholique de Louvain
Brussels, Belgium
Filippo Rossi Fanelli
University ‘La Sapienza’ Karel W. Hulsewe
Rome, Italy Academic Hospital
Maastricht, The Netherlands
Charles J. Foulks
Gaetano Iapichino
Texas A&M University Health Science
Universitá degli studi di Milano
Center
Milan, Italy
Temple, Texas
Katsuhisa Inoue
Peter Fürst Medical College of Georgia
University of Bonn Augusta, Georgia
Bonn, Germany
R. Jalan
Malliga E. Ganapathy University College Medical School and
Medical College of Georgia UCLH Hospitals
Augusta, Georgia London, United Kingdom

Vadivel Ganapathy Asker E. Jeukendrup


Medical College of Georgia University of Birmingham
Augusta, Georgia Birmingham, United Kingdom

Michael Gleeson Kenneth A. Kudsk


Loughborough University The University of Wisconsin–Madison
Leicestershire, United Kingdom Madison, Wisconsin
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Xavier M. Leverve Nathalie Neveux


Université Joseph Fourier Hôtel-Dieu Hospital
Grenoble, France Paris, France

Yvette Luiking Itzhak Nissim


Academic Hospital University of Pennsylvania School of
Maastricht, The Netherlands Medicine
Philadelphia, Pennsylvania
Michelle Mackenzie
University of Alberta Christiane Obled
Edmonton, Alberta, Canada Unité de Nutrition et Métabolisme
Protéique, INRA
Eric J. Mahoney Saint Genes Champanelle, France
Brown Medical School
Rhode Island Hospital Rudolf Oehler
Providence, Rhode Island University of Vienna
Vienna, Austria
Willy J. Malaisse
Brussels Free University Isabelle Papet
Brussels, Belgium Unité de Nutrition et Métabolisme
Protéique, INRA
Michael M. Meguid Saint Genes Champanelle, France
Upstate Medical University
Syracuse, New York Phillippe Patureau Mirand
Unité de Nutrition et Métabolisme
Alfred J. Meijer Protéique
Academic Medical Center Clermont-Ferrand, France
Amsterdam, The Netherlands
Puttur D. Prasad
Sidney M. Morris, Jr. Medical College of Georgia
University of Pittsburgh School of Augusta, Georgia
Medicine
Pittsburgh, Pennsylvania Danilo Radrizzani
Universitá degli studi
Laurent Mosoni Milan, Italy
Unité de Nutrition et Métabolisme
Protéique, INRA David K. Rassin
Clermont-Ferrand, France The University of Texas Medical Branch
at Galveston
Maurizio Muscaritoli Galveston, Texas
University ‘La Sapienza’
Rome, Italy D. Rémond
Unité de Nutrition et Métabolisme
K. Sreekumaran Nair Protéique
Mayo Clinic and Foundation Clermont-Ferrand, France
Rochester, Minnesota
Erich Roth
Thomas V. Nattakom University of Vienna
Memorial Medical Center Vienna, Austria
Las Cruces, New Mexico
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Gordon S. Sacks Yasuo Wakabayashi


The University of Wisconsin–Madison Kyoto Prefectural University of Medicine
Madison, Wisconsin Kyoto, Japan

Peter B. Soeters Stéphane Walrand


Academic Hospital Centre de Recherche en Nutrition
Maastricht, The Netherlands Humaine d’Auverfue
Clermont-Ferrand, France
Peter Stehle
University of Bonn Tomas Welbourne
Bonn, Germany Louisiana State University/HSC
Shreveport, Louisiana
John F. Tharakan
Massachusetts Institute of Technology Jan Wernerman
Cambridge, Massachusetts Huddinge University Hospital
Karolinska Institutet
Paul A.M. van Leeuwen Stockholm, Sweden
VU University Medical Center
Amsterdam, The Netherlands Guoyao Wu
Texas A&M University
Heikki Vapaatalo College Station, Texas
University of Helsinki
Helsinki, Finland Parveen Yaqoob
The University of Reading
Stephan vom Dahl Reading, United Kingdom
Heinrich Heine University
Düsseldorf, Germany Vernon R. Young
Massachusetts Institute of Technology
Anton J.M. Wagenmakers Cambridge, Massachusetts
Maastricht University and University
Hospital Thomas R. Ziegler
Maastricht, The Netherlands Emory University School of Medicine
Atlanta, Georgia
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Contents
Introduction ....................................................................................................................................1
John M. Kinney

Physiology and Physiopathology

Part I: Introduction to amino acid metabolism

Chapter 1 Measurement of amino acid concentrations in biological fluids


and tissues using ion exchange chromatography ...........................................17
Nathalie Neveux, Patrick David, and Luc Cynober

Chapter 2 Measurement of amino acid concentrations in biological fluids


and tissues using reversed-phase HPLC-based methods ..............................29
Birgit Alteheld, Peter Stehle, and Peter Fürst

Chapter 3 Approaches to studying amino acid metabolism: from quantitative


assays to flux assessment using stable isotopes ..............................................45
Dominique Darmaun and Luc Cynober

Chapter 4 Cellular uptake of amino acids: systems and regulation...............................63


Vadivel Ganapathy, Katsuhisa Inoue, Puttur D. Prasad, and Malliga E. Ganapathy

Part II: Physiology

Section A: Metabolism

Chapter 5 Amino acid metabolism and gluconeogenesis.................................................83


Xavier M. Leverve

Chapter 6 Contribution of amino acids to ketogenesis.....................................................97


Milan Holecek
ˇ

Chapter 7 Ureagenesis and ammoniagenesis: an update ............................................... 111


Alfred J. Meijer
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Chapter 8 Metabolism of branched-chain amino acids in man.....................................123


Anton J.M. Wagenmakers

Chapter 9 The glutamate crossway ....................................................................................135


Yasuo Wakabayashi

Chapter 10 Arginine metabolism in mammals...................................................................153


Guoyao Wu and Sidney M. Morris, Jr.

Chapter 11 Glutamine metabolism .......................................................................................169


Rudolf Oehler and Erich Roth

Section B: Control of and by amino acids

Chapter 12 Insulin and the regulation of amino acid catabolism and protein
turnover ................................................................................................................185
Jean-Pascal De Bandt

Chapter 13 Control of amino acid metabolism by counterregulatory hormones.........201


Jan Wernerman

Chapter 14 Nitric oxide...........................................................................................................211


Eric J. Mahoney and Jorge E. Albina

Chapter 15 Control of amino acid metabolism by lipids, ketone bodies,


and glucose substrates .......................................................................................241
Yves Boirie, Stéphane Walrand, and Bernard Beaufrère

Chapter 16 Amino acid signaling and the control of protein metabolism ....................253
Alfred J. Meijer and Peter F. Dubbelhuis

Chapter 17 The role of amino acids in the control of proteolysis ...................................275


Stephan vom Dahl and Dieter Häussinger

Chapter 18 Anabolic effects and signaling pathways triggered by amino acids


in the liver ............................................................................................................291
Louis Hue and Luc Bertrand

Chapter 19 Amino acids and immune function .................................................................305


Philip C. Calder and Parveen Yaqoob

Chapter 20 Amino acid-mediated insulin secretion ..........................................................321


Willy J. Malaisse
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Part III: Amino acid metabolism in disease

Chapter 21 Cancer-associated cachexia: altered metabolism of protein


and amino acids ..................................................................................................339
Michelle Mackenzie and Vickie E. Baracos

Chapter 22 Diabetes mellitus .................................................................................................355


Ketan Dhatariya and K. Sreekumaran Nair

Chapter 23 Acidosis and amino acid metabolism..............................................................375


Tomas Welbourne and Itzhak Nissim

Chapter 24 Muscle protein and amino acid metabolism with respect to


age-related sarcopenia ........................................................................................389
Stéphane Walrand and Yves Boirie

Chapter 25 Gastrointestinal disease......................................................................................405


Peter B. Soeters, Karel W. Hulsewe, Nicolaas E.P. Deutz, Yvette Luiking,
and Cornelis H.C. Dejong

Chapter 26 Amino acids and ammonia in liver disease ...................................................419


Cornelius H.C. Dejong, S.W.M. Olde Damink, R. Jalan, Nicolaas E.P. Deutz,
and Peter B. Soeters

Requirements and Supply

Part IV: Amino acid requirements

Chapter 27 Nutritional essentiality of amino acids and amino acid


requirements in healthy adults .........................................................................439
Vernon R. Young and John F. Tharakan

Chapter 28 Neonatal requirements for amino acids..........................................................471


David K. Rassin

Chapter 29 Amino acid requirements in the elderly .........................................................483


Phillippe Patureau Mirand, L. Mosoni, and D. Rémond

Chapter 30 Amino acid requirements in sport ...................................................................497


Michael Gleeson and Asker E. Jeukendrup
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Part V: Amino acid supply in diseases

Section A: Quantitative and qualitative aspects

Chapter 31 Quantitative and qualitative amino acid intake by the parenteral


route.......................................................................................................................519
Gaetano Iapichino, Danilo Radrizzani, and Luc A. Cynober

Chapter 32 Quantitative and qualitative aspects of nitrogen supply in enteral


nutrition in relation to free amino acids and peptides.................................529
George K. Grimble

Chapter 33 Branched-chain amino and keto acids in renal failure.................................557


Noël Cano

Chapter 34 Glutamine-supplemented diets in enteral nutrition .....................................577


Petra G. Boelens and Paul A.M. van Leeuwen

Chapter 35 The use of arginine in clinical practice............................................................595


Naji N. Abumrad and Adrian Barbul

Chapter 36 Glutamine and glutamine-containing dipeptides..........................................613


Peter Fürst and Peter Stehle

Chapter 37 Ornithine a-ketoglutarate ..................................................................................633


Luc A. Cynober

Section B: Formulas devoted to specific situations

Chapter 38 Amino acid support in patients with catabolic illness .................................649


Nicole M. Daignault, Daniel P. Griffith, Thomas V. Nattakom, and Thomas R. Ziegler

Chapter 39 Sulfur-containing amino acids and glutathione in diseases ........................667


Christiane Obled, Isabelle Papet, and Denis Breuillé

Chapter 40 Amino acid requirement in cancer...................................................................689


Maurizio Muscaritoli, Filippo Rossi Fanelli, Michael M. Meguid,
and Antonio C.L. Campos

Chapter 41 Amino acid solutions for acute renal failure..................................................705


Charles J. Foulks

Chapter 42 Amino acids to support gut function and morphology...............................717


Gordon S. Sacks and Kenneth A. Kudsk
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Section C: Nutraceutics

Chapter 43 L-arginine-enriched diets in cardiovascular diseases ...................................729


Marika Collin and Heikki Vapaatalo

Chapter 44 Taurine homeostasis and its importance for physiological functions........739


Svend Høime Hansen

Index .............................................................................................................................................749
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Introduction

John M. Kinney

A review of the history of proteins and amino acids at the beginning of the twentieth
century makes it apparent that the physiology of the protein was moving toward the
physiology of amino acids. The recognition of individual amino acids had developed for
over 100 years when Rose reported threonine in 1936.1 Yet the chemical identification of
individual amino acids involved an immense amount of time and skill, which usually
required the isolation of a material that could be weighed. This situation underwent major
changes in the 1940s and 1950s when new methodology changed the measurement of
amino acids to rapid and automated procedures.
Advances of the 1940s were associated with the stimulus of the wartime search for
antibiotics such as gramicidin. During this period came the discovery that certain bacteria
could be used quantitatively to measure the presence of specific amino acids. Fruton2 has
summarized the advances of this period. Martin and Synge introduced paper chromatog-
raphy with the use of ninhydrin. This was followed by the column chromatography of
Moore and Stein, using starch and then resins for ion exchange chromatography. This
technique was quickly enhanced by the addition of a fraction collector, producing an
automated system for separation of a mixture of amino acids. The next advance was high-
performance reversed-phase liquid chromatography (HPLC). Subsequent refinements and
advances in the measurement of amino acids are discussed in the early chapters of this
volume.
Balance studies to determine the amount of protein required to achieve nitrogen
balance in normal adults occupied much attention in the first third of the 1900s. However,
as attention turned to the necessary intake of amino acids rather than protein, the work
of W.C. Rose over 20 years was definitive in establishing the intake of individual amino
acids required for the growth and health of the laboratory rat.
This led to data in 1957 from balance studies in man that listed the essential amino
acid requirements for a healthy human diet.3 These studies included the identification of
threonine but did not include histidine and arginine, which were felt to be nonessential.
A consistent finding was that even when the estimates for amino acid requirement were
based on the highest levels of each essential amino acid indicated by previous studies,
the total was still quite low relative to estimates of the total protein needs.
Adult protein requirements had not been a high-priority area of research between
1920 and 1960 since protein needs seemed to be readily met in practice. However, the U.S.
Space Agency in 1960 became concerned about how astronauts remaining in space for
long periods should be fed. Remarkably detailed long-term studies were conducted at
Berkeley by Calloway and coworkers and at MIT by Scrimshaw and coworkers.1 Despite
some variability in results, there was general agreement that the requirements for nitrogen
balance considerably exceeded the theoretical estimate for optimum amino acid intake,

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© 2004 by CRC Press LLC 1
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2 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

suggesting that adults used even high-class proteins with no more than approximately
60% efficiency. The FAO/WHO/UNU report in 1985 concluded that 52.5 g of first-class
protein would meet the needs of 97% of the adult population. Various groups were unable
to obtain nitrogen balance on such small intakes.4
Vernon Young of MIT urged repeatedly that it was unsafe to accept the 1985 interna-
tional standards for adult amino acid requirements, based solely on the results of nitrogen
balance studies. Young and his colleagues began a major research program using an
approach that focused on an indirect measure of the balance of individual amino acids.
The details of these methods, which involved C13-labeled amino acids, have been carefully
described in Chapter 27.
A general conclusion from such studies was that the adult requirement for individual
essential amino acids (leucine, lysine, and threonine) appeared to be considerably greater
than the standards derived from earlier balance studies. Because of the errors in balance
studies and the complexity of the isotope studies, final conclusions on amino acid require-
ments have remained controversial, yet there has been growing acceptance that the former
estimates were too low.
Many hormones and cytokines are involved in the regulation of amino acid metabo-
lism. The leading anabolic hormone, insulin, continues to be of particular interest in
relation to muscle tissue. Insulin deficiency has long been known to be associated with
muscle wasting while visceral protein is affected less or not at all. However, the insulin
action on muscle tissue continues to revolve around whether protein synthesis is stimu-
lated or protein breakdown is inhibited.
Muscle protein synthesis has been shown in vitro to involve gene transcription, mes-
senger RNA translation, and activation of preexisting enzymes. This is followed by an
increase in tissue RNA content, of both ribosomal RNA and messenger RNA. In contrast
to in vitro findings, the in vivo studies in rats are less clear but generally support the effect
of insulin as stimulating muscle protein synthesis. The situation in man is even less clear.
Wolfe4 has summarized the errors in interpretation of using a tracer amino acid such as
phenylalanine in a limb balance study to determine synthesis and breakdown directly
from the rates of appearance and disappearance in the blood. He noted that combining a
direct measure of the fractional synthesis rate and the arteriovenous balance/intramus-
cular pool during hyperinsulinemia in the human leg revealed that insulin was associated
with more efficient reutilization of intracellular amino acids. The systemic infusion of
insulin causes a reduction in blood amino acid concentrations. In this circumstance, insulin
does not stimulate muscle protein synthesis. When this insulin-induced hypoaminoaci-
demia is avoided by an appropriate infusion of amino acids, insulin is seen to stimulate
muscle protein synthesis.
The ubiquitin–proteosome system is primarily responsible for protein breakdown in
resting muscle. An acute increase in insulin concentration, such as occurs after a meal,
has little effect on this system. This is in contrast to long-term regulation of the ubi-
quitin–proteosome pathway, where a stimulated production of mRNA encoding ubiliq-
uitin and proteosome subunits is associated with an increased transcription of the ubiq-
uitin gene.
In contrast to the ubiquitin–proteosome pathway, the lysosomal breakdown of protein
is responsive to acute changes in insulin concentration. Yet lysosomes do not normally
play an important role in myofibrillar breakdown. This is consistent with findings that
local hyperinsulinemia does not increase protein breakdown in resting muscle. Wolfe4
summarizes the actions of insulin on muscle protein as follows: at rest the basal insulin
concentration seems to play a role in curtailing the ubiquitin–proteosome pathway of
muscle protein breakdown, but acute increases in insulin (as after a meal) may have little
effect on muscle protein breakdown. This would be different if the individual is in a
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Introduction 3

stressed state or is studied immediately after exercise where the lysosomal pathway of
breakdown contributes significantly to the breakdown process and is therefore responsible
for the suppressive action of insulin.
The relative rates of growth and protein synthesis are higher during the neonatal
period than at any other stage of postnatal life, and more rapid gains occur in skeletal
muscle than in other parts of the protein mass. This feeding-induced stimulus of protein
synthesis is most dramatic in skeletal muscle and decreases profoundly with development.
Insulin is recognized as a key factor in the regulation of skeletal muscle protein synthesis.
Insulin sensitivity and responsiveness of amino acid disposal decrease with development.
The response of protein synthesis to insulin infusion declines with development in parallel
with the developmental decline in the stimulation of muscle protein synthesis by feeding.
However, whether insulin and amino acids interact at submaximal doses to stimulate
skeletal muscle protein synthesis remains uncertain.
O’Connor et al.5 utilized a pancreatic-amino acid clamp in fasted neonatal pigs to
block insulin secretion, while glucose and glucagon were maintained at fasting levels.
Insulin was infused at different amounts, and at each insulin dose, amino acids were
clamped at either the fasting or fed level. The results showed that insulin and amino acids
act independently to stimulate protein synthesis in skeletal muscle of the neonate. Davis
et al.6 used this model to show that exogenous insulin growth factor-1 (IGF-1) stimulates
protein synthesis in skeletal muscle and other insulin-sensitive tissues of the neonate,
presumably because IGF-1 acts on the same signaling pathway as insulin leading to
translation initiation.
Calbet and MacLean7 measured the insulin and glucagon responses to the rate of
appearance of amino acids after ingestion of different isonitrogenous solutions in humans.
The insulin response was closely related to the increase in plasma levels of leucine,
isoleucine, valine, phenylalanine, and arginine, regardless of the rate of gastric emptying.
Consumption of a protein-containing meal enhances the fractional rate of synthesis of
total mixed proteins in skeletal muscle. The feeding-induced stimulation of protein syn-
thesis requires the hormone insulin and an adequate supply of amino acids. The relative
contribution of these regulatory factors to the increase in protein synthesis is controversial,
particularly since some amino acids independently influence protein synthesis by enhanc-
ing insulin release. Anthony et al.8 have obtained evidence in the rat that transient increases
in serum insulin are permissive for the leucine-induced stimulation of protein synthesis
in skeletal muscle. This rise in insulin contributes to the hyperphosphorylation of trans-
lational factors and suggests that the signaling pathway involving mammalian target of
rapamycin (mTOR) may be a convergence point for both leucine- and insulin-mediated
effects on certain steps in translation initiation. However, unknown steps in mRNA trans-
lation appear to be rate controlling in the stimulus of protein synthesis by leucine.
Protein synthesis and degradation are closely regulated in vivo, and each is affected
by physiological and pathophysiological conditions such as fasting, feeding, exercise,
disease, and aging. Davis and Reeds9,10 have reviewed the advantages and problems of
different isotopic methods for quantifying protein turnover in vivo, considering the meth-
ods of tracer dilution in contrast to methods involving tracer incorporation. These authors
found that measurements made on the basis of labeling plasma and breath are well suited
for the measurement of body amino acid oxidation and balance, yet underestimate protein
turnover. They also note that leucine may be the most useful labeled amino acid, for
measuring both whole-body and muscle protein synthesis, because of the close isotopic
equilibrium between muscle-free and tRNA-bound leucine pools. The authors analyze the
use of tracer infusion and the flooding dose technique for measuring the incorporation of
tracer amino acids into tissue protein. The best estimate of the aminoacyl-tRNA precursor
pool for the constant infusion method will depend upon the organ or tissue under study.
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4 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Wolfe and Miller11 edited a group of papers devoted to the influence of the dietary
pattern and composition of ingested protein on protein metabolism. The rise in muscle
protein sysnthesis occurs relatively quickly with the rise in extracellular amino acid con-
centration. The latency and duration of amino acid stimulation of muscle protein were
reported to be important since the system becomes refractory after 2 h of constant elevation
of amino acid concentration. A study by Tipton et al.12 demonstrated that an acute response
of net muscle protein balance could reflect the 24-h balance after exercise and amino acid
ingestion.
In contrast to the rapid absorption and delivery after ingestion of free amino acids,
intact proteins are digested at variable rates, a factor that appears to independently
regulate postprandial protein gain. These differences are further modified by transient
elevations in substrates and hormones, as well as factors independent of the food source,
such as exercise, age, or metabolic stress. The splanchnic area, which sees very rapid
turnover of protein metabolism, seems to respond to almost all nutritional perturbations,
whereas the peripheral muscle protein mass, which is slowly renewed, appears to be only
slightly affected by acute dietary factors.
Liu and Barrett13 discuss what information can be obtained from whole-body protein
turnover studies, utilizing tracer infusion methods. A comparison of the flux data for
protein, triglycerides, and glycogen reveals that protein is unique in that there is no storage
form that is not already serving another significant purpose. Despite having a higher
energy requirement to synthesize a protein than to store energy as either carbohydrates
or fat, the turnover rate of the body’s protein pool is substantially higher than that of
either of the other two principal fuels. However, the fraction of amino acids liberated from
body protein via one or another proteolytic pathway that is subsequently oxidized fully
is substantially less than that fraction for either fat or carbohydrates. Thus, a selective
advantage appears to accrue to the organism when the building blocks of proteins are
used sparingly as oxidative fuel, whereas the protein pool per se turns over rapidly at
considerable energy cost to the body.
Protein synthesis and degradation are each regulated by multiple hormonal as well
as nutritional factors, and the protein balance of individual tissues, as well as the whole
body, changes constantly. Three peptide hormones, insulin, insulin growth factor-1, and
growth hormone, affect body protein metabolism acutely and have been studied exten-
sively. Recent findings for each hormone have been reviewed for the whole body and
individual organs by Liu and Barrett.13 These investigators note that for insulin there
appears to be a dissociation between the doses that affect protein synthesis vs. those that
affect protein degradation. It is suggested that proteolysis is more sensitive than synthesis
to small changes in plasma insulin within its physiological range. Inasmuch as insulin
and IGF-1 exert anabolic actions on both protein synthesis and proteolysis, evidence is
reviewed suggesting that the cellular signals that mediate insulin and IGF-1 action in
muscle, although similar, diverge significantly despite the fact that each hormone can exert
a major influence on both synthesis and degradative pathways. Growth hormone increases
lean body mass while decreasing fat mass, although the mechanisms remain unclear.
Biolo et al.14 have reviewed kinetic studies in clinical states to search for relationships
between rates of protein synthesis and breakdown. It appears that when protein synthesis
is primarily suppressed, protein degradation is found unchanged or even slightly
decreased. Where protein breakdown is primarily accelerated, the rate of synthesis is
unchanged or even increased. Apparent discrepancies among various studies of chronic
disease may arise from the many factors influencing protein metabolism. When the effects
of inflammatory mediators and stress hormones start to overwhelm the factors that tend
to decrease protein synthesis, the rate of turnover will increase along with a net increase
in the rate of protein breakdown.
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Introduction 5

The process of proteolysis has been recognized for many years. However, it was
formerly considered to be a nonselective process mainly involved in basal protein turnover,
the elimination of abnormal proteins, and perhaps the regulation of certain key enzymes.
More recently, the characterization and regulation of proteolysis have revealed the exist-
ence of three separate systems, the most prominent being the ubiquitin–proteosome sys-
tem.15 The details and metabolic steps of this system have been presented by Hasselgren.16
The ubiquitin–proteosome pathway is the major nonlysosomal process responsible
for the breakdown of most short- and long-lived proteins in mammalian cells. Abnormal
proteins, which are misfolded, oxidized, or mutant, are very good substrates of this system.
There are two main steps in the pathway: the covalent attachment of a polyubiquitin chain
to the substrate and the specific recognition of this signal and degradation of the tagged
protein by the 16S proteosome. This ubiquitinylation not only is a degradation signal but
also directs proteins to a variety of fates, including roles in DNA repair, protein translo-
cation, or modulating the structure or activity of the target proteins.17 In order to be
efficiently degraded, the substrate must be bound to a polyubiquitin degradation signal
that comprises at least four ubiquitin moieties.
Conaway and co-workers18 discuss new roles for ubiquitin in the regulation of RNA
polymerase II (Pol II) transcription that are being discovered at an accelerating pace. It
is evident that the integral role of ubiquitin in Pol II transcription also involves the
complex and multifaceted nature of ubiquitin’s participation in this process. These
authors emphasize that future investigations can be expected to reveal a fundamental
and perhaps general role for ubiquitin in some of the most basic aspects of transcription
activation and repression.
The proteosome is a self-compartmentalizing protease, as substrates must enter the
catalytic chamber within, in order to be degraded into peptides. Multiple active sites are
confined within the small chamber. The proteosome hydrolyzes most peptide bonds and
generates peptides that are typically 3 to 22 amino acids long and do not conserve
biological properties, except for antigen presentation. Recent evidence indicates that sev-
eral proteosome-dependent pathways can be involved in the breakdown of a single sub-
strate.19 This might explain how a cell could rapidly modulate the half-life of various
proteins in response to the cell environment.20 In order for the myofibrillar proteins actin
and myosin to be ubiquinated and degraded by the proteosome, the myofilaments must
first be released from the sarcomere by the calcium-dependent protease calpain.19
Muscle cachexia has been induced by severe injury, sepsis, and cancer where there is
increased gene expression and activity of the calcium/calpain and ubiqitin–proteosome
proteolytic pathways. Despite certain exceptions, most proteins require ubiquination
before catabolism. In cancer cachexia, this process is controlled by a tumor-produced
sulfated glycoprotein.21 A proteolysis-inducing factor (PIF) detected in the urine of weight-
losing cancer patients has been detected in gastrointestinal tumors.22 In vivo studies have
shown that PIF induces catabolism of skeletal muscle, while visceral protein reserves are
preserved. PIF also appears to be involved in the inflammatory response observed in
cachexia, related to increased production of interleukin (IL)-6, IL-8, and C-reactive protein
and a decreased production of transferrin.23
Eicosapentaenoic acid (EPA) has been shown to decrease protein catabolism with
activation of the ubiquitin–proteosome pathway. Animal evidence of reducing protein
catabolism by EPA in starvation suggests that a similar mechanism may be involved in
conditions other than cancer.24
The cascade of various neurologic stimuli that can contribute to loss of appetite and
the catabolism of starvation has been reviewed by Nandi and co-workers.25 Recent studies
have focused on the interactions between Ca++ and the proinflammatory cytokines (in
particular tumor necrosis factor-a) and the activation of transcription factors such as
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6 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

nuclear factor-kB for the stimulation of major proteolytic pathways in cachexia.


Langhans26 has reviewed interrelationships between hypermetabolism and the catabolism
of lipid and protein in cachexia, emphasizing steps that hold promise for future pharma-
ceutical therapy.
Proteins are synthesized by translation of their mRNAs, a process carried out by the
ribosome. The process is divided into stages: translation, elongation of the nascent peptide,
and termination of peptide synthesis. The rate of elongation and termination is determined
by the amount of aminoacylated tRNA available to decode the mRNA and by the activities
of various translation factors. However, evidence is emerging that the nascent peptide
itself, within the ribosome that is making it, can profoundly affect mRNA translation.27
A supply of a complete complement of essential amino acids is a prerequisite for
maintenance of optimal rates of protein synthesis in both liver and skeletal muscle.28
Degradation of even a single essential amino acid causes a decrease in the synthesis of
essentially all cellular proteins through an inhibition of the initiation phase of mRNA
translation. However, the synthesis of all proteins is not repressed equally. Specific subsets
of proteins, in particular those encoded by mRNAs containing an F-terminal oligopyrim-
idine (TOP) motif, are affected to a much greater extent than most proteins. The specific
decrease in TOP mRNA translation is a result of an inhibition of the ribosomal protein S6
kinase, S6K1, and a concomitant decline in S6 phosporylation. Interestingly, many TOP
mRNAs encode proteins involved in mRNA translation, such as elongation factors eEFIA
and eEF2, as well as the ribosomal proteins. Thus, deprivation of essential amino acids
not only directly and rapidly represses global mRNA translation but also potentially
results in a reduction in the capacity to synthesize protein.
Over the past several decades, biologists have unraveled some of the ways cells
communicate with each other. The long-distance messages that the cells exchange involve
hundreds of different proteins, such as hormones and growth factors. The receivers are a
multitude of specialized receptors on the surface of target cells. Beyond these fundamen-
tals, however, the picture of cellular communication gets sketchy. There are very few cases
in which scientists understand all the intricate biochemical steps that flow from message
received to action inside the cell.28
Protein kinases and phosphorylation play a key role in many intracellular signaling
pathways. Because kinases catalyze the addition of a phosphate group to another protein
or enzyme, investigators often look for proteins that are rapidly phosphorylated, in the
hope that they will be part of a cell’s response to an extracellular message.
The presence and importance of amino acid transport and receptor proteins have been
biochemically identified for some years. However, it is only in the past decade that
definitive reports have appeared regarding the structure and function of these proteins.
cDNAs have recently been reported that encode proteins capable of the activities of the
Na+-dependent systems (A and N) and the Na+-independent systems (T and asc). This
new information will make possible the exploration of how individual transport proteins
in apical and basolateral peptide transport activities are coordinated with other transport
factors to achieve cellular, tissue, and whole-body amino acid flux capacity.29
Major progress has been made in the potential role of the excitatory receptors AMPA
(preferentially gated low-conductance ion channels permeable to Na and K and voltage
independent) and NMDA (large-conductance ion channels permeable to the Ca ion).
Characterization of these channels is important for both molecular composition and turn-
over in the synaptic membrane. Hypoxic-ischemic brain injury in animals is followed by
the lethal overstimulation of glutamate receptors. This research holds great promise for
understanding and treating hypoxic-ischemia encephalopathy in particular, as well as
addictive behavior and other neurologic disorders.
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Introduction 7

Amino acids serve as substrates for many functions besides the obvious role of protein
synthesis. These include gluconeogenesis and ureagenesis. However, they can also serve
as regulators of cell metabolism. Chapter 16 in this volume discusses ways in which amino
acid signaling can influence protein metabolism. The focus begins with the discovery of
Haussinger et al.30 that cell swelling, associated with the Na+-dependent influx of some
amino acids into cells or with the intracellular accumulation of amino acid catabolites,
such as glutamate and aspartate, exerts both anabolic and anticatabolic effects.
Later work has revealed the existence of an amino acid-stimulated signal transduction
pathway that shares components and acts in concert with a signaling pathway that is
stimulated by insulin. It is this pathway that simultaneously controls autophagic protein
degradation and protein synthesis, but in opposite directions. A central player in the
intracellular pathways discussed by these authors is mTOR. The mechanism by which
amino acids activate mTOR remains unknown, although several possibilities are presented.
The fact that amino acid signaling can behave with insulin-like actions in regard to
protein synthesis and also promote autophagic protein degradation provides a unique
challenge. The authors point out that the amino acid signaling pathway differs from
glutamate signaling via glutamate receptors in the central nervous system. Here these
receptors gate cation channels, and their activation causes depolarization and an increase
in cytosolic Ca ++ , together with interacting with phosphatidylinositol 3-kinase
(PI 3-kinase).
In amino acid signaling, the authors speculate that mTOR may also sense amino acid-
induced increases in cell volume. The importance of defining this pathway may shed light
on cancer growth and perhaps lead to treatment with agents such as rapamycin and its
analogues, which might not only inhibit protein synthesis but at the same time accelerate
autophagic protein degradation.
The anabolic effects of amino acids are further explored in Chapter 18. Hue and
Bertrand observe that in addition to their mass effect on protein synthesis, certain amino
acids inhibit autophagy in the liver. They contrast the effects of insulin, glutamine, and
leucine on protein synthesis in isolated liver cells. The central role of mTOR in the control
of protein synthesis by nutrients and energy is such that it has been proposed to act as
an ATP sensor of the cell. After presenting the detailed anabolic response of glutamine
associated with Na+ movement, they report that leucine transport does not depend on
Na+ and therefore does not cause cell swelling or glutamate-activated protein phosphatase
(GAPP) activation. The work of these authors suggests that two separate phosphatases
are involved, one upstream of mTOR and one downstream. They also note that AMPK,
a protein kinase activated by hypoxia, is able to inactivate other enzymes in the anabolic
pathway, perhaps explaining the inhibition of protein synthesis observed during oxygen
deprivation.
In recent years the intestine has received increasing attention as an organ of unique
metabolic activity involving particular amino acids. Chapter 42 discusses the intestine
from the separate perspectives of the mucosa, the local immune system, and the control
of the vasculature. This presentation focuses on the dietary provision of glutamine,
glutamate, ornithine, arginine, and glutathione and the importance of each for intestinal
integrity.
Evidence is presented that enteral glutamine is important to maintain mucosal thick-
ness, villus height, and cell count, as well as normal permeability. Of particular interest
is the idea that while increased permeability can be measured with lactulose, mannitol,
or xylose, this does not imply increased permeability to bacteria or endotoxin molecules,
which are significantly larger in size. Oral glutathione (GSH) as well as GSH monoesters
provide effective transport and conversion into intracellular GSH, while intact GSH is
poorly transported into enterocytes.
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8 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Secretory immunoglobulin A (sIgA) is produced through the interaction of sensitized


B and T cells in the lamina propria below the mucosa and moves to the mucosal surfaces
of the gastrointestinal and respiratory tracts to prevent bacterial and viral adherence to
epithelial cell layers and mucosal penetration. Glutamine has direct effects in supporting
the function of B and T cells within the mucosa to provide defenses against intraluminal
infectious agents. Provision of intravenous nutrition that lacks glutamine has shown that
the intestinal changes have implications on extraintestinal sites like the respiratory tract
as well. Thus, the concept of a common mucosal immune system (MALT) has been
proposed, which is active in defense against both bacterial and viral infections. Evidence
is presented that intracellular stores of GSH promote the production of interleukin (IL)-2
and interferon (IFN)-gamma, thus affecting IgA synthesis and mucosal protection.
Sacks and Kudsk present evidence that changes occur in an intracellular adhesion
molecule (ICAM)-1, which is responsible for adhesion between the endothelium and
polymorphonuclear neutrophils (PMNs). This can produce deleterious effects as the gut
serves as a priming bed for circulating neutrophils. Both IL-10 and IL-4 normally inhibit
ICAM-1 expression, and both cytokines decrease in response to parenteral nutrition. A
study in mice involved administration of isotopic ICAM-1 antibody and myeloperoxidase
(MPO), an enzyme found primarily in PMNs. While on intravenous nutrition, the expres-
sion of intestinal ICAM-1 significantly increased along with increases in MPO levels and
was quickly reversed with chow refeeding.
Isotopic albumin was used to measure vascular permeability in various tissues and
lung, while PMN accumulation was assessed with MPO. A significantly higher vascular
permeability was seen in lung and liver while on intravenous nutrition. Pulmonary tissues
showed marked increases in expression of CD18, a marker for PMN cell priming, while
no increases were seen in the enterally fed mice. Glutamine (GLN)-supplemented intra-
venous nutrition produced major increases in survival for 72 h after 15 min of mesenteric
artery occlusion compared to those not receiving GLN. The results of GLN were thought
to possibly reflect prevention of mucosal changes associated with increased permeability
or a decreased production of oxygen free radicals by PMNs. Other studies have suggested
that ischemia-reperfusion activates protein kinase cascades that regulate the expression
of proinflammatory genes. Excessive gene activation can lead to decreased gut-associated
lymphoid tissue (GALT) and ultimately multiple organ failure. The amino acid GLN is of
special interest since it has been shown to have beneficial effects on all three aspects —
mucosa, immunity, and vascularity — of the intestine.
Some benefits of GLN may result from an influence on arginine production. Arginine
has been reported to facilitate mucosal recovery after ischemia-reperfusion by preventing
the reduction in mucosal blood flow, presumably by providing a source of nitric oxide
(NO).
Inflammation is the response of the immune system to the damage caused by chem-
icals, physical insults, or pathogenic organisms. Although painful, inflammation is usually
a healing response. This response can spiral out of control, leading to shock and death,31,32
or it can proceed to a chronic state associated with debilitating disease. Only recently has
the great significance of inflammation been recognized in the etiology of atherosclerosis.33
The points of control in inflammation involve signaling pathways between circulating
white cells and their inflammatory products, such as hormones, cytokines, and others.34
Some biochemical agents can serve as important signals while under other circumstances
they may play deleterious roles. Notable in this regard is the molecule NO, which arises
from the amino acid arginine.
Chapter 14 on nitric oxide separates the biology and mechanism of action from a
detailed discussion of pathophysiology in mammalian cells. The sheer number and diver-
sity of roles attributed to NO are astounding: the control of blood pressure, emptying of
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Introduction 9

the stomach, penile erection, release of neurotransmitters and hormones, learning and
memory, pain sensation, and protection of cells from intracellular parasites are some of
the roles. Many of the reports seem contradictory. NO is reported to both promote and
suppress apoptosis; it is both an oxidant and an antioxidant; it is cytostatic yet promotes
tumor growth. What can explain this?
Lane and Gross35 have reviewed many of the factors that contribute to this remarkable
diversity of function. NO is unlike any other cellular messenger. It is a lipophilic, reactive,
free radical, gaseous molecule, representing the combination of two of the most abundant
atoms in our atmosphere. It is a free radical with an unpaired electron in its outer orbit,
yet it does not react readily. This slow reactivity, combined with its lipophilic nature,
allows NO to diffuse rapidly through most cells and tissues with little consumption or
reaction. Instead of binding selectively to a specific receptor, it engages in chemical reac-
tions with multiple protein targets. These may be enzymes, receptors, structural proteins,
or transcription factors. Whether the net effect of NO in a given biological system is
beneficial or deleterious to an organism is determined by the relative availability of
alternative protein targets and the duration and amount of NO produced.
The large diversity of functions may be further explained by NO being an array of
three interchangeable species (NO*, NO+, and NO–), each with a distinct spectrum of
chemical and biological activities. Synthesis of NO is mediated by a family of three
mammalian gene products termed nitric oxide synthases (NOSs). All three catalyze the
oxidation of one of the guanidinonitrogens of L-arginine, yielding NO and L-citrulline.
Each of the three NOS isoforms differs in its tissue distribution, subcellular localization,
and mode of regulation. The two constitutive forms (neuronal and endothelial) are regu-
lated predominantly by changing levels of intracellular Ca++. The remaining isoform is
the inducible NOS (iNOS), resulting from exposure to immunostimulants, which is con-
tinuous and high output, and Ca++ independent. It is regulated at the transcriptional
level, and once the protein is expressed, a large continuous flux of NO ensues that is
limited only by substrate availability.
Asymmetrical dimethylarginine (ADMA) is an endogenously produced inhibitor of
nitric oxide synthase, whereas symmetrical dimethylarginine (SDMA) competes with
arginine for transport. The metabolism of these two compounds is largely unknown.
However, ADMA is subject to enzymatic degradation by an enzyme that is highly
expressed in the liver. Nijveldt et al.36 have conducted studies in the rat demonstrating
that the liver plays an important role by removing ADMA from the systemic circulation.
Stuhlinger et al.37 present evidence indicating that ADMA is elevated in many disorders
and appears to be associated with endothelial dysfunction. There is great interest in
whether therapy that improves insulin resistance will also improve endothelial function
while lowering ADMA.38 Nijveldt et al.39 have reported that high plasma ADMA concen-
tration is an independent risk factor of mortality in intensive care patients.
Professor Cynober deserves special commendation for assembling modern contribu-
tions to such an ever-broadening field. The subject of the book embraces science in the
depths of the cell, the integration of tissues and organs, and, ultimately, the future of
optimum nutrition in human health and disease.

References
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Oxford, 2001, 30, 304 pp.
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3. Carpenter, K.J., Vitamins and amino acids 1910–1950, in Protein and Energy: A Study of
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regulation, Sci. Compass, 296, 1254–1258, 2002.
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Introduction 11

26. Langhans, W., Peripheral mechanisms involved with catabolism, Curr. Opin. Clin. Nutr.
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29. Matthews, J.C. and Anderson, K.J., Recent advances in amino acid transporters and excitatory
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30. Haussinger, D., Hallbrucker, C., vom Dahl, S., et al., Cell volume is a major determinant of
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31. Cohen, J., The immunopathogenesis of sepsis, Nature, 420, 885, 2002.
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33. Libby, P., Inflammation in atherosclerosis, Nature, 420, 868, 2002.
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36. Nijveldt, R.J., Terrlink, T., Siroen, M.P.C., Vanlambalgen, A.A., Rauwerda, J.A., and
VanLeeuwen, P.A.M., The liver is an important organ in the metabolism of asymmetrical
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Physiology and Physiopathology


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Part I

Introduction to amino acid metabolism


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chapter one

Measurement of amino acid


concentrations in biological fluids
and tissues using ion exchange
chromatography
Nathalie Neveux
Hôtel-Dieu Hospital
Patrick David
Hôtel-Dieu Hospital
Luc Cynober
Hôtel-Dieu Hospital

Contents
Introduction....................................................................................................................................18
1.1 Sampling and storage of the biological material for analysis ......................................19
1.1.1 Blood..........................................................................................................................19
1.1.2 Urine ..........................................................................................................................19
1.1.3 Other biological fluids and tissues .......................................................................20
1.2 Deproteinization of biological samples ............................................................................20
1.3 Principle of ion exchange chromatography.....................................................................20
1.3.1 Factors influencing the separation........................................................................21
1.3.1.1 Resin............................................................................................................21
1.3.1.2 Buffers .........................................................................................................22
1.3.1.3 pH ................................................................................................................22
1.3.1.4 Temperature ...............................................................................................22
1.3.2 Detection ...................................................................................................................22
1.3.3 Data processing, internal standards, and calibration solutions.......................24
1.4 Interferences ..........................................................................................................................24
1.5 Conclusion .............................................................................................................................25
References .......................................................................................................................................26

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 17
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18 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Introduction
Quantification of free amino acids present in biological fluids and tissues is an important
tool in biomedical and nutritional research and in the diagnosis of various disease states,
especially metabolic deficiencies. The main diagnostic application of free amino acid
profiling is for blood, urine, and amniotic fluid1–3 (Table 1.1). Other biological fluids such
as breast milk, saliva, synovial fluid, cerebrospinal fluid, and tears are analyzed much less
frequently.2,7 Amino acids can also be measured in numerous cells and tissues (e.g., in
liver and muscles).2
The classical amino acid analysis techniques involve separation of amino acids by ion
exchange chromatography (IEC), followed by postcolumn continuous reaction with nin-
hydrin. Originally, the analysis was performed at a constant temperature on a two-column
system, each column requiring a separate sample injection; the acidic and neutral amino
acids were separated using two elution buffers on one column, and the basic amino acids
using a single buffer on the other column.
Since the first reports8,9 describing the separation of plasma amino acids on a sul-
fonated polystyrene resin column, followed a few years later by the first automated amino
acid analyzer developed by Spackman et al.,10 many improvements have been made,
essentially to obtain faster and more sensitive analysis. Thus, time required for a complete
amino acid analysis of physiological fluids using an automatic amino acid analyzer has
decreased considerably, from 4 days in 195811 to less than 2 h today (including the column
regeneration time). In parallel, the sensitivities of these analyses have gradually increased,
and thresholds of 50 to 100 pmol and 1 pmol of amino acid have been reached using

Table 1.1 Amino Acid Concentrations in Adult Human Plasma, Urine, and Cerebrospinal
Fluid (CSF) Determined by Ion Exchange Chromatography
Amino Acids Plasma [4]a Urine [5]b CSF [6]a
Taurine 55 ± 13 16–180 (72) 6.8 ± 1.7
Aspartate 3 ±1 2–7 (4) 0.6 ± 0.3
Threonine 140 ± 33 7–29 (13) 27.7 ± 4.7
Serine 114 ± 19 21–50 (30) 24.5 ± 4.4
Asparagine 41 ± 10 5.4 ± 1.4
Glutamate 24 ± 15 <12 11.3 ± 6.4
Glutamine 586 ± 84 20–76 (36) 444 ± 84
Proline 168 ± 60 <9
Glycine 230 ± 52 43–173 (107) 4.7 ± 1.5
Alanine 333 ± 74 16–68 (30) 23.2 ± 5.1
Citrulline 38 ±8 <4 1.5 ± 0.5
Valine 233 ± 43 3–13 (5) 15 ± 2.8
Cysteine 52 + 11 6–34 (13) 0.1 + 0.1
Methionine 25 ±4 2–16 (6) 1.9 ± 0.7
Isoleucine 62 ± 14 <4 3.9 ± 1.0
Leucine 123 ± 25 2–11 (5) 10.1 ± 2.1
Tyrosine 59 ± 12 2–23 (10) 6.4 ± 1.5
Phenylalanine 57 ±9 2–19(7) 6.5 ± 1.2
Ornithine 55 ± 16 <5 3.7 ± 1.0
Histidine 82 ± 10 26–153 (79) 11.9 ± 1.7
Lysine 188 ± 32 7–58 (17) 21.7 ± 3.7
3-MH 3 ±2 19–47 (32)
Arginine 80 ± 20 <5 18.3 ± 3.2
a Mean ± SD, mmol/l.
b Range (and mean), mmol/mol of creatinine.
3-MH: 3-Methylhistidine.
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Chapter one: Measurement of amino acid concentrations in biological fluids and tissues 19

colorimetric detection and fluorescence, respectively. Further improvements have followed


the development of automatic sample loading, flow-cell design and electronic stabilization
of the detectors, reduction of the signal-to-noise ratio, function timing, and data process-
ing. Most often, a computer is used not only to operate the apparatus but also to carry
out diagnostic tests such as measuring pressure limits or monitoring buffer volumes.

1.1 Sampling and storage of the biological material for analysis


1.1.1 Blood
There are differences between some amino acid concentrations in plasma and serum,
probably linked to platelet disruption during clotting,1 and plasma samples are generally
preferred. The choice of the anticoagulant is important; heparin appears to be most often
used, although when in excess, it can cause hemolysis and consequently release ninhydrin-
positive substances from red blood cells.12 Problems may also arise with EDTA, which can
contain contaminants that cause the appearance of unusual peaks.12–14
After blood centrifugation, the plasma must be immediately sampled to avoid con-
tamination by intracellular amino acids.12,15 The plasma then needs to be deproteinized as
quickly as possible (within 1 h of blood sampling).16,17 The deproteinized sample can be
stored at –70˚C until analysis without any loss of amino acid, for several months2 to at
least 1 year.18
However, if the deproteinization cannot be carried out rapidly after blood samples
have been obtained, an alternative consists of immediately freezing the separated plasma
at –30˚C for 30 h. The frozen plasma can then be stored at –70˚C for 4 weeks before protein
removal and analysis.19 If the specimens cannot be stored at –70˚C but only at –20˚C, it is
preferable to deproteinize them, because at –20˚C amino acids are more stable in depro-
teinated plasma than in native plasma.20,21 Other reports22,23 have also recommended that
samples be stored at neutral as well as deproteinized and deep-frozen.
Capillary blood sampling from newborns is made at the heel or finger. After removal
of the first drop, at least three spots are taken on paper cards. The cards are then dried at
room temperature, and disks of controlled size are punched out and placed in diluent
buffer. After centrifugation, the supernatant, which contains free amino acids, is treated
and analyzed as for plasma.24 Blood collected on filter paper can be stored for up to 2 weeks
at room temperature (providing it is completely dry) and for at least 21 weeks at +4 to
–70˚C.25

1.1.2 Urine
As urinary amino acid content undergoes circadian variation, 24-h urine must be used for
quantitative analysis of amino acids.2 It is essential to avoid fecal and bacterial contami-
nation, which may increase or decrease the concentrations of many amino acids in urine.25
It is usual to add a preservative such as chloroform or thymol to the container. The sample
should be stored at temperatures not exceeding 4˚C, but it is preferable to freeze it at –20˚C
or below, especially if analysis cannot be performed within 24 h. Repeated freezing and
thawing increases amino acid levels and should be avoided.2 When the sample has to be
transported, particularly at ambient temperature, changes in the amino acid composition
may occur if no bactericidal agent is added. In normal situations, the low level of urinary
proteins does not interfere with amino acid profiling. In pathological states, if the presence
of an abnormal amount of proteins is measured or suspected, samples need to be depro-
teinized. In practice, it is better to deproteinize in any case, simply because this ensures
that samples have a constant pH.
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20 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

1.1.3 Other biological fluids and tissues


The same general recommendations as those for plasma are made for the storage of other
biological fluids: if the sample contains proteins, it should be rapidly deproteinized and
stored at –70˚C if it cannot be analyzed immediately.
Amino acid determination in cerebrospinal fluid is carried out first to diagnose non-
ketotic hyperglycinemia and second to measure the neurotransmitters g-aminobutyric acid
(GABA), aspartic, and glutamic acids in research studies. In the latter case, it may be better
not to deproteinize, because the neurotransmitters are present predominantly as conju-
gates and may be degraded during protein removal.26
Tissues and biopsies should be placed in liquid nitrogen immediately after sampling.

1.2 Deproteinization of biological samples


Proteins must be removed from biological fluids before they are applied to an ion exchange
resin column, because they stick to the resin, causing peak spreading and increased column
back pressure.
Numerous techniques can be used to deproteinize biological fluids before amino acid
analysis. Among these, physical techniques such as high-speed centrifugation, dialysis,
and ultrafiltration are currently not often used because, although they can achieve 100%
recovery,27 they do not completely remove proteins.28 Ultrafiltration procedures, although
simple, are not widely applied in practice because they decrease the retention times during
amino acid separation by ion exchange chromatography.2 This results in distorted sepa-
rations of critical pairs of amino acids, particularly serine–threonine and tyrosine–phenyl-
alanine.
The most widely used method for the deproteinization of a biological sample (plasma,
urine, cerebrospinal, and amniotic fluids) before ion exchange analysis is chemical pre-
cipitation with sulfosalicylic acid (SSA). It is preferable to use as little of the precipitating
reagent as possible. This represents 20 to 50 mg of the substance per milliliter of sample.
When larger amounts are used, the ion exchange separation of amino acids is distorted.29
SSA is added directly either as a powder or as a solution. Samples containing SSA should
be stirred immediately and, after 10 min, centrifuged to remove the precipitated proteins.
After SSA treatment, the acid does not have to be removed from the supernatant before
analysis, and the pH, between 1.0 and 2.0, is almost ideal for subsequent ion exchange
chromatography. Because precipitation may cause amino acid losses (from 2 to 20%), it is
important to add an internal standard.20,27 With picric acid, recoveries are better, but the
acid has to be removed before analysis by ion exchange chromatography.9,12
Deproteinization with perchloric acid or trichloracetic acid30 is usually used for tissues
because they are homogenized in these acids, but this is also possible with SSA (10:1,
w/v).31

1.3 Principle of ion exchange chromatography


The free amino acids are separated by high-performance ion exchange chromatography
with postcolumn derivatization. The resolution is based on differential interaction with a
negatively charged stationary phase (cation exchanger). The amino acids are eluted with
a gradient of acidic buffers applied stepwise. As they elute from the column, amino acids
are mixed with the derivatizing solution and sent through a reaction coil, and the reaction
products are detected continuously. Amino acids are identified and quantified by relating
the peaks to an internal standard and to a standard mixture analyzed at the start of every
new analysis series (defined as the use of a new ninhydrin solution; see below).
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Chapter one: Measurement of amino acid concentrations in biological fluids and tissues 21

Buffers Valves

Buffer Sample
pump loader
3

Sample

4
Chart recorder/
data handling
5
Column

Regeneration
buffer

Ninhydrin Ninhydrin Mixing Reaction Detector


reservoir pump manifold coil

Figure 1.1 Schematic representation of a cation exchange amino acid analyzer.

The main components of a cation exchange amino acid analyzer are set out in
Figure 1.1.

1.3.1 Factors influencing the separation


To obtain a good chromatographic separation, it is necessary to have the right combination
of resin, buffers, pH, and temperature.

1.3.1.1 Resin
The properties of the resin largely influence the performance of the analysis and especially
the separation of the amino acids. The resin consists of small beads of polystyrene, sul-
fonated to provide a negative electrical charge, and reacted with divinylbenzene to achieve
around 8 to 10% cross-linkage.32,33 The optimal cross-linkage is about 8%.8,34 At higher
values, the resolution is poor, while at lower values, the resin distorts under pressure.
The charged groups (SO3–) are associated with mobile counter-ions, either Na+ or, more
frequently, Li+. Amino acids are injected in the column at an acidic pH (about 2.2) at which
ionization of the carboxyl group is suppressed and most amino acids have a net positive
charge. They enter the pores of the resin and displace some of the bound Li+. Separation
is primarily due to differences in the pKa of amino acids and follows the general order:
acidic, neutral, basic.8,35 At pH 2.2, the most basic amino acids (histidine, lysine, and
arginine) are bound most strongly, and the most acidic (aspartic and glutamic acids) least
strongly. Increasing the pH of the mobile phase toward the isoelectric point of a given
amino acid causes loss of charge and desorption of that amino acid. Increasing the strength
of the eluting buffers decreases the interaction with the resin and also causes desorption.
Particle size of the resin is also a critical factor because it determines the diffusion
time needed to reach equilibrium. Since 1958 the advantages of decreasing particle size
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22 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

for increased speed and resolution in amino acid analysis have been demonstrated.36 In
practice, the use of increasingly fine resin beds has allowed the operating temperature,
column length, and diameter to be reduced, which in turn affords greater sensitivity and
speed of analysis. The particles are spherical with diameters ranging from 5 to 10 mm.

1.3.1.2 Buffers
To obtain a satisfactory analysis of physiological fluids, four or five buffers are required.
Currently, the most frequently used buffers are lithium citrate buffers, which have super-
seded sodium citrate buffers and have allowed the resolution of asparagine, glutamic acid,
and glutamine.37 Thiodiglycol is added as an antioxidant to prevent loss of methionine.28
Addition of organic solvents such as propanol improves the resolution of threonine and
serine.25
Chemical contamination is a recurring problem, arising from impurities in
commercial38 and extemporaneous buffers, water, and glassware, the major source of
contamination being the water used to prepare the buffer solutions.28 The use of a water
purification system is essential for the preparation of buffers, which must be membrane-
filtered before use.38 Microbial synthesis is another possible source of amino acid contam-
ination: adding a preservative such as phenol or caprylic acid inhibits bacterial growth.
The introduction of precolumn filters and buffer refrigeration in modern analyzers also
limits contamination of the column.2,38
Another problem linked to the buffer solutions is the presence of high ammonia
concentrations that may affect the baseline. This problem can be solved by introducing
an extra column between buffer outlets and the column inlet.39
The flow rate of the buffers is also critical: if it is too fast, there is asymmetry and
tailing of the peaks, loss of resolution, and increased back pressure. Precise control of both
timing of buffer change and flow rates is included in the modern analyzer.

1.3.1.3 pH
pH is critical for resolution. When it is too high, the amino acid peaks elute prematurely,
with poor resolution, and if it is too low, they elute late and are wider. The most sensitive
amino acids are those first eluted (see Figure 1.2). To avoid errors of identification with
automatic integration, the pH of the sample must be close to that of the calibrating standard
mixture.

1.3.1.4 Temperature
The separation of amino acids is dependent on temperature, partly because the pH of
citrate buffers increases with temperature, and partly because of altered affinity of amino
acids for the resin. Generally, the temperature of the column is held below 40˚C until
glutamine has eluted (see Figure 1.2) to minimize its loss and is subsequently increased
stepwise.1

1.3.2 Detection
The classical amino acid analysis technique involves separation of amino acids by cation
exchange chromatography, followed by postcolumn derivatization by a continuous reac-
tion with ninhydrin.40 The column effluent is mixed with an acetate buffer containing
reduced ninhydrin (hydrindantin) and then heated to 130 to 135˚C in a Teflon® reaction
coil. Primary amino acids form a purple chromophore and the imino amino acids (proline
and hydroxyproline) a yellow complex with absorbance peaks at 570 and 440 nm, respec-
tively. Absorbance is measured at both wavelengths, which can help in peak identification
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Chapter one: Measurement of amino acid concentrations in biological fluids and tissues 23

Figure 1.2 Chromatographic chart of amino acids using IEC-based method. (A) Calibration solution.
(B) Plasma profile. (C) Muscle profile. (D) 3-methylhistidine (short program). All these chromato-
grams were recorded on a Jeol AminoTac JLC-500V machine, except for D, which was recorded on
a Hitachi L-8500A machine.
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24 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

because the ratio A570/A440 is specific to each amino acid. Inspection of this ratio helps to
detect interference, e.g., by antibiotics (see below). As the reaction is sensitive to oxygen,
buffers and reagent must be maintained under nitrogen. To avoid the problem of ninhydrin
reagent instability, which limits its use to 1 to 2 weeks after preparation, extemporaneous
automatic mixing of the detection reagent is available on some modern apparatus.4
Postcolumn derivatization using fluorescent reactions offers an alternative to the use
of ninhydrin. Both orthophthalaldehyde (OPA) and fluorescamine have been shown to
react rapidly with primary amines to form highly fluorescent products.41–43 However, OPA
is most frequently used because it has the best fluorescent yield and is soluble and stable
in the aqueous reaction buffer (unlike fluorescamine).44 For primary amino acids, OPA is
at least 10 times more sensitive than ninhydrin, but there is a low response to cysteine.2
Hence, this type of derivatization is of particular interest for the analysis of small (i.e.,
50 mg) human biopsies (e.g., from muscles). However, except for its increased sensitivity
(1 pmol), postcolumn derivatization with OPA offers no improvement over the classical
ion exchange ninhydrin procedure. Furthermore, secondary amines do not form fluores-
cent derivatives with OPA unless oxidative reagents are present in the reaction mixture,
which necessitate two postcolumn pumps.

1.3.3 Data processing, internal standards, and calibration solutions


Computerized data processing is an essential feature of modern analyzers. Data are col-
lected and integrated, and amino acids are identified by comparison of their retention
times with a calibration solution. Results may be displayed, stored, and retrieved.
A major problem in amino acid analysis is the reliability of the commercially prepared
standard solutions used for calibration. In these solutions, concentration of several amino
acids may be significantly different from the stated concentrations.18,45 Preparation of a
standard by dissolving salts of individual amino acids can solve this problem, and for
maximum accuracy, use of calibration solutions from several sources is recommended. It
is noteworthy that glutamine is not incorporated into commercial standards because of
its instability in solution. Thus, an aqueous solution of this amino acid must be prepared
fresh for each analytical batch, and an aliquot added to the calibration mixture before
analysis.1
Precision and accuracy are improved by adding an internal standard that serves as a
control for the analytical procedure and allows correction for losses.46 By definition, the
substance chosen as the internal standard must not be present at a detectable level in the
physiological fluid being analyzed. Also, the retention time of the internal standard(s)
must not interfere with the retention time of the physiological amino acids. Since charac-
teristics of resin, buffers, etc., vary from one analyzer to another, the most suitable internal
standards may be different. For example, with our Hitachi L-8500A we use acetyl–lysine,
whereas with our Jeol AminoTac JLC-500V we use glucosaminic acid and aminoethyl–cys-
teine as internal standards.
Also, for specific diagnosis, we may be interested in a single amino acid, e.g., plasma
homocysteine as a marker of cardiovascular risk47 or urinary 3-methylhistidine (3-MH) as
a marker of muscle myofibrillar protein breakdown.48 Thus, short programs are used (so
as not to wait 1 h for 3-MH). In that case, retention times of all amino acids are modified,
and the appropriate internal standard may be different.

1.4 Interferences
Ninhydrin-positive drugs or drug metabolites are responsible for interfering peaks in
both plasma and urine samples, and these are most troublesome in urine. Among the
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Chapter one: Measurement of amino acid concentrations in biological fluids and tissues 25

Table 1.2 Performance (Between-Run Reproducibility) of IEC-Based Apparatus over the


Last 20 Years
1980 1995 2000
Chromakon 500 Hitachi L-8500A Jeol AminoTac
Amino Acids [51] [4] JLC-500Va
Taurine 11.1 1.5 2.3
Aspartate 12.5 12.0 9.5
Threonine 9.3 3.1 2.3
Serine 9.5 2.3 2.2
Asparagine 5.1 1.2
Glutamate 10.2 7.9 3.2
Glutamine 11.1 4.7 1.6
Proline 17.6 1.5 3.6
Glycine 9.3 1.8 1.9
Alanine 7.9 2.8 1.9
Citrulline 10.0 7.5 4.3
Valine 9.4 1.8 1.9
Cysteine 2.1 2.2
Methionine 10.3 1.4 3.8
Isoleucine 10.0 1.5 1.9
Leucine 8.2 1.4 1.8
Tyrosine 11.6 1.4 2.3
Phenylalanine 8.9 1.9 1.8
Ornithine 11.6 1.3 2.3
Histidine 9.6 3.0 4.0
Lysine 12.7 1.8 2.7
Arginine 7.6 7.3 4.4

Note: Data are expressed in CV %.


a Neveux et al., unpublished data.

numerous drugs involved are antibiotics (e.g., benzylpenicillin, cefotaxime, ampicillin)


and therapeutic amines or amino acid derivatives such as N-acetylcysteine, dopamine,
or D-penicillamine.2,28,49

1.5 Conclusion
It is now almost 50 years since the first automated procedure for the analysis of amino
acids by ion exchange chromatography was described.8 Although other methods have
attempted to rival this system, it still remains the most common method used for quan-
titative amino acid profiling,45,50 the analytical performance having improved dramatically
in recent years (Table 1.2). However, some problems persist independently of the instru-
mentation. These arise in sample preparation, treatment, and storage, which are frequently
neglected preanalytic stages where artifacts or specious results may originate. Cation
exchange chromatography is the method of choice when precise profiling of amino acid
in biological fluids and tissues and quantitative data are required. The separation of
underivatized amino acids is carried out by means of a stepped series of lithium citrate
buffers with ninhydrin or OPA detection. Automation and computerization have been
largely developed and are applied to automated sample delivery, automated and com-
puterized gradient formation, and quantification of the data obtained. The alternative is
reversed-phase high-performance liquid chromatography (RP-HPLC) methods with pre-
column derivatization (see Chapter 2). Advantages and drawbacks of ion exchange chro-
matography compared with reversed-phase HPLC methods are given in Table 1.3. It is
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26 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 1.3 Advantages and Drawbacks of IEC-Based Apparatus Compared to RP-HPLC with
Precolumn Derivatization

Advantages
• Allows the measurement of all amino acids
• Well standardized
• Best analytical performance (lowest within-run and between-run reproducibility CVs)
• Excellent after-sales service and methodological assistance from the supplier
• Low cost per assay (a column allows 2000 analyses and buffers are cheap)

Drawbacks
• High cost of the machine (at least twice the price of an HPLC system)
• Longer analysis time (although decreasing, it is still twice the time required with RP-HPLC)
• Fully dedicated machine (cannot be used for other applications)

clear that a fully automated apparatus based on the ion exchange method is the best option
for a laboratory with a high amino acid analysis throughput, such as in a hospital.

References
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protein metabolism, in Methods for Investigation of Amino Acid and Protein Metabolism, El-
Khoury, A.E., Ed., CRC Press, Boca Raton, FL, 1999, p. 147.
32. Yokoyama, Y., Watanabe, M., Horikoshi, S., and Sato, H., Sulfoacylated macro-porous poly-
styrene-divinylbenzene low-capacity cation exchanger selective for amino acids, Anal. Sci.,
18, 59, 2002.
33. Klingenberg, A. and Seubert, A., Sulfoacylated poly(styrene-divinylbenzene) copolymers as
resins for cation chromatography: comparison with sulfonated, dynamically coated and silica
gel cation exchangers, J. Chromatogr. A, 946, 91, 2002.
34. Long, C.L. and Geiger, J.W., Automatic analysis of amino acids: effect of resin cross-linking
and operational variables on resolution, Anal. Biochem., 29, 265, 1969.
35. Ersser, R.S. and Davey, J.F., Liquid chromatographic analysis of amino acids in physiological
fluids: recent advances, Med. Lab. Sci., 48, 59, 1991.
36. Benson, J.R., Improved ion-exchange resins, Meth. Enzymol., 47, 19, 1977.
37. Vega, A. and Nunn, P.B., A lithium buffer system for single-column amino acid analysis,
Anal. Biochem., 32, 446, 1969.
38. James, L.B., Amino acid analysis: buffers and artifacts, J. Chromatogr., 436, 80, 1988.
39. Bergström, J., Fürst, P., Noree, L.-O., and Vinnars, E., Intracellular free amino acid concen-
tration in human muscle tissue, J. Appl. Physiol., 36, 693, 1974.
40. Samejima, K., Dairman, W., and Udenfriend, S., Condensation of ninhydrin with aldehydes
and primary amines to yield highly fluorescent ternary products. 1. Studies on the mecha-
nism of the reaction and some characteristics of the condensation product, Anal. Biochem.,
42, 222, 1971.
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28 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

41. Roth, M., Fluorescence reaction for amino acids, Anal. Chem., 43, 880, 1971.
42. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., and Weigele, M., Fluores-
camine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the
picomole range, Science, 178, 871, 1972.
43. Stein, S., Bohlen, P., Stone, J., Dairman, W., and Udenfriend, S., Amino acid analysis with
fluorescamine at the picomole level, Arch. Biochem. Biophys., 155, 202, 1973.
44. Benson, J.R. and Hare, P.E., O-phthalaldehyde: fluorogenic detection of primary amines in
the picomole range: comparison with fluorescamine and ninhydrin, Proc. Natl. Acad. Sci.
U.S.A., 72, 619, 1975.
45. Parvy, Ph., Bardet, J., Rabier, D., Gasquet, M., and Kamoun, P., Intra- and inter-laboratory
quality control for assay of amino acids in biological fluids: 14 years of the French experience,
Clin. Chem., 39, 1831, 1993.
46. Gardner, M.L.G., A comparison of internal and external standardization in amino acid
analysis, Anal. Biochem., 150, 174, 1985.
47. Malinow, M.R., Plasma homocyst(e)inemia and arterial occlusive disease: a mini-review, Clin.
Chem., 41, 173, 1995.
48. Ballard, F.J. and Tomas, F.M., 3-Methylhistidine as a measure of skeletal muscle protein
breakdown in human subjects: the case for its continued use, Clin. Sci., 65, 209, 1983.
49. Parvy, P., Modification des concentrations des acides aminés plasmatiques et urinaires in-
duites par des thérapeutiques, Ann. Biol. Clin., 40, 23, 1982.
50. Rattenbury, J.M. and Townsend, J.C., Establishment of an external quality-assessment scheme
for amino acid analyses: results from assays of samples distributed during two years, Clin.
Chem., 36, 217, 1990.
51. Cynober, L., Coudray-Lucas, C., Ziegler, F., and Giboudeau, J., High performance ion-ex-
change chromatography of amino-acids in biological fluids using Chromakon 500: perform-
ance of the apparatus, J. Autom. Chem., 7, 201, 1985.
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chapter two

Measurement of amino acid


concentrations in biological fluids
and tissues using reversed-phase
HPLC-based methods
Birgit Alteheld
University of Bonn
Peter Stehle
University of Bonn
Peter Fürst
University of Bonn

Contents
Introduction....................................................................................................................................29
2.1 Analytical techniques for plasma and other tissue material ........................................30
2.2 Measurement of urinary amino acids...............................................................................34
2.3 Determination of protein-bound glutamine ....................................................................36
2.4 Quality control ......................................................................................................................36
2.5 Analytical pitfalls to consider ............................................................................................36
2.6 Reference ranges...................................................................................................................38
2.7 Conclusion .............................................................................................................................40
References .......................................................................................................................................41

Introduction
More than 50 years ago, Moore and Stein reported quantitative analyses of amino acids
by using sulfonated polystyrene resin columns;1,2 the method was automated some years
later by Spackman et al.3 It was stated, “a protein free filtrate corresponding to 4 mL of
plasma constitutes an appropriate sample size for an analysis,” and further “quantitative
determination of amino acids is made simpler and more rapid, the more complex mixtures
of blood plasma, urine and mammalian tissues can be analysed in 2 days.”3 Since these
statements, much has happened. Better resins, automated sample injectors, steering with

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 29
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30 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

microprocessors, computing integrators, and computer evaluations facilitate ultrarapid


analyses in less than 13 min in 10 mL of ultrafiltrate.4
At present, many laboratories still use the original cation exchange method, yet mod-
ified and modernized.5,6 In Chapter 1 the classical procedure for amino acid analysis is
described; it involves separation of amino acids by ion exchange chromatography followed
by derivatization with ninhydrin. The disadvantage of this method includes the
low-mobile-phase flow rate, thereby limiting the rate of sample analyses. A further draw-
back is the relatively poor sensitivity for free amino acids. Sensitive detection methods
are highly desirable, considering the remarkable achievements related to research on
amino acid metabolism of individual tissues and cells. The substantial progress made in
the field of analytical biochemistry is exemplified by the explosion of new information
about the potential uses of reversed-phase high-performance liquid chromatography (RP-
HPLC). Numerous reports emphasize the use of RP-HPLC in amino acid analysis. Indeed,
the application of RP-HPLC reduces the time required for analysis and increases the
sensitivity for quantification of amino acids.7
There are other methods, employing gas chromatography, combined gas chromato-
graphy–mass spectrometry, and mass spectrometry, used mainly for the tandem determi-
nation of individual amino acids or small groups of amino acids and related metabolites.
Capillary zone electrophoresis is an extremely sensitive method that is until later applied
only to protein hydrolysates, though its implication in biological fluids is certainly prom-
ising.8 The interested reader is referred to available reviews in order to deal with these
exceptional and auspicious methods.9 The present compilation is restrictively devoted to
describing recent advances with RP-HPLC of free amino acids, especially in biological
material.

2.1 Analytical techniques for plasma and other tissue material


According to our experience, five automated or semiautomated precolumn derivatization
methods are generally used for determination of free amino acids in biological fluids,
including muscle tissue, by using 9-fluorenylmethyl chloroformate (FMOC-Cl), phenyl
isothiocyanate (PITC), o-phthaldialdehyde (OPA), 1-dimethylaminonaphthalene-5-sul-
phonyl chloride (dansyl-Cl), and ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate
(SBD-F) for thiols. The superior sensitivity and practicability favors the use of three of
them, namely, OPA, dansyl-Cl, and SBD-F.
Because of instability of the OPA adducts, automated on-line derivatization is required
when using this method in general practice. Reliable automated assessments of plasma,
muscle, and liver free amino acids are facilitated with the method in 250 mL of plasma or in
biopsy specimens of about 1 mg of tissue in about 40 min.4,10 A representative chromatogram
is given in Figure 2.1. The major disadvantage of the OPA method lies in the fact that only
primary amines form adducts. This means measurements of proline and hydroxyproline are
not feasible with this method. Cyst(e)ine also cannot be measured with this derivatization
because of quenching of the fluorescence (Table 2.1).
OPA derivatization also allows detection of the rare amino acid N-6-trimethyllysine
(TML) that is formed by posttranslational methylation of lysine. A detection limit of 3.5
pmol could be achieved that requires a volume of 10 mL of plasma for the sample
preparation procedure for TML extraction.11
An ultrarapid and sensitive OPA/3-mercaptopropionic acid method has been devel-
oped by using 3-mm particle-size reversed-phase columns, enabling separation of 26 major
tissue free physiological amino acids in the lower picomole range in 12.7 min (still the valid
world record).4 Ultrasensitive applications are the micro- and narrow-bore methods,
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Chapter two: Measurement of amino acid concentrations in biological fluids and tissues 31

Figure 2.1 Typical chromatograms of OPA derivatized (A) amino acid standard and (B) human plasma.
Chromatographic conditions: column, Spherisorb ODS II (3 mm) (150 ¥ 4.5 mm, I.D.); fluorescence
detection, lex = 330 nm, lem = 450 nm; injection volume, 20 mL; standard, 10 pmol/amino acid.

employing RP-HPLC columns with internal diameters of 1.0 and 1.8 mm, respectively. This
method requires special and individual care and thus is not suitable for routine applications.
The sensitivity of the analysis is about 25 fmol and 1 pmol of amino acid per injection,
respectively, and the interassay reproducibility and reliability range between 4 and 8%
(C.V.). The separation of 23 major free amino acids can be accomplished in 22 min. The
limit of detection is about 5 and 150 fmol at a signal-to-noise ratio (S/N) of 2.5, respectively.
For the narrow-bore application, a tissue specimen of 1 mg wet weight is an appropriate
sample size. The great advantage of narrow-bore chromatography compared to conven-
tional column technology is the considerably reduced consumption of expensive and pol-
luting organic solvents, the actual use of such reagents with the narrow-bore method being
only 15 to 20% of that with conventional RP-HPLC (Figure 2.2) (see Fürst et al.7).
Precolumn derivatization with FMOC-Cl permits the fluorimetric detection of primary
and secondary amino acids as stable FMOC adducts, while determination of free
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32 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 2.1 HPLC Analyses of Free Amino Acids and Thiols: Comparison of Five Derivatization
Methods
SBD-F
Parameter OPAa FMOC-Clb PITCa Dansyl-Cla (Thiols)c
Limit of sensitivity, 0.8 0.2 5.0 1.5 0.1–0.3
pmol (S/N = 2.5)
Error of the method 1.0–4.7 0.7–4.9 3.6–7.0 1.7–4.5 2.0–3.2
(C.V., %)
(based on duplicate
determinations)
Reproducibility 0.4–2.2 0.9–3.8 2.6–5.5 1.5–4.1 2.0–6.6
(interassay, C.V., %)
Stable adducts N Y Y Y Y
Detection of N/N Y/N Y/Y Y/Y N/Y
secondary
amines/cystine
Laborious – +++ ++ + +
Problematic amino Asp/Trp Trp, His Orn, Trp, His, His, Asn
acids Cystine

Note: Y = yes; N = no; – = not; + = slightly; ++ = moderate; +++ = very.


a Reprinted from Fürst, P. et al., J. Chromatogr., 499, 557–569, 1990.
b Reprinted from Bank, R.A. et al., Anal. Biochem., 240, 167–176, 1996.
c Reprinted from Kuhn, K.S. et al., Clin. Chem., 46, 1003–1005, 2000.
Source: Reprinted from Fürst, P. et al., J. Chromatogr., 499, 557–569, 1990; Bank, R.A. et al., Anal. Biochem., 240,
167–176, 1996; Kuhn, K.S. et al., Clin. Chem., 46, 1003–1005, 2000.

tryptophan and cyst(e)ine is not possible with FMOC-Cl, because the fluorescence of the
adducts is quenched. The FMOC-Cl method suffers from the disadvantage that an excess
of strong fluorescent reagent has to be extracted manually with pentane as FMOC-OH, in
order to stop the derivatization reaction and to avoid spontaneous hydrolysis of the FMOC
adducts. This laborious manual extraction procedure prevents the wide acceptance of this
method. This shortcoming, however, might be overcome by using specially designed
autosamplers or a combination of the FMOC-Cl and OPA methods.7
A recent modification in the FMOC-Cl method improved recovery of the so far critical
amino acid histidine by using a different pH for the derivatization (pH 11.4 instead of the
originally described pH 7.7). A threefold improvement in sensitivity could be achieved
by the selection of a higher-emission wavelength (630 nm instead of 313 nm) (Table 2.1).12
Application of the PITC method, although less sensitive, is useful in clinical chemistry,
where sample availability is rarely a problem. Determination of free cyst(e)ine is not
practicable because of poor linearity and reproducibility (Table 2.1). In addition, we
observed rapid deterioration of the column when analyzing tissue material (e.g., muscle).
In our experience, a maximum of 150 physiological analyses per column could be per-
formed in spite of the use of rigorous sample preparation and suitable guard columns
containing the same resin.7 This is a serious shortcoming of the PITC method.
Dansyl-Cl is a well-known fluorogenic reagent for the determination of primary and
secondary amines. Adducts are formed at room temperature in the dark. In contrast to
the PITC method, the dansyl-Cl technique shows excellent linearity for cyst(e)ine and also
for cyst(e)ine-containing short-chain dipeptides (Figure 2.3). Hence, the dansyl-Cl method
appears to offer a suitable quantitative approach for measuring free and peptide-bound
cyst(e)ine in biological material by RP-HPLC techniques.
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Chapter two: Measurement of amino acid concentrations in biological fluids and tissues 33

Figure 2.2 Narrow-bore HPLC of (A) a standard mixture and (B) TCA-precipitated rat plasma
sample after derivatization with OPA-3-MPA. Chromatographic conditions: column, Spherisorb
ODS II (3 mm) (200 ¥ 1.8 mm, I.D.); fluorescence detection, lex = 330 nm, lem = 450 nm; injection
volume, 1 mL; standard, 1 pmol/amino acid.

Furthermore, derivatization with dansyl-Cl enables the determination of the thiol


glutathione (Mercaptopropionic acid) in reduced (GSH) and oxidized (GSSG) forms, which
are important intracellular antioxidants and measured for calculation of redox potentials.13
Red blood cells contain an approximately 500 times higher GSH concentration than
plasma, so that minor hemolysis (0.1 to 1%) can result in erroneously high plasma values.
GSH concentrations might change in the sample due to oxidation or degradation by
l-glutamyltranspeptidase. Samples should therefore be preserved by addition of
serin–borate (inhibits enzymatic degradation), bathophenanthroline disulfonate (BPDS)
(inhibits oxidation), and iodacetic acid for alkylation of GSH.13 Some thiols like homo-
cysteine, mixed disulfides, and, to a lesser extent, cysteine are unstable when stored
without prior deproteinization.14
Glutathione can be measured alternatively with SBD-F derivatization (vide infra). This
method is especially suitable for assessment for tissue glutathione.
Derivatization with SBD-F was first used for determination of homocysteine.15 Line-
arity was shown up to 500 mmol/L with a detection limit of 0.3 pmol per injection. Total
imprecision was determined with plasma samples and revealed a coefficient of variation
of 3.0%; the within-run component of imprecision was 2.3%. Recently, SBD-F has also been
used to analyze glutathione both in plasma and in tissue samples.16 SBD-F possesses major
advantages for measuring thiols: high reactivity with the thiol compounds, a low detection
limit, high stability of the derivatives, and lack of native fluorescence or fluorescent by-
products.17 A routinely manageable method for the separation of GSH, Cys, g-Glu-Cys,
and Cys-Gly in small tissue specimens is reported, with detection limits of 0.3 pmol for
Cys, 0.2 pmol for g-Glu-Cys, and 0.1 pmol for Cys-Gly and GSH (Table 2.1).16
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34 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 2.3 Elution profile of a human plasma sample after derivatization with dansyl-Cl. Chromato-
graphic conditions: column, Spherisorb ODS II (3 mm) (125 ¥ 4.5 mm, I.D.); fluorescence detection,
lex = 330 nm, lem = 550 nm; injection volume, 20 mL.

The results obtained with HPLC methods compare favorably with those derived from
conventional ion exchange amino acid analyses (Table 2.2). According to our experience,
the separation remains satisfactory for at least 600 OPA and FMOC-Cl, 300 dansyl-Cl, and
150 SBD-F analyses. Careful control of factors and limitations inherent to the various
methodologies is a prerequisite for proper identification and appropriate quantitation. The
sensitivity, errors of the methods, advantages and disadvantages, and problems with
certain “difficult” amino acids are summarized in Table 2.1.

2.2 Measurement of urinary amino acids


There are few reports of controlled analyses of amino acids in urine by HPLC. Precolumn
OPA derivatization has been applied to urine.18 Due to the presence of numerous primary
and secondary amines and aromatic compounds, the baseline is usually severely disturbed
and quantitative evaluation of the data is difficult because of the large range of concen-
trations within the sample. PITC methods have also been used, yet aromatic compounds
with absorption at 254 nm cause interferences, especially in the front part of the chromato-
gram.19,20 A separate extraction on ion exchange resin20 or electrochemical detection may
eliminate disturbing interferences,21 but this is at the expense of increased baseline noise
and instrument instability. The major drawback concerning assessment of urinary free
amino acids is that there is substantial background noise resulting from interfering sub-
stances and the columns can only be used for a rather small number of analyses.
How to collect the sample is an essential question. The excretion rate of amino acids
varies independently of creatinine over 24 h. Therefore, when using creatinine as a reference
in random urine samples, a higher variation is to be expected than in 24-h collections.22 It
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Chapter two: Measurement of amino acid concentrations in biological fluids and tissues 35

Table 2.2 Free Amino Acid Concentrations in Human Plasma (mmol/L) Determined by HPLC
(Precolumn Derivatization with OPA, FMOC-Cl, and PITC) and Ion Exchange Chromatography
(Ninhydrin Postcolum Derivatization)
HPLC
Ion exchange OPA
Amino acid (ninhydrin) (automated) FMOC-Cl PITC
Glu 23.0 ± 8.4 16.4 ± 4.7 19.2 ± 4.0 11.6 ± 3.9
Asn 38.4 ± 7.1 38.4 ± 10.9 41.2 ± 7.0 43.8 ± 9.5
Ser 119.9 ± 10.6 99.4 ± 26.6 103.3 ± 18.3 102.0 ± 13.5
Glu 574.5 ± 31.8 556.7 ± 31.5 547.3 ± 51.1 493.1 ± 56.9
Gly 262.2 ± 46.6 214.5 ± 34.7 207.1 ± 29.5 191.8 ± 23.4
Thr 114.7 ± 30.3 96.5 ± 30.1 102.1 ± 20.3 116.6 ± 20.8
His 85.7 ± 23.2 85.5 ± 16.4 69.7 ± 20.5 79.8 ± 20.3
Ala 353.5 ± 105.1 319.0 ± 99.4 303.7 ± 70.7 339.9 ± 51.5
Tau 60.7 ± 8.8 38.9 ± 9.8 38.7 ± 4.4 59.4 ± 16.8
Arg 84.8 ± 7.2 74.3 ± 29.9 74.9 ± 19.6 77.5 ± 11.6
Tyr 66.8 ± 17.2 48.1 ± 17.0 51.4 ± 11.1 54.7 ± 5.0
Val 198.7 ± 36.9 209.1 ± 40.6 185.3 ± 43.2 222.2 ± 28.3
Met 28.7 ± 12.4 35.0 ± 7.5 25.3 ± 7.0 25.9 ± 2.8
Ile 54.2 ± 12.1 51.1 ± 6.6 50.4 ± 13.8 68.7 ± 10.3
Phe 53.7 ± 12.9 52.2 ± 12.3 47.5 ± 8.2 60.3 ± 4.8
Trp 45.8 ± 17.4 34.5 ± 7.6 — —
Leu 114.9 ± 24.7 112.7 ± 24.2 104.2 ± 29.2 141.6 ± 16.8
Orn 80.0 ± 23.2 81.4 ± 15.1 78.5 ± 10.8 —
Lys 195.9 ± 44.7 182.7 ± 39.4 178.1 ± 40.7 202.7 ± 43.6
Pro 205.3 ± 40.8 — 193.3 ± 50.6 240.8 ± 48.1

Note: Results are means ± S.D. (n = 10).


Source: Reprinted from Fürst, P. et al., J. Chromatogr., 499, 557–569, 1990. With permission.

is important to avoid fecal and bacterial contaminations since they increase concentrations
of many amino acids.23,24 It is recommended that a suitable preservative be added to the
specimen container, like toluene, chloroform, or thymol. Samples should be stored at –20˚C
or lower until analysis. Repeated freezing and thawing increase amino acid concentra-
tions.25
Special attention should also be paid to the excretion of 3-methylhistidine (3-MeHis),
which has been suggested as a suitable marker amino acid for measurements of myofibril-
lar protein breakdown.26 An automated method for the determination of urinary 3-MeHis
has been recently described.27 There is considerable controversy on the interpretation of
3-MeHis excretion rates and whether nonmuscle or smooth muscle sources of 3-MeHis
may contribute significantly to urinary 3-MeHis.28,29 This suggests not only the need for
considerable caution in the interpretation of 3-MeHis data but also the need for further
investigation. Meanwhile, its value for monitoring the response to treatment in severe
trauma and acute illnesses has been encouraging.29,30
Urinary hydroxyproline (Hyp) might be a good marker of bone turnover, since it is
released during collagen breakdown. Thus, increased Hyp excretion indicates enhanced
bone turnover, like in Paget’s disease and malignancies.31,32 RP-HPLC methods have been
reported by using derivatizing agents PITC, dansyl-Cl, OPA, and FMOC-Cl.33,34 Recently,
a new procedure has been developed for quantification of Hyp by prior elimination of all
primary amino acids with nitrous acid to the corresponding hydroxyl acids. Amino acids
are transformed into N-nitroso derivatives that are treated in a following step with HBr
for denitrosation. Finally, the remaining secondary amino acids, including Hyp, are deriva-
tized preferentially with dansyl-Cl.
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36 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Pyridinoline and deoxypyridinoline are 3-hydroxypyridinium derivatives, present


only in mature collagen. Urinary excretion of these pyridinoline cross-links determined
are increased during normal growth and in disorders with increased bone resorption.34–36

2.3 Determination of protein-bound glutamine


Glutamine is considered to be a conditionally essential amino acid during episodes of
catabolic stress and malnutrition. Knowledge of glutamine contents of natural proteins is
thus of utmost importance. Quantitative assessment of protein-bound glutamine is, how-
ever, hampered by glutamic acid formation during acid hydrolysis, invalidating subse-
quent distinction between glutamine and glutamic acid residues.
Reliable assessment of the true glutamine content might be obtained using laborious
and cost-intensive biotechnological methods (cDNA technology) or might be acquired
from sequence analyses (purified protein fragments are requested).
Recently, an easy and rapid procedure for the determination of glutamine in isolated
proteins has been developed. It involves a prehydrolysis reaction of glutamine residues
with bis-(1,1-trifluoroacetoxy)-iodobenzene (BTI) to yield acid-stable L-2,4-diaminobutyric
acid (DABA), protein hydrolysis using the microwave technique.37 Subsequent amino acid
analyses are performed with dansyl-Cl derivatization of amino acids as described above.
The RP-HPLC analysis of a human muscle specimen is shown in Figure 2.4. DABA and
the proteic amino acids could be simultaneously measured with high sensitivity (2.0
pmol/injection; S/N = 3.1) and good reproducibility (C.V. = 2.3%, measured as interassay
variability of the derivatization plus chromatographic procedure from analyses of 20
standard solutions). The linearity between DABA formation and glutamine concentration
was excellent (r = 0.9996).

2.4 Quality control


The important issue of the quality of analytical results is the scope of discussion in Chapter
1. It is, however, pertinent to note that the majority of efforts concerning quality assurance
are devoted to ion exchange with ninhydrin. The French external scheme operative since
1978, including 49 laboratories, reports on intra- and interlaboratory quality control in
biological fluids in 94% from ion exchange and in 6% from gas chromatography methods.38
A quality assurance scheme has been operating in the U.K.; the combined results from 26
(23 used ion exchange and 4 HPLC) laboratories were published. ERNDIM is the European
Research Network for Evaluation and Improvement of Screening, Diagnosis and Treat-
ment of Inherited Disorders of Metabolism; it reflects a system of quality control of
laboratory measurements. ERNDIM was founded in 1994 in Maastricht, The Netherlands.
The section Quantitative Amino Acids includes 160 participants, mainly working with ion
exchange chromatography. Indeed, it is desirable to establish a European quality assurance
scheme for amino acid analyses in biological fluids with HPLC as soon as possible.
Considering the concerted action of research efforts as harmonized by the European Union,
this would be a highly realistic approach. Validation and recovery data for useful com-
parisons are warranted.

2.5 Analytical pitfalls to consider


Successful normalization or partial correction of an abnormal amino acid pattern is often
taken as a guarantee for adequate amino acid utilization. However, the limitations inherent
in methodology and in data interpretation are sadly often neglected. There are many
factors that influence amino acid concentrations. Particularly important points to consider
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Chapter two: Measurement of amino acid concentrations in biological fluids and tissues 37

Cys-Gly
240

MPG
M illivolts 180

GSH
Cys

Hcy
120

Glu-Cys
60

1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50

Cys Time (min)

B
960

720
M illivolts

Cys-Gly

480
Hcy

MPG
Glu-Cys

GSH

240

1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50

Time (min)

Figure 2.4 Typical chromatogram of (A) a standard mixture of thiols and (B) human plasma sample
after derivatization with SBD-F. Chromatographic conditions: column, Hypersil ODS II (3 mm) (150
¥ 4.6 mm, I.D.); fluorescence detection, lex = 385 nm, lem = 515 nm; injection volume, 20 mL.

are when to take the sample, how to perform the sampling, and what kind of sample will
be analyzed.
Diurnal variations in amino acid levels should be contemplated.39 It is important to
emphasize that alterations in amino acid rhythmicity are easily induced by the onset of
an acute illness or during chronic disease states.39 Thus, adequate control observations are
especially pertinent during any study of amino acid absorption after feeding a test meal
or an amino acid load or infusion.
The state of nutrition must also be considered. Many patients are first seen when acutely
ill, often at times of diminished food intake. Under such conditions it is necessary to
segregate the amino acid imbalance caused by secondary illness from that associated with
the primary disease and the effects of nutritional supplementation.
Apparent steady state: In most cases samples are collected after an overnight fast. Slight
variations in sampling time after an overnight fast may not seriously affect plasma amino
acid concentrations.40 Postprandial sampling or sampling during ongoing parenteral nutri-
tion during a given phase of treatment may provide a more adequate picture of the amino
acid steady state. Postprandial changes in total homocysteine (tHcy) concentrations
include a modest decrease in the first few hours, followed by an increase after 8 h; therefore,
fasting blood samples are recommended to minimize any effects of meals.41,42
Physical activity40 — rarely controlled — as well as alterations that have been reported
throughout the menstrual cycle43 might also be considered.
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38 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 2.5 The amino acid concentrations in plasma and muscle as a function of sex and age in chronic
uremic patients compared with data derived either from mixed healthy controls or from age- and sex-
matched controls. (Results adapted from Alvestrand, A. et al., Clin. Nephrol., 18, 297–305, 1982.)

Technical errors: Repeated venipuncture lowers the values of taurine and glutamic acid
and arginine since arginase is released during the clotting process.44 The majority of amino
acid levels in serum are not only considerably higher than in plasma but also more
variable.40 The choice of anticoagulant can also be important,45 as discussed in Chapter 1.
Immediate and adequate deproteinization is important to avoid certain errors and artifacts,
such as enhanced deamidation of glutamine46 and significant losses of disulfide amino
acids.44,47 Glutamic acid and aspartatic acid rise slowly and glutamine and asparagine fall
equivalently in samples stored at –20˚C.40,46–48 Additional loss of cyst(e)ine is reported.40,46–48
Contamination with sweat,49 from platelets and leukocytes44,45 or hemolysis,40,44 will result
in errors and artifacts that can jeopardize the reliability of analyses.
Age and sex are two further important factors affecting amino acid concentrations.50,51
Measurements of free amino acids are usually made at a single point in time, and it is
upon such a determination that one needs to decide whether a subject manifests normal
or abnormal amino acid metabolism. Thus, an adequately controlled database is of the
utmost importance in evaluating the results. As exemplified in Figure 2.5, patient data
may reveal a completely normal level of an amino acid when compared with healthy
controls, but the value is, as a matter of fact, significantly and markedly reduced when
the comparison is made with adequate age- and sex-matched controls. In contrast, an
apparently increased value is in reality completely normal when age- and sex-matched
controls are used for comparison.

2.6 Reference ranges


Reference data for plasma, muscle, and erythrocytes are presented in Table 2.3 simulta-
neously obtained from healthy subjects. As emphasized, age and sex are important factors,
Table 2.3 Simultaneous Measurements of Free Amino Acid Patterns of Plasma, Muscle, and Erythrocytes in 27 Healthy Human Subjects
Plasma Muscle RBC Gradient Gradient Gradient
mmol/L PlW mmol/L ICW mmol/L ICW RBC/Plasma Muscle/plasma Muscle/RBC
Chapter two:

Essential
Histidine 87± 3 592 ± 54 120 ± 18 1.3 ± 0.05 6.4 ± 0.51 5.0 ± 0.44
Isoleucine 63 ± 3 68 ± 4 71 ± 3 1.0 ± 0.03 1.0 ± 0.05 1.1 ± 0.05
Leucine 120 ± 5 133 ± 6 137 ± 6 1.1 ± 0.04 1.1 ± 0.04 1.0 ± 0.05
Lysine 195 ± 9 994 ± 77 177 ± 5 0.9 ± 0.04 5.0 ± 0.42 5.6 ± 0.45
Methionine 25 ± 1 41 ± 6** 20 ± 3 0.8 ± 0.04 1.6 ± 0.28** 2.0 ± 0.26**
Phenylalanine 53 ± 2 62 ± 3 62 ± 2 1.1 ± 0.05 1.1 ± 0.06 1.0 ± 0.05
Threonine 128 ± 5 571 ± 31 157 ± 6 1.1 ± 0.06 4.3 ± 0.2 4.0 ± 0.24
1382_C02.fm Page 39 Tuesday, October 7, 2003 6:01 PM

Tyrosine 60 ± 4 87 ± 4 82 ± 5 1.3 ± 0.06 1.4 ± 0.07 1.1 ± 0.04


Valine 220 ± 8 253 ± 11 248 ± 9 1.1 ± 0.03 1.1 ± 0.04 1.1 ± 0.03

Non-essential
Alanine 316 ± 17 2249 ± 96 419 ± 16 1.3 ± 0.05 6.8 ± 0.3 5.5 ± 0.2
Arginine 86 ± 3 633 ± 46 258 ± 23 2.8 ± 0.28 6.8 ± 0.5 4.3 ± 1.2
Asparagine 47 ± 2 266 ± 11 155 ± 4 3.1 ± 0.10 5.4 ± 0.3 1.8 ± 0.07
Citrulline 34 ± 1 170 ± 13 47 ± 2 1.3 ± 0.05 4.7 ± 0.4 3.8 ± 0.31
Glutamic acid 32 ± 4 4015 ± 249 446 ± 17 14.0 ± 2.65 123 ± 14 8.6 ± 1.42
Glutamine 655 ± 17 20050 ± 514 758 ± 15 1.1 ± 0.04 29 ± 0.9 26.9 ± 0.8
Glycine 248 ± 13 1304 ± 75 544 ± 21 2.2 ± 0.11 5.0 ± 0.3 2.5 ± 0.14
Ornithine 66 ± 4 493 ± 50 271 ± 18 4.4 ± 0.42 5.3 ± 0.8 2.2 ± 0.16
Serine 114 ± 4 584 ± 24 211 ± 6 1.8 ± 0.05 5.0 ± 0.2 2.9 ± 0.12
Taurine 49 ± 3 19194 ± 676 196 ± 28 4.0 ± 1.14 385 ± 17 184 ± 22
Carnosine 6130 ± 370
 EAA 857 ± 27 2831 ± 91 1034 ± 23 1.1 ± 0.04 3.1 ± 0.15 2.8 ± 0.15
 NEAA* 1446 ± 38 29101 ± 696 2345 ± 41 1.5 ± 0.05 18.1 ± 0.56 11.8 ± 0.38
 BCAA 402 ± 15 457 ± 19 456 ± 16 1.1 ± 0.04 1.1 ± 0.04 1.0 ± 0.04
 AA 2303 ± 58 31902 ± 729 3380 ± 53 1.4 ± 0.04 12.6 ± 0.43 9.1 ± 0.25

Note: Values are means ± SEM. ** in 5 subjects only; PLW, plasma water; ICW, intracellular water; RBC, erythrocytes; Â, sum of; EAA, essential amino
Measurement of amino acid concentrations in biological fluids and tissues

acids; NEAA, non-essential amino acids; BCAA, branched-chain amino acids. * Taurine, citrulline and ornithine are not included in the sum.
Source: Reprinted from Divino Filho, J.C. et al., Clin Nutr., 16, 299–305, 1997.
39
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40 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 2.4 Free Amino Acids in Plasma and in Muscle in Young and Elderly Males
Plasma amino acids Muscle amino acids
(mmol/L) mmol/L of intracellular water)
Elderly men Young men Elderly men Young men
(n = 10) (n = 14) (n = 10) (n = 12)
Taurine 99.2 ± 13.7 84.1 ± 8.6 19.46 ± 2.20 15.34 ± 1.03
Aspartate 7.4 ± 0.6 9.6 ± 2.1 1.83 ± 0.13 1.18 ± 0.13**
Serine 136.2 ± 9.7 120.0 ± 7.4 1.16 ± 0.13 1.45 ± 0.28
Asparagine 53.0 ± 2.7 63.4 ± 6.1 0.36 ± 0.06 0.57 ± 0.11
Glutamate 46.2 ± 6.8 34.1 ± 3.5 4.84 ± 0.47 3.93 ± 0.21
Glutamine 808.4 ± 56.0 696.5 ± 34.3 23.62 ± 2.55 18.47 ± 1.45
Proline 235.2 ± 45.0 192.4 ± 15.2 1.22 ± 0.19 1.04 ± 0.11
Glycine 256.1 ± 25.4 258.2 ± 15.1 2.21 ± 0.29 1.18 ± 0.19
Alanine 396.0 ± 28.5 346.7 ± 30.2 3.57 ± 0.44 2.51 ± 0.28*
Citrulline 48.6 ± 5.7 39.7 ± 7.6 0.25 ± 0.06 0.05 ± 0.01**
Ornithine 116.3 ± 7.4 96.9 ± 8.1 0.56 ± 0.07 0.40 ± 0.06
Histidine 106.9 ± 10.5 77.0 ± 5.2** 0.61 ± 0.08 0.44 ± 0.03*
Arginine 113.7 ± 19.8 81.6 ± 7.3 0.89 ± 0.07 0.51 ± 0.04***
Threonine 162.9 ± 18.6 129.9 ± 9.8 0.89 ± 0.07 0.81 ± 0.04
Valine 279.5 ± 25.0 205.6 ± 22.2* 0.44 ± 0.03 0.39 ± 0.04
Methionine 28.2 ± 2.2 31.9 ± 5.1 0.05 ± 0.01 0.05 ± 0.01
Isoleucine 70.1 ± 5.7 62.2 ± 5.3 0.11 ± 0.01 0.09 ± 0.01
Leucine 143.7 ± 9.2 121.3 ± 11.6 0.22 ± 0.01 0.17 ± 0.02*
Tyrosine 99.1 ± 7.7 60.0 ± 5.0** 0.05 ± 0.01 0.06 ± 0.01
Phenylalanine 62.4 ± 4.5 62.6 ± 5.4 0.07 ± 0.02 0.06 ± 0.01
Lysine 220.2 ± 18.5 165.9 ± 14.1* 1.49 ± 0.13 0.88 ± 0.09***
ÂNEAA 2.43 ± 0.16 2.15 ± 0.09 45.33 ± 3.02 31.03 ± 1.87***
ÂEAA 0.97 ± 0.07 0.78 ± 0.06* 3.18 ± 0.22 2.52 ± 0.13*
ÂNEAA/ÂEAA 2.59 ± 0.10 2.82 ± 0.16 18.61 ± 1.11 12.54 ± 0.79***
Tyr/Phe 1.60 ± 0.06 1.03 ± 0.12*** 0.83 ± 0.11 1.31 ± 0.34
Gly/Val 0.96 ± 0.12 1.55 ± 0.26 5.06 ± 0.58 5.29 ± 0.55

Note: Young, 20–36 years; elderly, 52–77 years. Mean values ± SE are shown. * 0.01 < P < 0.05; ** 0.001 < P
< 0.01; *** P < 0.001. EAA and NEAA, essential and non-essential amino acids, respectively.
Source: Reprinted from Möller, P. et al., Clin. Sci. (Lond.), 56, 427–432, 1979.

significantly affecting amino acid concentrations. Table 2.4 and Table 2.5 present repre-
sentative data for plasma and muscle in young and elderly females and males, respectively.
Great caution should be exercised at interpretation of normal ranges. Physiological
deviations are to be considered since amino acid concentrations are affected by many
variables. As pointed out previously, an adequately controlled database, i.e., age- and sex-
matched control material, is of essential importance for adequate data interpretation.

2.7 Conclusion
Quantitative analysis of amino acids in biological fluids is a critical issue: success depends
upon fastidious attention to details not only concerning analytical technique, but also the
tiniest details related to sampling and preparation procedures. The presented HPLC meth-
ods have many practical advantages over the classical ion exchange method; reduced
analysis time and cost, improved sensitivity, and more robust instrumentation are a few.
Along with good separation of amino acids, data interpretation of abnormalities is a major
task that requires considerable experience and knowledge not only in analytical chemistry
but also in the fields of pathophysiology and medicine. Keeping with this background,
1382_C02.fm Page 41 Tuesday, October 7, 2003 6:01 PM

Chapter two: Measurement of amino acid concentrations in biological fluids and tissues 41

mmol/L) and in Muscle (mmol/L Intracellular Water) in


Table 2.5 Free Amino Acids in Plasma (m
Young and Elderly Females
Plasma Muscle
Elderly females Young females Elderly females Young females
n = 91 n = 11 n = 91 n = 11
65.6 ± 1.8 years) 27.9 ± 1.65 years 65.6 ± 1.8 years 27.9 ± 1.65 years
Taurine 108.2 ± 11.1 108.2 ± 15.4 21.20 ± 2.02 24.00 ± 2.40
Aspartate 19.1 ± 1.8 17.9 ± 1.9 1.98 ± 0.20 2.45 ± 0.21
Serine 177.2 ± 8.6 188.2 ± 13.5 1.15 ± 0.11 1.42 ± 0.19
Asparagine 64.9 ± 4.7 63.8 ± 5.3 0.52 ± 0.05 0.46 ± 0.06
Glutamate 31.2 ± 1.9* 42.6 ± 4.1 4.97 ± 0.34 5.15 ± 0.36
Glutamine 707.4 ± 32.4 637.2 ± 31.6 21.47 ± 1.38 20.88 ± 1.04
Proline 225.1 ± 30.6 233.1 ± 25.0 0.80 ± 0.07* 1.47 ± 0.24
Glycine 363.4 ± 39.4 331.7 ± 32.4 1.86 ± 0.17 2.33 ± 0.27
Alanine 449.5 ± 38.2 388.1 ± 36.1 2.85 ± 0.10 3.58 ± 0.38
Citrulline 65.0 ± 7.2* 35.8 ± 3.5 0.12 ± 0.03 0.13 ± 0.03
Ornithine 114.1 ± 7.7 114.8 ± 12.9 0.45 ± 0.05 0.44 ± 0.04
Histidine 100.6 ± 2.8* 113.8 ± 3.5 0.35 ± 0.02* 0.58 ± 0.06
Arginine 88.0 ± 7.8 72.4 ± 6.7 1.05 ± 0.37 0.95 ± 0.07
Threonine 188.2 ± 6.0 208.8 ± 12.9 0.84 ± 0.04* 1.06 ± 0.09
Valine 263.4 ± 15.5 264.2 ± 9.6 0.24 ± 0.01 0.30 ± 0.04
Methionine 30.0 ± 1.7 28.9 ± 2.1 0.02 ± 0.01 0.05 ± 0.01
Isoleucine 63.0 ± 4.5 69.3 ± 3.9 0.08 ± 0.01 0.11 ± 0.02
Leucine 145.3 ± 9.0 159.3 ± 8.2 0.13 ± 0.01* 0.22 ± 0.03
Tyrosine 91.8 ± 5.6 84.6 ± 5.6 0.14 ± 0.01 0.15 ± 0.01
Phenylalanine 77.1 ± 4.5 68.0 ± 4.0 0.7 ± 0.01 0.10 ± 0.01
Lysine 202.4 ± 9.5 223.3 ± 13.6 1.08 ± 0.10* 1.63 ± 0.19
ÂTAA, mmol/L 3.57 ± 0.14 3.42 ± 0.17 40.20 ± 1.89 43.54 ± 2.59
ÂNEAA, mmol/L 2.43 ± 0.11 2.31 ± 0.14 37.61 ± 1.80 39.93 ± 2.39
ÂEAA, mmol/L 1.14 ± 0.05 1.14 ± 0.04 2.59 ± 0.12+ 3.61 ± 0.32
ÂNEAA/ÂEAA 2.13 ± 0.05 1.96 ± 0.05 14.28 ± 0.94* 11.11 ± 1.43
ÂBCAA, mmol/L 0.47 ± 0.03 0.49 ± 0.02 0.45 ± 0.03 0.62 ± 0.09

Note: Young, 20–35 years; elderly, 57–75 years. Mean values ± SE are shown. * p < .05; + p < .01. ÂTAA =
sum of total amino acids; ÂNEAA = sum of nonessential amino acids; ÂEAA = sum of essential amino
acids; ÂBCAA = sum of branched-chain amino acids.
1 No biopsy specimens obtained from patient 1.
Source: Reprinted from Moller, P. et al., Gerontology, 29, 1–8, 1983.

there would be a strong case to establish centralized specialist laboratories containing both
analytical and interpretative expertise at the same place.

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1382_C03.fm Page 45 Tuesday, October 7, 2003 6:08 PM

chapter three

Approaches to studying amino acid


metabolism: from quantitative
assays to flux assessment using
stable isotopes
Dominique Darmaun
Hôtel-Dieu Hospital, Nantes
Luc Cynober
Hôtel-Dieu Hospital, Paris

Contents
Introduction....................................................................................................................................46
3.1 Plasma amino acid concentrations and the significance of their variations ..............46
3.1.1 Interpreting normoaminoacidemia.......................................................................47
3.1.2 Interpreting hyperaminoacidemia ........................................................................47
3.1.3 Interpreting hypoaminoacidemia .........................................................................48
3.2 Measurement of arteriovenous differences......................................................................48
3.2.1 Blood flow measurements......................................................................................49
3.2.2 Regional balances of amino acids.........................................................................50
3.3 Use of stable isotopes to assess amino acid metabolism in vivo ..................................51
3.3.1 Assessment of whole-body protein kinetics .......................................................51
3.3.1.1 The 15N-glycine method ...........................................................................51
3.3.1.2 The L-13C-leucine method ........................................................................51
3.3.1.3 The phenylalanine–tyrosine method .....................................................55
3.3.2 Use of stable isotopes to assess protein metabolism in specific tissues.........56
3.3.2.1 Assessing the fractional uptake of amino acids in the
splanchnic bed...........................................................................................56
3.3.2.2 Combined use of stable isotopes and arteriovenous differences .....56
3.3.2.3 Assessing the fractional synthesis rate of specific proteins...............56
3.3.2.4 Future developments................................................................................57
References .......................................................................................................................................57

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46 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Introduction
Critical illness is accompanied by vast alterations in protein turnover resulting in wasting
of lean body mass. Body protein indeed contributes 10 to 13% of weight loss in these
situations.1 Although the amount of body protein that can be lost without serious delete-
rious effects is not known,2 a loss of 8 to 17% of body protein results in a loss of fitness
in healthy persons,1 and a 50% loss is fatal.3 Most of this nitrogen loss arises from skeletal
muscle,4,5 with alanine and glutamine accounting for 35 to 46% of peripheral nitrogen
output. Alanine is used by the liver for gluconeogenesis. Glutamine is used by several
visceral organs. It is a source of ammonia for the kidney to aid in acid–base balance
homeostasis. In intestinal mucosa, glutamine serves as a primary oxidizable fuel source.
Glutamine also serves as an energy source for immunologically active cells and other
rapidly proliferating cells such as fibroblasts.5,6 More-recent evidence points to a major
role of glutamine as a source of carbon for gluconeogenesis as well.7,8 Concomitant with
this efflux of amino acids from muscle is an increase in the rate of hepatic synthesis of
various plasma proteins.
Classically, nitrogen (N) balance has been used as a tool to study protein metabolism
and assess nitrogen requirements under steady-state conditions. N balance is calculated
as the difference between the nitrogen in and the nitrogen out. This method, however, is
plagued by several intrinsic flaws: (1) The most important of these relates to the fact that
illness is usually associated with non-steady-state conditions, and hence many of the
assumptions used for estimating nitrogen balance are not applicable. (2) N intake is likely
to be overestimated because of inadequate assessment of the food consumed. (3) The
component of nitrogen out is likely to be underestimated because of methodological
problems in collecting and measuring nitrogen losses from the body.9 This includes esti-
mates of losses through the urine, feces, and integuments (skin, hair, nails, sweat, etc.).
For example, under normal physiologic conditions, the integumental losses are usually
very small and relatively insignificant. After severe burn injury, however, these losses
increase dramatically and were estimated by Waxman et al.10 to be equivalent to 0.19 ¥
body surface area ¥ percent burn size. Thus, in an adult man suffering a 50% burn, this
loss would amount to 9.0 g of nitrogen per day in the first week and 4.5 g per day
subsequently.11 (4) Furthermore, nitrogen balance does not yield any insight into the
dynamics of protein turnover (e.g., protein synthesis and breakdown, as well as amino
acid oxidation). A positive nitrogen balance is indicative of a relative excess in the rate of
protein synthesis over that of breakdown. A negative nitrogen balance suggests that
synthesis is lower than breakdown, while a nil nitrogen balance indicates that the subject
is in equilibrium with synthesis relatively equivalent to breakdown.5 Finally, nitrogen
balance does not shed any light on the relative contribution of the different organs to
nitrogen output.

3.1 Plasma amino acid concentrations and the significance of their


variations
Plasma amino acids can be measured by various methods, the most popular being ion
exchange chromatography and reversed-phase high-performance liquid chromatography
(see Chapters 1 and 2, respectively).
Although there is considerable literature dealing with the plasma concentrations of
amino acids under various physiological and pathological conditions, it is frequently
stated that interpreting plasma amino acid concentrations is difficult, or even impossible.
The basis for this statement is that the plasma pool of free amino acids is very small
compared to the intracellular pool of free amino acids, which is in turn of little importance
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Chapter three: Approaches to studying amino acid metabolism 47

with regard to the protein-bound amino acid pool, the three being in equilibrium. In
addition, some amino acids, especially the acidic ones, are present in large amounts in
erythrocytes. The contribution of this pool to tissue exchanges remains a subject of debate.12
Finally, amino acids are subject to multiple interorgan exchanges, which further confuses
the interpretation of static plasma values.
However, catheterization and stable isotope-based techniques have clearly indicated
(see infra) that the concentration of a given amino acid in plasma is the end result of its
rate of appearance (Ra) in plasma minus its rate of disappearance (Rd), where Ra is mainly
a function of muscle production æ provided it is measured at a time when no amino acids
arise from the intestinal absorption of a meal or from amino acid infusion æ and Rd
depends on uptake by splanchnic tissues, mainly the liver.

3.1.1 Interpreting normoaminoacidemia


There are many situations where plasma amino acid concentrations are normal with Ra
= Rd, such as the interprandial state where Ra and Rd are normal, the mild catabolic states
where Ra and Rd are increased equally,13 and the intermediate stage of starvation where
they are both equally decreased.14 In these situations, it can be considered that the pro-
duction of amino acids matches their consumption, and thus, the determination of plasma
amino acids gives little information.

3.1.2 Interpreting hyperaminoacidemia


Hyperaminoacidemia can result from a decrease in Rd with Rd < Ra or from an increase
in Ra with Ra > Rd.
The decrease in Rd is typically observed during multiorgan failure; in the terminal
phase of septic shock, hyperalaninemia is observed, resulting primarily from a collapse
in the uptake of alanine by the liver.15
An excessive rise in Ra can be observed during the administration of an inappropriate
parenteral nutrition regimen. Hyperaminoacidemia can be global as the result of a too
rapid perfusion rate, or more or less selective when the solution is qualitatively inadequate
for the patient. It is reasonable to assume that the most appropriate solution for a given
patient is that which induces the least imbalance in plasma amino acid concentrations,
reflecting a correct utilization of the amino acids perfused.16 The study of the pharmaco-
kinetics of perfused amino acids is probably one of the most promising fields for the
future, as described in Chapter 31 and in recent reviews.17,18
Hyperphenylalaninemia merits further discussion because, in the context of clinical
nutrition, it can result from either an increase in Ra or a decrease in Rd. As a matter of
fact, phenylalanine is poorly metabolized in peripheral tissues and is mainly metabolized
in the liver so that any increase in its plasma concentration reflects either protein hyper-
catabolism or hepatocellular insufficiency. In burn patients, plasma phenylalanine corre-
lates positively with 3-methylhistine excretion and negatively with N balance.19 Therefore,
the plasma phenylalanine level can be considered a suitable marker of N turnover in
situations of stress. Its determination offers the advantage of not requiring urine collection,
which is always difficult even in intensive care units (ICUs). When liver function is altered,
plasma phenylalanine increases sharply. This has been exploited in the context of liver
transplantation; the occurrence of hyperphenylalaninemia in the first hour following rep-
erfusion is associated with complications (primary graft dysfunction, cellular necrosis) in
the 48 h following the transplantation.20
The use of variations in the concentrations of a given amino acid as a marker of organ
function could be expanded. For example, since kidneys actively metabolize citrulline into
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48 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

arginine, hypercitrullinemia is a constant feature of chronic renal failure21; however, to the


best of our knowledge, this type of approach has not been explored. In the same way,
tumor consumption of amino acids can specifically modify the plasma amino acid pattern.
This could be of use in the diagnosis and follow-up of cancer patients (see Chapter 40 for
more details).

3.1.3 Interpreting hypoaminoacidemia


Hypoaminoacidemia can result from an increase in Rd or a decrease in Ra.
An increase in Rd is typical of stress situations, resulting from the increase in hepatic
uptake, primarily of gluconeogenic amino acids.22 In this situation, Ra is also increased,
but this is insufficient for maintaining amino acid homeostasis and Ra < Rd. For example,
in burn patients, plasma alanine decreases by 50% and glutamine by even more.23,24 In
addition, the drop in plasma alanine is more profound in patients with sepsis or those
who subsequently die than in those who survive.25 The same has been found for
glutamine.23 This underlines the relationship between morbidity and metabolic exhaus-
tion, i.e., the body’s incapacity to mobilize the required stores to cope with stress.
A decrease in Ra can result either from a decrease in protein intake or from a decrease
in mobilizable amino acid stores. This is typical of states of chronic undernutrition and
mainly concerns essential amino acids.
Also, a decreased Ra may be observed in the case of organ failure in a tissue that plays
a major role in the production of a given amino acid. For instance, Crenn et al.26 found
that plasma citrulline is low in patients with short bowel syndrome, compared with control
subjects, with a correlation between residual intestinal mass and plasma citrulline level.
Similar observations have been reported during the rejection of intestinal transplants27 or
after intestinal irradiation.28
Finally, a decrease in Ra can result from therapeutic manipulations. For example,
Cynober et al.29 have shown that ornithine a-ketoglutarate (OKG) administration to burn
patients results in a significantly greater decline in the plasma amino acid concentrations,
compared to burn patients receiving the control regimen. In a further trial,30 it was shown
that OKG decreases muscle amino acid output (see Chapter 37 for more details). Similarly,
Mjaaland et al.31 demonstrated that human growth hormone (hGH) therapy decreases
both the concentration of amino acids in venous plasma and their efflux from the forearm.
The correct interpretation of this data is that the decrease in plasma concentrations is due
to a decrease in muscle output in response to OKG or hGH, without any significant change
in their splanchnic uptake. Obviously, in this case a single determination of plasma amino
acids cannot be interpreted and even confuses the issue.

3.2 Measurement of arteriovenous differences


This section describes some of the methods utilized in the laboratory of Abumrad et al.
for the past decade in the conscious dog model.32–34 This approach offers several advan-
tages. The size of the animal is such that it allows for easy surgical preparation and for
withdrawal of adequate amounts of blood without untoward effects. These measurements
can be easily combined with determinations of hemodynamic status. Finally, ethical con-
siderations for the use of invasive techniques in humans limit the ability of the investiga-
tors to control for all the necessary variables to eliminate bias in the experimental design.
Details regarding animal preparations, preoperative care, operative procedures, and
postoperative care have already been published.33 In brief, the animals undergo placement
of silastic catheters in any or all of the various arteries (femoral, carotid, or vertebral) or
veins (femoral, portal, hepatic, internal jugular, coronary, etc.) under general endotracheal
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Chapter three: Approaches to studying amino acid metabolism 49

anesthesia at least 3 weeks prior to the day of the study. Catheters are filled with heparinized
saline, knotted, and placed in subcutaneous pouches. When needed, the animals are also
fitted with tracheostomy or with placement of intracerebroventricular catheters in either the
third or fourth ventricles. Simultaneously, Doppler flow cuffs of various diameters (e.g.,
6 to 7 mm for the portal vein, 3 mm for the hepatic artery, 5 to 6 mm for the iliac artery,
etc.) are usually placed around the vessels of interest, and the leads are then tunneled and
secured to the abdominal incision as those of the silastic catheters. On the day of the study,
the catheter ends are usually exteriorized by making a small skin incision using local
anesthesia (1% lidocaine). The catheters are flushed with saline and used for blood sampling.

3.2.1 Blood flow measurements


Blood flow measurements are carried out in one of several methods. The first, Doppler
technique, is based on the recorded shift in a pulsed sound signal according to the method
of Hartley et al.35 In this method, blood volume flow is estimated by the equation

Volume flow (Q) = Vavg ¥ A (3.1)

where Vavg is the average velocity of the blood and A is the average diameter of the
vessel lumen. This method has two major distinct advantages. The first relates to the
ability to make separate estimates of arterial and venous blood flows. The second relates
to the consistency in the measurements across specific organs that can be obtained.
A second method is based on the use of dye dilution techniques. Both indocyanine
green (ICG) and para-aminohippurate (PAH) dyes can be utilized. ICG may be used for
estimating splanchnic blood flows according to the method of Leevy et al.36 Continuous
infusion of ICG is given at 0.1 mg/m2/min, blood samples are taken any time after 45 min
(after establishment of steady-state levels), and plasma ICG is estimated by spectropho-
tometric methods at 810 nm. Similarly, we have utilized PAH for estimation of kidney
plasma flow. This method involves the use of a primed (0.3 mg/kg), continuous
(0.3 mg/kg/min) infusion of PAH. Estimate of organ plasma flow is estimated according
to the equation

Infusion rate
organ plasma flow = (3.2)
artery dye - vein dye

Another technique frequently used for estimation of pulmonary plasma (or blood
flows) is the use of thermodilution according to the method of Fegler.37 Modifications of
this method entail the manual injection of 5.0 ml of cold saline (0.9%) into the right atrium
and recording the change in blood temperature in the pulmonary artery. This method
bears a high coefficient of correlation with dye dilution techniques and electromagnetic
flow meter. The use of radiolabeled microspheres has been proposed for estimates of
regional blood flow in the hindlimbs, kidneys, brain, etc., as was described by Heyman
et al.38 Several radiolabeled microspheres have been used: 141cerium, 85strontium, and
46scandium are usually injected into the left atrium. Blood is drawn at a constant rate via

the femoral artery before, during, and after the microsphere injections. At the end of the
experiment, tissues are sampled for radioisotope determination using a multichannel
pulse-height analyzer. All samples are counted on the same day. Regional blood flow is
then estimated according to

(regional # microspheres in cpm / g) ¥ (reference blood flow in ml / min)


(3.3)
reference blood number of micropheres in cpm
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50 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Several assumptions need to be satisfied for appropriate estimates of blood flow using
this method. Several studies, however, have shown good coefficients of correlation with
other established methods such as electromagnetic flow meter, dye dilution, etc.

3.2.2 Regional balances of amino acids


In general, the net balance of any amino acid across an organ can be estimated by the
following:

net balance = (arteryamino acid – veinamino acid) (3.4)

As would be anticipated, a positive net balance indicates net retention of the amino
acid, while a negative balance indicates net release. In none of these circumstances, how-
ever, do the methods allow the differentiation between a significant change in either
protein synthesis over that of protein breakdown. Such a shortcoming can be overcome
by the constant infusion of a labeled (either radioactive or stable isotope) amino acid.
Although several amino acids have been utilized, those that are carbon labeled are pre-
ferred, as they allow the determination of the rate of production of either 14CO2 or 13CO2
across the organ in question. Estimates of the net uptake or release of the labeled amino
acid across the organ will allow for the estimation of the unidirectional entry (utilization,
estimate of protein synthesis) or exit (loss, protein breakdown) according to the following
formulas:

(artery labeled a.a. - vein labeled a.a ) ¥ blood flow


Unidirectional entry of amino acid = (3.5)
SA labeled a.a

( vein *CO2 - artery *CO2 ) ¥ blood flow


Net oxidation of amino acid = (3.6)
SA labeled a.a

Rate of protein breakdown across an organ = net balance – unidirectional utilization (3.7)

In summary, it is quite clear that the combined use of labeled radio- or stable isotopes
in conjunction with arteriovenous techniques allows the investigator to estimate the rel-
ative regional contribution of protein synthesis and breakdown and amino acid oxidation
to the overall metabolism of the specific amino acid in question during periods of stress,
illness, etc.
Such methods have been used for estimating the relative contribution of various
organs to overall metabolism of various organs to amino acid metabolism. Several studies
by Nair et al. in man,39 Helland et al. in pigs,40 and Lobley et al. in cattle41 estimate the
contribution of muscle protein synthesis to the overall rate of whole-body protein synthesis
to be approximately 25 to 30%. Previous studies from the Abumrad33,34 laboratory showed
that the splanchnic organs are responsible for about 30% of the total flux of leucine. Most
of these studies have utilized state-of-the-art methodologies for estimating arteriovenous
differences of amino acids in combination with various techniques for measurements of
blood flow as outlined above.
Finally, measurement of arteriovenous differences has resolved the problem of the
origin of 3-methylhistidine; 3-methylhistidine comes mainly from muscle. In trauma
patients, the increase in the urinary output of this amino acid is solely due to increasing
muscle myofibrillar protein catabolism.42,43
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Chapter three: Approaches to studying amino acid metabolism 51

3.3 Use of stable isotopes to assess amino acid metabolism in vivo


3.3.1 Assessment of whole-body protein kinetics
Because metabolism is a dynamic process, tracer methods are theoretically ideally suited
to follow the fate of amino acids in vivo.

3.3.1.1 The 15N-glycine method


15
The N-glycine method was the first stable isotope method used for quantitation of protein
turnover in vivo.44,45 Briefly, when 15N-glycine is administered at a constant rate (i), the 15N
content (enrichment) in urinary urea reaches steady state after 48 to 72 h. It is assumed
that glycine’s nitrogen (N) is evenly distributed in the body’s nitrogen metabolic pool and
that the fraction of the 15N dose administered that is eventually excreted equals the fraction
of the nitrogen pool turnover excreted as urea; whole-body N turnover (Q) is quantitated
by measuring steady-state N enrichment in urea (Eurea):

Q = i/Eurea

Assuming that the body N pool is at steady state, the amount of N entering the pool,
i.e., the sum of dietary N intake (I) and N released from protein breakdown (B), equals
the amount leaving the pool and directed into either protein synthesis (S) or oxida-
tion/excretion to urea (U). Thus,

B = Q – I and S = Q – U

The method relies on robust analytical techniques — precise determination of low 15N
enrichments (down to <0.01%) on large amounts (several micromoles) of urinary urea N
by gas isotope ratio mass spectrometry (IRMS) after conversion to N2 gas45 — and is
noninvasive. Yet it shares some of the practical difficulties of the N balance technique and
relies on a host of unproven assumptions. For instance, there is no such thing as a nitrogen
metabolic pool since there are both anatomical and biochemical barriers to glycine: (1)
because glycine is poorly transported across certain cell membranes, it does not freely
enter all tissues of the body,46,47 and (2) its a-amino N does not end up evenly distributed
among the 20 free amino acids.47 Some of these obstacles may be overcome by using a
mixture of 15N-labeled amino acids.48 In addition, the ideal end product in which 15N
determination should be performed æ urinary urea, urinary ammonium, or both æ
remains a matter of debate.49 Finally, in order to shorten the measurement period, a
technique involving the single administration of a single tracer bolus has been proposed.49

3.3.1.2 The L-13C-leucine method


The l-13C-leucine method50 emerged when new developments in gas chromatogra-
phy–mass spectrometry (GCMS) made it possible to determine relatively high isotopic
enrichments (>0.2%, usually 1 to 5%) not in micromoles of end products in liters of urine,
but rather in nanomolar amounts of precursors, that is, amino acids themselves, in micro-
liters of plasma.45 When pure L[1-13C]-leucine is infused (Figure 3.1) at a constant rate (i),
a plateau of 13C-leucine enrichment is reached in 2 to 3 h in plasma (Ep), and the appear-
ance rate (Ra) of leucine can be calculated from the dilution of the labeled leucine in
plasma at steady state:

Ra = i [Ei/Ep – 1]
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52 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 3.1 The 13C-leucine model.

where Ei is the isotopic enrichment in the tracer infused. As leucine is an essential amino
acid, its only sources are protein breakdown (B) and dietary intake (I):

Ra = B + I

If the total rate of respiratory CO2 excretion (V CO2) and the 13C enrichment of breath
CO2 are quantitated using indirect calorimetry and IRMS, respectively, the rate of leucine
oxidation (Ox) is measured. Under conditions of steady state, leucine Ra equals its disap-
pearance rate (Rd). Since leucine may only be oxidized or utilized for protein synthesis,
the nonoxidized fraction of leucine Rd is thus attributed to leucine incorporation into
protein synthesis (S):

S = Rd – Ox

Leucine fluxes can be extrapolated to whole-body protein fluxes, based on the average
leucine content of body protein (8 g of leucine per 100 g of protein).50
The model relies on several assumptions, many of which have been tested and
validated:

1. There is no reason to believe that any enzymatic system or membrane carrier


distinguishes between 13C- and 12C-leucine. For instance, although the red blood
cell membrane is poorly equipped with amino acid carriers, 13C-leucine freely
equilibrates between erythrocytes and plasma.46
2. 13C-Leucine, once incorporated into protein, may be released into plasma over the
course of the experiment. This tracer recycling51 results in artificially high plasma
leucine enrichments and, consequently, underestimation of leucine Ra, causing as
much as a 30% error for a 24-h-long infusion, but is thought to be minimal when
infusions are less than 4 to 8 h.51
3. It is now proven that plasma 13C-leucine enrichment overestimates intracellular
leucine enrichment. Because a-ketoisocaproate (KIC) is solely derived from intra-
cellular transamination of leucine (Figure 3.2) and since transamination is extreme-
ly fast within cells, several authors have proposed to measure the 13C enrichment
of plasma free KIC instead of leucine.52,53 Plasma 13C-KIC enrichment is indeed a
good estimate of 13C enrichment in the muscle tissue tRNA-bound leucine, from
which leucine is drawn for protein synthesis.54 Moreover, the steady-state 13C
enrichment of the leucine incorporated in plasma apolipoprotein B — a protein
with a very fast turnover rate — was similar to that of plasma KIC during an
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Chapter three: Approaches to studying amino acid metabolism 53

Figure 3.2 Relationships between leucine and a-ketoisocaproate (KIC) during an infusion of L-[l-13C]
leucine. (From Matthews, D.E. et al., Metabolism, 31, 1105, 1982. With permission.)

infusion of 13C-leucine.55 Plasma 13C-KIC enrichment usually reaches 80% of


plasma 13C-leucine enrichment; the difference is attributed to the fraction of leucine
that is released by protein breakdown inside cells but does not appear in the general
circulation.
4. Recovery of metabolically produced 13C is not quantitative, due to loss of labeled
bicarbonate in hidden, slowly overturning pools such as bone or utilization of CO2
in the gluconeogenic pathway. Recovery is affected by rates of energy expenditure
and total CO2 production — e.g., rising from 74 to 82% in the transition from the
fasted to the fed state — but not by the route of tracer delivery.56 Ideally, recovery
of 13CO2 in breath should be determined in any given experimental setting. Several
groups have shown that an infusion of 13CO3HNa can be performed in the few
hours immediately preceding the infusion of labeled leucine. An additional ad-
vantage of this approach is the fact that indirect calorimetry is no longer necessary
to quantify overall CO2 production, since the rate of CO2 production can be deter-
mined using isotope dilution equations.57–59
5. As leucine is an essential amino acid, B = Ra is the fasting state. Rates of protein
breakdown (B) are calculated by subtracting intake (I) from total rate of appearance
(B = Ra – I), when I is accurately known in a parenterally fed patient. When a
patient is fed orally, however, part of dietary leucine undergoes first-pass extraction
in the splanchnic bed and never reaches systemic blood. This problem can be
circumvented by adding a second (e.g., 2H3-labeled) leucine tracer to the enteral
regimen.60 Yet this approach assumes that the leucine present as bound residues
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54 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

in peptides/protein in the regimen has the same fate as the free leucine tracer in
the intestinal lumen. This may not be the case, as recent studies using intrinsically
13C-labeled proteins have found that proteins with different rates of hydrolysis

in the intestinal lumen clearly have different effects on whole-body protein


metabolism.61,62
6. Although leucine and phenylalanine have different target organs in the body —
leucine is predominantly oxidized in muscle and phenylalanine in liver — their
Ra values yield similar estimates of whole-body protein breakdown, even in situ-
ations such as cortisol-induced hypercatabolism,63 which supports the use of leu-
cine Ra as a valid index of whole-body proteolysis.
7. The assessment of protein synthesis is indirect, derived from the difference between
two measured parameters (total Ra and oxidation) and is therefore the weak point
of the method. However, inhibition of protein synthesis using the drug emetine
resulted in nearly complete suppression of nonoxidative disposal in dogs.64

Overall, the 13C-leucine method has survived two decades of intense scrutiny and is
now considered the method of reference to obtain fair estimates of whole-body protein
metabolism.
A major strength of this method is that it uses a stochastic model and relies on simple,
robust, mathematical analysis. Although compartmental models have been developed for
leucine metabolism,65 they have not been widely used, presumably because of the com-
plexity of their mathematical analysis.
However, the 13C-leucine method provides no insight into other important aspects of
protein metabolism:

1. Although the model has only been validated under conditions of steady state,
many physiological situations (such as oral food intake) cannot be described in
terms of steady state, and the constraints of the model have forced most investi-
gators to use a simplified experimental design such as continuous feeding.50
2. The leucine method does not explore the metabolism of nonessential amino acids.
This can, however, be achieved by a simultaneous infusion of 13C-leucine and a
labeled nonessential amino acid tracer, e.g., L [2- 15 N ]-glutamine.63 Because
glutamine is a nonessential amino acid, two endogenous sources contribute to its
Ra in the postabsorptive state: (1) protein breakdown and (2) de novo synthesis.
The contribution of proteolysis to glutamine Ra can be estimated based on leucine
Ra — an index of proteolysis — and the average glutamine and leucine content
of body protein (0.5 mol of glutamine for each mole of leucine). This approach
relies on the assumption that the release of amino acids through protein breakdown
is directly proportional to their relative abundance in body protein. Protein break-
down indeed only accounts for 13% of glutamine Ra.66 The fraction of glutamine
Ra not accounted for by proteolysis is attributed to de novo synthesis,63 with >30%
of its N derived from branched-chain amino acids in postabsorptive humans.67
Although this approach has yielded further insight into glutamine interorgan
exchange, it should be borne in mind that the measured glutamine Ra only mea-
sures a fraction of whole-body glutamine production, due to the compartmentation
of glutamine between the extra- and intracellular milieus.68,69 Similarly, although
the endogenous rates of production of most essential amino acids correlate with
their relative abundance in body protein,70 the rates of appearance of several
nonessential amino acids were found to be paradoxically low, barely exceeding
what can be accounted for by protein breakdown. This is particularly true for
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Chapter three: Approaches to studying amino acid metabolism 55

glutamate,68 arginine,71 or proline.72 The relatively low de novo synthesis rates


measured for these presumably nonessential amino acids imply either (1) that the
model used for measuring their turnover rate is overly simplistic, or (2) alterna-
tively, that these amino acids indeed are not truly nonessential and can become
conditionally essential under some circumstances.73
3. While the human genome codes for the synthesis of several thousand different
proteins, the 13C-leucine method only assesses the overall picture, i.e., the end
result of the synthesis and breakdown of a host of proteins with widely different
half-lives, ranging from a few minutes for hepatic enzymes to several weeks in
the case of myofibrillar proteins. This can be overcome by measuring the synthesis
rate of specific tissue proteins (see below).

3.3.1.3 The phenylalanine–tyrosine method


As stated above, the leucine method is applicable to other essential amino acids as well,
among which is phenylalanine. One shortcoming of the 13C-leucine method is that it
requires collection of both blood and breath and three analytical instruments: GCMS,
IRMS, and indirect calorimetry for plasma 13C-leucine, breath 13CO2, and CO2 production,
respectively. In contrast, the first, irreversible step in phenylalanine oxidation produces
tyrosine rather than CO2. Upon constant infusion of 2H5-phenylalanine, oxidation of the
tracer will produce 2H4-tyrosine, provided the deuteriums are located on the phenyl ring.74
Both labeled phenylalanine and labeled tyrosine can be quantitated by GCMS. Phenyl-
alanine oxidation can be calculated based on (1) the appearance of 2H4 in tyrosine and
(2) total tyrosine Ra, which requires the simultaneous infusion of another tracer of tyrosine
(e.g., 13C-tyrosine), just like measurement of total CO2 production is needed when leucine
oxidation is quantitated. As conversion to tyrosine and incorporation into protein are the
only significant routes of phenylalanine disposal, the nonoxidized fraction of phenyl-
alanine Ra is attributed to phenylalanine incorporation into protein.75
Moreover, labeled phenylalanine has been used in combination with measurement of
arteriovenous gradients to assess muscle protein metabolism.75,76 Indeed, contrary to leu-
cine, phenylalanine cannot be oxidized in muscle. Therefore, its extraction in skeletal
muscle solely reflects its utilization for muscle protein synthesis. Thus, if catheters are
inserted into the artery and a deep vein of the forearm, and if blood flow (F) through the
forearm is measured, then the extraction coefficient (ER) of phenylalanine can be deter-
mined as the fraction of the labeled phenylalanine disappearing between the arterial and
venous sites:

ER = (*Phea – *Phev)/*Phea)

where *Phea and *Phev are the concentrations of 2H5-labeled phenylalanine in arterial and
deep venous blood, respectively. The total rate of phenylalanine release from the forearm
equals Phea ¥ F, where Phev is the concentration of unlabeled phenylalanine in venous
plasma. This rate of release is the sum of the phenylalanine that crosses the forearm
unaltered, i.e., phenylalanine that is not extracted,

F ¥ Phea ¥ (1 – ER)

plus the phenylalanine released by muscle proteolysis (Bm). Thus,

Phev ¥ F = (1 – ER)(Bm + F ¥ Phea)


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56 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

It follows that

Bm = [Phev ¥ F/(l – ER)] – [F ¥ Phea]

In turn, the arteriovenous balance for unlabeled phenylalanine measures the net
difference between local proteolysis and local protein synthesis (Sm), which can therefore
be calculated as:75

Sm = F (Phea – Phev) – Bm

3.3.2 Use of stable isotopes to assess protein metabolism in specific tissues


3.3.2.1 Assessing the fractional uptake of amino acids in the splanchnic bed
As the splanchnic bed plays a prominent role in the disposal of most amino acids, several
groups have used a dual-label approach to quantify the fraction of a given amino acid
that undergoes uptake in the splanchnic territory. For instance, an intravenous infusion
of deuterium-labeled leucine (2H3-leucine) has been combined with the concomitant infu-
sion of 13C-leucine through a nasogastric tube to assess the rate of first-pass splanchnic
extraction in the splanchnic bed. This approach relies on the assumptions that (1) 100%
of the enterally administered labeled amino acid undergoes intestinal absorption, and
(2) 2H3- and 13C-labeled leucine are handled similarly in the body. Both of these assump-
tions have been validated. This method was used to demonstrate that approximately 25%
of enterally administered leucine is taken up in the first pass in the splanchnic bed of
healthy adult humans in the postabsorptive state77,78 (with only 2 to 3% of the leucine
oxidized in the first pass). In contrast, a much higher fraction (ª42 to 50%) of dietary
leucine is taken up in the splanchnic territory in premature infants79 or elderly subjects.80
Fractional splanchnic uptake varies considerably from one amino acid to the next, as it
amounts to 50 to 70% for glutamine81,82 and >90% for glutamate.83

3.3.2.2 Combined use of stable isotopes and arteriovenous differences


Although the combined use of stable isotope infusion and arteriovenous gradient methods
has been applied less extensively in humans than in animals, this approach has, for
instance, been used to demonstrate that muscle contributes over 50% of the overall endog-
enous production of alanine and glutamine in healthy humans.84 Similarly, the contribution
of the human kidney in the production of glucose from glutamine carbon was quantitated
by this approach.85

3.3.2.3 Assessing the fractional synthesis rate of specific proteins


In the last decade, the measurement of labeled amino acid incorporation into specific
proteins has been greatly facilitated by the advent of a new technique combining the ease
of separation of GCMS with the high sensitivity of IRMS, namely, gas chromatogra-
phy–mass spectrometry–combustion–isotope ratio mass spectrometry (GC-C-IRMS),
whereby the leucine peak eluted from the gas chromatograph is combusted on line to CO2
in a furnace and subsequently analyzed for 13CO2/12CO2 by an on-line IRMS.86 In this
approach, the isotope enrichment is measured both in the precursor pool (e.g., intracellular
free leucine) and product, i.e., the protein of interest. The fractional synthesis rate (FSR)
of the protein of interest is calculated as

FSR = 24 ¥ 100 ¥ (Eboundt2 – Eboundt1)/(Eprecursor¥ Dt)


1382_C03.fm Page 57 Tuesday, October 7, 2003 6:08 PM

Chapter three: Approaches to studying amino acid metabolism 57

where Eboundt2 and Eboundt1 are the isotope enrichments (mole percent excess) measured
in protein-bound leucine at times t2 and t1 (hours), respectively, over the course of the
isotope infusion study; Eprecursor is the steady-state isotope enrichment in the precursor
amino acid pool used for protein synthesis; Dt is the time interval (hours) between the
two sampling points t2 and t1; and 24 and 100 convert FSR to percent per day. The major
difficulty in this approach lies in the choice of the appropriate precursor pool. Although
tRNA-bound leucine would be the ideal precursor pool, determination of isotope enrich-
ment in the tRNA pool has been hampered by technical difficulties54 and is therefore rarely
performed. The use of plasma KIC may be an alternative for liver and muscle87 but is not
optimal in other tissues, such as small intestine, where free leucine enrichment should be
measured.88,89
This approach has been applied to circulating plasma proteins such as albumin,
fibrinogen, or apolipoproteins,55 or to the synthesis rate of a nutritionally significant pep-
tide abundant in erythrocyte such as glutathione.90 In vivo studies have, for instance, shown
that insulin deficiency decreases albumin FSR, while it stimulates fibrinogen FSR.91 This
illustrates the fact that a given regulatory factor can affect various proteins in opposite
directions and the need for measurement of the synthesis of specific proteins.
The performance of a muscle biopsy has allowed the determination of the FSR of
mixed muscle protein, as well as of specific contractile proteins such as myosin heavy
chain in both young and elderly subjects.92,93 Similarly, the performance of a duodenal or
colonic biopsy, using an endoscopic procedure at the end of a 4- to 6-h infusion of labeled
leucine, allows for the determination of intestinal protein synthesis. This approach showed
gut protein synthesis to be extremely rapid (ª40 to 80% per day), compared with muscle
protein synthesis (ª1% per day).88,89,94

3.3.2.4 Future developments


In the long run, developments in the techniques of nuclear magnetic resonance (NMR)
may allow amino acid metabolism to be followed live and in situ in a specific tissue and
in a nonsampling fashion. Such a noninvasive approach of protein kinetics has only been
accomplished in isolated tissues in vitro95 so far.

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chapter four

Cellular uptake of amino acids:


systems and regulation
Vadivel Ganapathy
Medical College of Georgia
Katsuhisa Inoue
Medical College of Georgia
Puttur D. Prasad
Medical College of Georgia
Malliga E. Ganapathy
Medical College of Georgia

Contents
Introduction....................................................................................................................................63
4.1 Classification of amino acid transport systems...............................................................64
4.2 SLC 1 family..........................................................................................................................68
4.3 SLC 6 family..........................................................................................................................69
4.4 SLC 7 family..........................................................................................................................70
4.5 SLC 16 family........................................................................................................................71
4.6 SLC 38 family........................................................................................................................71
4.7 Heterodimeric amino acid transporters ...........................................................................73
4.8 Conclusions ...........................................................................................................................74
Acknowledgments ........................................................................................................................75
References .......................................................................................................................................75

Introduction
Amino acids perform a variety of functions in mammalian cells. They not only serve as
the building blocks for protein synthesis but also play additional important roles in
neurotransmission, production and storage of metabolic energy, nitrogen metabolism, and
synthesis of hormones, purine and pyrimidine nucleotides, and glutathione. The intra-
cellular pool of amino acids is derived not only from endogenous production (biosynthesis
as well as protein degradation) but also from transfer across the plasma membrane. Due

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64 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

to their ionic nature, amino acids do not permeate biological membranes by diffusion to
any great extent. Transfer across biological membranes therefore requires participation of
specific transport proteins. This transfer process does not necessarily have to serve only
the entry of amino acids into cells. This process also mediates the exit of certain amino
acids in a tissue-specific manner. In a number of cases, the transfer involves exchange
across the membrane, a process that couples the entry of certain amino acids into cells
and the exit of different amino acids from the cells. A multitude of transport systems is
known to facilitate the transfer of amino acids across the plasma membrane in mammalian
cells. These transport systems exhibit variable but in many instances overlapping substrate
specificity and are not expressed uniformly in all cell types. The differential expression of
amino acid transport systems coupled with their variable substrate specificity has physi-
ological significance because different tissue types vary markedly in their requirements
for specific amino acids to suit their specific physiological functions. In addition to the
differences in substrate specificity, the amino acid transport systems also differ in their
energetics and hence in their transport mechanism. Some transport systems are passive,
functioning either as uniporters or exchangers without involving any driving force,
whereas others are active, driven by transmembrane ion gradients. In many cases, multiple
ion gradients are involved in the energization process, thus enhancing the concentrative
capacity of the transport systems.

4.1 Classification of amino acid transport systems


Prior to the cloning era, the nomenclature of amino acid transport systems was dominated
by the classification system developed by Halvor Christensen.1–3 Even though most of the
work from Christensen’s laboratory involved amino acid transport in nonepithelial cells,
his original classification system was later expanded by him and other investigators in
the field to include amino acid transport in polarized epithelial cells as well.4–6 Polarized
cells such as the intestinal and kidney epithelial cells and placental syncytiotrophoblast
utilize amino acid transport systems not only to modulate the intracellular pool of amino
acids, but also to effect vectorial transfer of amino acids from one side of the cell to the
other. This means that while different types of nonepithelial cells express different amino
acid transport systems in their plasma membrane, polarized cells express different amino
acid transport systems within the two domains of the plasma membrane, namely, the
apical membrane and the basolateral membrane. This adds a new dimension to the
complexity of amino acid transport systems in polarized epithelial cells. The classification
system developed by Christensen relied primarily on substrate specificity. The system was
later improved to include information on whether a transmembrane Na+ gradient plays
a role in the function of the transport systems.7 According to this newer nomenclature,
the Na+ gradient-dependent transport systems are denoted by uppercase letters, whereas
the Na+-independent transport systems are denoted by lowercase letters (Table 4.1 to
Table 4.3). In many instances, the names of the transport systems themselves provide clues
to the identity of the substrates handled by the transport systems, and the notations in
superscript indicate the ionic nature of the amino acid substrates. For example, system A
refers to a Na+ gradient-dependent transport system that prefers alanine and other small
neutral amino acids as substrates, and system B0,+ refers to a Na+-dependent transport
system that exhibits a broad substrate specificity, including neutral (indicated by 0 in the
superscript) as well as cationic (indicated by + in the superscript) amino acids as substrates.
On the other hand, system l (originally known as system L) refers to a Na+-independent
transport system that shows preference for leucine and other hydrophobic bulky amino
acids, system t (originally known as system T) refers to a Na+-independent transport
system that accepts tryptophan and other aromatic amino acids as substrates, and system
Table 4.1 Amino Acid Transporters Coded by Single Genes
Solute Carrier Christensen Alternative Ion Transport
Classification Classification cDNA Identity Names Dependence Mechanism
Chapter four:

SLC1 ASC ASCT1 SAAT1 Na+ 1 Na+–AA symport/1Na+–AA antiport


ASCT2 ATB0
X–AG EAAT1 GLAST Na+, K+, H+ 3 Na+–1 H+–AA
symport/1 K+ antiport
EAAT2 GLT-1
EAAT3 EAAC1
EAAT4
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EAAT5

SLC6 B0,+ ATB0,+ Na+, Cl¯ 2 Na+–1 Cl––AA symport


b TAUT 2 or 3 Na+–1 Cl––AA symport
GLY GLYT1 2 Na+–1 Cl––AA symport
GLYT2 3 Na+–1 Cl––AA symport

SLC7 y+ CAT1 None AA uniport


CAT2
CAT3

SLC16 t (T) TAT1 None AA uniport


Cellular uptake of amino acids: systems and regulation

SLC38 A ATA1 GlnT, SAT1, NAT2, SA2 Na+ 1 Na+–AA symport


ATA2 SAT2, SA1
ATA3 NAT3
N SN1 NAT1 Na+, H+ Na+–AA symport/H+ antiport
SN2
PAT1 LYAAT-1 H+ 1 H+–AA symport
PAT2

Note: AA = amino acid.


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66 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 4.2 Substrate Specificity and Gene Locus of Single-Gene Amino Acid Transporters
cDNA Identity Human Gene Locus Primary Physiological Substrates
ASCT1 2p13–15 Ala, Ser, Cys
ASCT2 19q13.3 Ala, Ser, Cys, Thr, Gln, Asn

EAAT1 5p13 Anionic amino acids (Glu, Asp)


EAAT2 11p13 Anionic amino acids (Glu, Asp)
EAAT3 9p24 Anionic amino acids (Glu, Asp)
EAAT4 19p13.1 Anionic amino acids (Glu, Asp)
EAAT5 1p32 Anionic amino acids (Glu, Asp)

ATB0,+ Xq23–24 Neutral and cationic amino acids

TAUT 3p25 Taurine, b-Ala

GLYT1 1p33 Gly, Sarcosine


GLYT2 11p15.1–15.2 Gly

CAT1 13q12 Cationic amino acids (Arg, Lys, Orn)


CAT2 8p22 Cationic amino acids (Arg, Lys, Orn)
CAT3 Xq12 Cationic amino acids (Arg, Lys, Orn)

TAT1 6q21–22 Aromatic amino acids (Trp, Phe, Tyr)

ATA1 12q12 Linear neutral amino acids (Pro)


ATA2 12q12 Linear neutral amino acids (Pro)
ATA3 12q12 Linear neutral amino acids (Pro)

SN1 3p21.3 Gln, Asn, His


SN2 Xp11.23 Gln, Asn, His, Ser, Gly

PAT1 5q33.1 Gly, Ala, Pro


PAT2 Not known Gly, Ala, Pro, Ser

x-c refers to a Na+-independent transport system that prefers the anionic form of the
disulfide-containing amino acid cystine as the substrate. Some systems transport certain
amino acids in a Na+-independent manner but other amino acids in a Na+-dependent
manner. The newly improved classification method also allows for accurate identification
of such a system. For example, system y+L refers to a transport system that transports
cationic amino acids in a Na+-independent manner and neutral amino acids in a Na+-
dependent manner.
Successful cloning of several of the plasma membrane amino acid transporters in
recent years has led to the expansion of Christensen’s original classification to incorporate
the new information on the structure, substrate specificity, and transport mechanism of
these transporters. Interestingly, the new information on the molecular nature of these
amino acid transporters has not weakened in any way the original classification. What is
new is the realization that many of the transport systems, which were once thought to be
single transport systems according to the original classification, based on substrate spec-
ificity and ion coupling, actually consist of several subtypes. These subtypes, coded by
separate genes, are expressed differentially in different tissues but mediate similar trans-
port function in terms of substrate specificity and ion coupling. Another new revelation
as a result of cloning studies is that in many cases, transport systems that are distinct in
Chapter four:

Table 4.3 Heterodimeric Amino Acid Transporters Coded by Two Different Genes
Light Chain (SLC7) Heavy Chain (SLC3)
Christensen cDNA Human cDNA Human Amino Acid Ion Transport
Classification Identity Gene Locus Identity Gene Locus Substrates Dependence Mechanism
l (L) LAT1 16q24.3 4F2hc (CD98) 11q12 Neutral AA None AA exchange
LAT2 14q11.1 4F2hc (CD98) 11q12 Neutral AA None AA exchange
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y+L y+LAT1 14q11.1 4F2hc (CD98) 11q12 Neutral and Na+ (only for neutral Na+–AAo/AA+
cationic AA AA) exchange
y+LAT2 16q21 4F2hc (CD98) 11q12 Neutral and Na+ (only for neutral Na+–AAo/AA+
cationic AA AA) exchange

bo,+ bo,+AT 19q12–13.1 rBAT (NBAT, D2H) 2p21 Neutral and None AA exchange
cationic AA

x–c xCT 4q28 4F2hc (CD98) 11q12 Cystine, Glu None Cystine/Glu
exchange

asc Asc1 19q12–13.1 4F2hc (CD98) 11q12 Ala, Ser, Cys None AA exchange
Asc2 — Not known Ala, Ser, Cys None Not known
Cellular uptake of amino acids: systems and regulation

ag AGT1 8q21.1 Not known Asp, Glu None Not known

Note: AA = amino acid; AAo = zwitterionic amino acid; AA+ = cationic amino acid.
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68 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

substrate specificity and ion coupling fall within a single group based on homology in
amino acid sequence of the transport proteins. Thus, within a given single group consisting
of members of similar amino acid sequences, the members differ in substrate specificity,
ion coupling, and transport mechanism. This new nomenclature based on the structure
of the transporter proteins rather than on the transport function is called solute carrier
classification. Recent cloning studies have also led to the discovery that while many of
the transport systems do consist of single-gene products, a growing number of transport
systems function as heterodimers whose subunits are coded by separate genes. Interest-
ingly, based on amino acid sequence, the heavy chains of these heterodimeric transporters
fall into one solute carrier group, whereas the light chains fall into another solute carrier
group. According to this solute carrier classification, the plasma membrane amino acid
transporters thus far identified in mammalian cells at the molecular level are grouped into
six distinct families: SLC (solute carrier) 1, SLC 3, SLC 6, SLC 7, SLC 16, and SLC 38 (Table
4.1 to Table 4.3). Of these, SLC 1, SLC 6, SLC 16, and SLC 38 comprise solely amino acid
transporters coded by single genes. In contrast, SLC 7 comprises amino acid transporters
coded by single genes as well as the light chains of amino acid transporters that function
as heterodimers. SLC 3 consists solely of the heavy chains of the heterodimeric amino acid
transporters. Tables 4.1 to 4.3 summarize the available information on solute carrier clas-
sification, Christensen’s original classification, identity of the cloned amino acid trans-
porter cDNAs, alternative names, substrate specificity, ion coupling, transport mechanism,
and chromosomal localization of the genes for the plasma membrane amino acid trans-
porters identified thus far in mammalian cells at the molecular level. Readers are referred
to recent reviews for a detailed analysis of the structure, function, and regulation of various
amino acid transporters in mammalian cells.5,8–15 In this chapter, we provide a short
overview of these transporters.

4.2 SLC 1 family


The SLC 1 family consists of transport systems referred to as ASC and X-AG according to
Christensen’s classification. ASC is a Na+-coupled transport system expressed widely in
mammalian tissues that accepts alanine, serine, and cysteine as the preferred substrates.
Two subtypes of this transport system have been identified thus far, namely, ASCT1 and
ASCT2.16–19 Both subtypes function as Na+-dependent amino acid exchangers rather than
as uniporters. ASCT1 is specific for alanine, serine, and cysteine, whereas ASCT2 accepts,
in addition to these three amino acids, glutamine and asparagine as substrates.19 Since
ASCT2 accepts glutamine as a high-affinity substrate and is also capable of mediating the
influx as well as the efflux of this amino acid depending on the metabolic profile of the
cell types, it has been proposed that this transporter may play a critical role not only in
glutamine release in the brain, placenta, and liver as a part of the glutamine–glutamate
cycle that occurs in these tissues, but also in glutamine uptake in tumor cells to support
cell proliferation.12 An interesting aspect of ASCT2 that is independent of its transport
activity is its function as a receptor for certain types of retroviruses.20,21 ASCT2 is also
referred to as ATB0 because the functional characteristics of ASCT2 resemble those of system
B0 described in the brush border membrane of intestinal and kidney epithelial cells.19
Successful cloning of ASCT2 from intestine and cell lines of intestinal origin22 and dem-
onstration of localization of this transporter in the brush border membrane of intestinal
and kidney epithelial cells23 support this notion. However, the notion of ASCT2 being
identical to system B0 is not universally accepted. Many investigators in the field believe
that system B0 still remains to be identified at the molecular level.11 One of the primary
reasons for the lack of widespread acceptance of the notion is that ASCT2 behaves as a
Na+-dependent amino acid exchanger, whereas system B0 is believed to be a Na+-dependent
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Chapter four: Cellular uptake of amino acids: systems and regulation 69

amino acid uniporter. Thus, the issue of whether ASCT2 is identical to system B0 remains
unresolved. The function and expression of ASCT2 in some tissues and cell lines is up-
regulated by epidermal growth factor,24 growth hormone,25 and protein kinase C.26
System X-AG mediates the cellular influx of anionic amino acids (glutamate and aspar-
tate) in a Na+-coupled manner. Since glutamate is an excitatory amino acid in the brain,
the transporters responsible for X-AG activity are referred to as EAATs (excitatory amino
acid transporters). There are five subtypes of X-AG (EAAT1 to EAAT5). All of these subtypes
are highly concentrative with the ability to accumulate glutamate inside the cells several
hundred-fold higher than outside. This is facilitated by transmembrane gradients of Na+,
K+, and H+ and membrane potential as driving forces. The transport mechanism involves
the influx of 3 Na+, 1 H+, and 1 glutamate, coupled to the efflux of 1 K+.27 Each EAAT
subtype is expressed differentially in a tissue-specific manner. EAAT1, EAAT2, and EAAT4
are mainly brain specific, and EAAT5 is almost exclusively expressed in the retina. EAAT3
is expressed in the brain as well as in peripheral tissues, including the absorptive cells of
the intestine and kidney. In the brain, EAAT1 and EAAT2 are present primarily in glial
cells, whereas EAAT3, EAAT4, and EAAT5 are present in neurons. The function of EAAT3
is subject to regulation by osmotic stress and amino acid deprivation.28 Exposure of
EAAT3-expressing cells to hyperosmotic conditions leads to a large increase in glutamate
influx associated with enhanced expression of EAAT3 mRNA. In contrast, amino acid
deprivation enhances EAAT3 activity by a process not linked to changes in EAAT3 mRNA
levels. The remarkable ability of EAAT3 to concentrate glutamate inside the cells makes
this transporter ideally suited for osmoregulation. The cells exposed to hyperosmotic
conditions up-regulate the expression of EAAT3 to increase the intracellular concentrations
of glutamate and consequently reverse the cell volume changes induced by hyperosmotic
conditions. The concentrative ability is not unique to EAAT3. Other subtypes of EAAT
also possess this characteristic. But it remains to be seen whether other EAAT subtypes
also respond to hyperosmotic insult in a similar manner. The transport function of EAATs
is also subject to regulation by different isoforms of protein kinase C, but without involving
changes in the steady-state levels of respective mRNA.29,30 The effects of protein kinase C
are due to changes either in the cell surface expression of the transporter protein or in the
catalytic efficiency of the transporter. Interestingly, the activation of protein kinase C does
not have the same effect on all EAAT subtypes. EAATs are also subject to regulation by
phosphatidylinositol 3-kinase (PI 3-kinase).30 Recent studies have unraveled a new mode
of regulation of certain subtypes of EAAT.31,32 This occurs through protein–protein inter-
action involving specific intracellular proteins, known as GTRAPs (glutamate transporter-
associated proteins). The transport function of EAAT3 is enhanced by GTRAP3 to
GTRAP18, and the process involves an increase in the substrate affinity of the transporter.31
GTRAP41 and GTRAP48 increase the transport activity of EAAT4 either through an
increase in the catalytic rate of the transporter or through an increase in the cell surface
availability of the transporter.32

4.3 SLC 6 family


The SLC 6 gene family is also known as the neurotransmitter transporter gene family
because it consists of transporters for neurotransmitters such as serotonin, dopamine,
norepinephrine, and g-aminobutyrate. With regard to amino acids as substrates, systems
B0,+, b, and GLY belong to this family. B0,+ is a transporter that mediates the cellular
uptake of a broad spectrum of zwitterionic amino acids and cationic amino acids in a
Na+- and Cl--coupled process. Na+ as well as Cl- are obligatory for the transport function.
The cloned transporter, known as ATB0,+, exhibits exactly the same functional charac-
teristics with regard to substrate specificity and ionic requirements as those described
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70 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

originally for systems B0,+.33 ATB0,+ is expressed primarily in the lung, breast, and colon.
The Na+:Cl-:amino acid stoichiometry for the transport process is 2:1:1. An interesting
feature of ATB0,+ is its ability to transport D-amino acids.34,35
System b refers to a Na+- and Cl--coupled amino acid transport activity that is specific
for taurine and other b-amino acids. The cloned transporter, TAUT, exhibits functional
features that are identical to those described for system b.36 TAUT is expressed widely in
mammalian tissues, especially in those tissues with very high levels of intracellular taurine
(e.g., retina, heart). TAUT is also responsible for the intestinal absorption of dietary taurine
and for the renal reabsorption of taurine from the glomerular filtrate. The most notable
characteristic of TAUT is the regulation of its expression in response to hyperosmotic
stress.37 One of the well-recognized biological functions of taurine is its role as an osmolyte.
TAUT operates with a Na+:Cl-:taurine stoichiometry of 2 to 3:1:1, which empowers the
transporter thermodynamically to mediate uphill transport of taurine. The remarkable
ability of TAUT to concentrate taurine inside the cells against a concentration gradient
makes this transporter ideal for osmoregulation. As is the case with EAAT3, the expression
of TAUT is up-regulated when cells are exposed to hyperosmotic conditions. The resultant
increase in intracellular levels of taurine aids in the reversal of cell volume changes induced
by the hyperosmotic conditions. The expression of TAUT is also up-regulated by nitric
oxide38 and tumor necrosis factor-a,39 but down-regulated by the tumor repressor protein
p53.40
System GLY refers to a transport activity that mediates the Na+- and Cl--dependent
uptake of glycine. Cloning studies have revealed that this system consists of two subtypes,
GLYT1 and GLYT2.41 Both subtypes are obligatorily dependent on Na+ and Cl- for their
transport function, and both accept glycine as a substrate. However, the two subtypes can
be differentiated based on their interaction with sarcosine (N-methylglycine). GLYT1
accepts sarcosine as a substrate, whereas GLYT2 does not. GLYT1 is expressed in the brain
as well as in peripheral tissues, including liver, lung, kidney, and placenta. In contrast,
the expression of GLYT2 is restricted to brain. In the brain, GLYT1 is present in glial cells
and GLYT2 is present in neuronal cells. In addition to these differences in substrate
selectivity and tissue distribution pattern, GLYT1 and GLYT2 also differ in transport
mechanism in terms of coupling to Na+.42 GLYT1 operates with a Na+:Cl-:glycine stoichi-
ometry of 2:1:1, and therefore the transport process results in the transfer of one positive
charge into the cells per transport cycle. In contrast, GLYT2 operates with a Na+:Cl-:glycine
stoichiometry of 3:1:1, and this results in the transfer of two positive charges into the cells
per transport cycle. Therefore, GLYT2 is much more concentrative than GLYT1. This may
have physiological relevance in terms of the handling of glycine in the brain by the glial
cells vs. the neuronal cells. The mode of glycine transport via GLYT1 can be reversed from
influx to efflux even under physiological conditions if intracellular concentrations of
glycine and Na+ build up to a certain level. Such a reversal in the direction of glycine
transport may not occur in the case of GLYT2. This means that under certain conditions,
glycine may be released from glial cells via GLYT1 and transferred into neurons via GLYT2.

4.4 SLC 7 family


This family consists of system y+, which transports the cationic amino acids arginine and
lysine. Ornithine is also a substrate for this system. There are three subtypes within this
family for which transport function has been demonstrated. These subtypes are referred
to as CAT (cationic amino acid transporter) 1, CAT2, and CAT3.5,8,9 All three subtypes
mediate the cellular uptake of cationic amino acids by a process that is facilitated by the
inside-negative membrane potential. The expression of CAT1 is widespread in mammalian
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Chapter four: Cellular uptake of amino acids: systems and regulation 71

tissues with the notable exception of liver. CAT2 consists of two alternative splice variants,
CAT2A and CAT2B. CAT2A is specifically expressed in the liver, and CAT2B is expressed
primarily in immune cells such as macrophages. CAT3 shows species-dependent varia-
tions in tissue expression.43 In the rat, CAT3 expression is brain specific, whereas in humans
this subtype is expressed not only in the brain but also in various peripheral tissues. In
terms of transport function, CAT1, CAT2B, and CAT3 exhibit high affinity for their sub-
strates. In contrast, CAT2A shows a 10-fold lower affinity comparatively toward its sub-
strates. There are also significant differences between CAT2A and CAT2B in terms of
voltage dependence.44 There has been a long-standing interest in CATs because of their
involvement in the supply of arginine to nitric oxide synthases for generation of nitric
oxide.45 CAT1 plays an essential role in nitric oxide production in endothelial cells where
this transporter appears to function in concert with endothelial nitric oxide synthase
(NOS3). Similarly, CAT2 is functionally coupled to inducible nitric oxide synthase (NOS2)
in activated macrophages.46 CAT1 is linked to cell proliferation, and its expression is
induced in regenerating liver and in proliferating immune cells.5,8,9,45 Thus, normal liver
expresses only CAT2A, but regenerating liver expresses high levels of CAT1. The relation-
ship between cell proliferation and induction of CAT1 may be related to the role of arginine
as the precursor for polyamine biosynthesis. There is also evidence that CAT1 activity is
modulated by protein kinase C47 and cytoskeletal proteins.48 On the other hand, the
expression of CAT2 is induced by bacterial lipopolysaccharide and proinflammatory
cytokines such as interleukin-1, tumor necrosis factor-a, and interferon g.45 These agents
are known to enhance nitric oxide production in macrophages, and accordingly, they
induce the expression of CAT2 and NOS2 in a coordinated manner. Recent findings
indicate that CAT2 expression is enhanced in peripheral blood mononuclear cells from
patients with sepsis, a pathological condition associated with increased production of
nitric oxide.49 Antiinflammatory cytokines such as interleukin-10 suppress the expression
of CAT2 induced by proinflammatory cytokines.50

4.5 SLC 16 family


This family consists of monocarboxylate transporters that handle lactate and pyruvate.
Recent studies have shown that the amino acid transport system t belongs to this gene
family.51,52 System t refers to a Na+-independent amino acid transport system that mediates
the cellular uptake of tryptophan and other aromatic amino acids such as phenylalanine
and tyrosine. It was originally described in human erythrocytes.4 The recently cloned
amino acid transporter TAT1 exhibits functional characteristics resembling those of system
t.51,52 TAT1 mediates the cellular uptake of aromatic amino acids. It is expressed in the
intestine, kidney, liver, and placenta. Interestingly, even though TAT1 is structurally related
to monocarboxylate transporters that transport their substrates in a H+-coupled manner,
the amino acid transport via TAT1 is not associated with H+ cotransport.

4.6 SLC 38 family


The SLC 38 family consists of amino acid transport systems A and N. System A refers to
a Na+-coupled transport activity that prefers alanine and other small neutral amino acids
as substrates, whereas system N refers to a Na+-coupled transport activity that prefers as
substrates those amino acids containing nitrogen in their side chain (glutamine, aspar-
agine, and histidine). There are three subtypes within system A, referred to as ATA (amino
acid transporter A) 1, ATA2, and ATA3.53–58 A unique characteristic of system A is its ability
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72 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

to transport the amino acid model substrate a-(methylamino)isobutyric acid (MeAIB). All
three subtypes of system A mediate the transport of MeAIB in a Na+-coupled manner
with a Na+:MeAIB stoichiometry of 1:1. H+ is not a cotransported ion for ATAs, but the
function of these transporters is markedly influenced by pH. The physiological substrates
of ATAs include alanine and other small linear neutral amino acids. Glutamine is also an
excellent substrate for ATAs. Bulky neutral amino acids such as leucine are excluded by
system A subtypes. ATA1 and ATA2 cannot be differentiated simply based on transport
characteristics because they exhibit similar substrate selectivity, substrate affinity, Na+
dependence, and sensitivity to pH. ATA3 differs from ATA1 and ATA2 in substrate affinity
and substrate selectivity. ATA3 transports not only neutral amino acids but also cationic
amino acids. Furthermore, ATA3 exhibits lower affinity for its substrates than ATA1 and
ATA2. All three subtypes of ATA differ markedly in their tissue distribution pattern. The
expression of ATA1 is restricted to the brain and placenta, whereas ATA2 is expressed in
almost every tissue. In contrast, ATA3 is expressed most exclusively in the liver.
System N consists of two subtypes, SN1 and SN2.59–63 Both subtypes transport
glutamine, asparagine, and histidine in a Na+-dependent manner. Interestingly, a trans-
membrane H+ gradient also plays a role in the transport mechanism. The transport of
amino acid substrate and Na+ occurs in one direction, whereas the transport of H+ occurs
in the opposite direction. Even though the Na+:amino acid:H+ stoichiometry is 2:1:1 and
the transport process is associated with membrane depolarization, it appears that the
actual mechanism of the transport process is more complex than originally thought.64 The
actual transport process itself is electroneutral, involving the movement of one Na+ and
the amino acid substrate in one direction, coupled to the movement of one H+ in the
opposite direction. Irrespective of the ion coupling, the transport process mediated by
SN1 and SN2 leads to intracellular alkalization when these transporters catalyze the influx
of amino acid substrates. However, SN1 and SN2 can also function in the reverse mode,
in which Na+ and amino acid substrate move out of the cell and H+ moves into the cell.
In this mode, the efflux of amino acid substrates via these transporters is associated with
intracellular acidification.
The physiological and clinical significance of systems A and N is partly related to the
ability of these two systems to mediate the transport of glutamine. This amino acid plays
a crucial role in the metabolic functions of the liver, brain, skeletal muscle, and placenta.
The different subtypes of systems A and N function in the cellular uptake or release of
glutamine as a part of the glutamine–glutamate cycle and glutamine cycle that occur
between or within these organs. SN1 and SN2 are expressed in all of these organs. ATA1
is expressed in the brain and placenta, ATA2 is expressed ubiquitously, and ATA3 is
expressed primarily in the liver. The potential for the reversal of the direction of glutamine
transport via SN1and SN2 makes them suitable to participate in the intercellular glutamine
cycle that occurs between the periportal and perivenous hepatocytes in the liver and in
the glutamine release from the astrocytes associated with the glutamine–glutamate cycle
in the brain. In the liver, periportal hepatocytes take up glutamine, whereas perivenous
hepatocytes release glutamine. Since glutaminase activity is high in periportal hepatocytes
and glutamine synthetase activity is high in perivenous hepatocytes, the direction of
glutamine concentration gradient differs between these two cell populations, favoring
glutamine uptake in the former and glutamine release in the latter. In addition, the
magnitude of the H+ gradient also differs between these two cell populations. Periportal
hepatocytes synthesize urea, which requires not only ammonia but also HCO3-. These
cells thus have an effective means of HCO3- disposal. In contrast, perivenous hepatocytes
remove ammonia via glutamine synthesis rather than urea synthesis, and this process
does not involve HCO3-. Thus, perivenous hepatocytes do not have an effective means of
HCO3- disposal. Therefore, it is likely that the intracellular pH in periportal hepatocytes
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Chapter four: Cellular uptake of amino acids: systems and regulation 73

is acidic compared to the intracellular pH in perivenous hepatocytes. An acidic intracel-


lular pH in periportal hepatocytes is expected to facilitate the transport function of SN1
and SN2 in the direction of glutamine uptake. Similarly, an alkaline intracellular pH in
perivenous hepatocytes is expected to facilitate the transport function of SN1 and SN2 in
the direction of glutamine release. In the brain, glutamine release from astrocytes is
mediated by SN1 and SN2. In these cells, there exists a high inside-to-outside concentration
gradient for glutamine because of the abundant glutamine synthetase activity in these
cells. In addition, synaptic transmission results in an increase in intracellular pH in astro-
cytes. The resultant outward-directed gradients for glutamine and pH favor glutamine
release coupled to H+ influx. In glutamatergic neurons, extracellular glutamine is the
immediate source of glutamate. The presynaptic glutamatergic neurons take up glutamine
that is released from the astrocytes and convert it into glutamate. This uptake step is
mediated by ATA1. Since ATA1-mediated transport occurs only in the influx mode, this
transporter is uniquely suited for this purpose. Thus, the amino acid transport systems A
and N participate in the brain in the glutamine–glutamate cycle. Similar functions of
various subtypes of systems A and N are likely in the glutamine–glutamate cycle that
occurs between placenta and fetal liver and in the uptake and release of glutamine that
occurs in the skeletal muscle.
The activity of system A is regulated by various hormones.15 However, most of the
work on the regulation of this transport system was carried out prior to the molecular
identification of the transporters responsible for this transport activity. Recent studies
show that ATA2 is the subtype that is subject to regulation by hormones such as insulin65
and by amino acid deprivation66,67 and hyperosmolality.68 There is also evidence indicating
that the three known subtypes of system A are affected differentially by cAMP.69
The SLC38 gene family also consists of at least two amino acid transporters whose
transport function is coupled to cotransport of H+. These transporters, known as PAT1
and PAT2, are expressed differentially in mammalian tissues. They mediate electrogenic
transport of small neutral amino acids into the cells.70–72 The H+:amino acid stoichiometry
for the transport process is 1:1. PAT1 is expressed predominantly in the intestinal tract.
Moderate expression is evident in the brain, colon, kidney, liver, lung, placenta, and testis.
PAT2 is expressed mainly in the heart and lung. Both transporters recognize small neutral
amino acids (glycine, alanine, and proline) as substrates, but with variable affinity. PAT1
is a low-affinity transport system compared to PAT2. A H+-coupled transport activity for
small neutral amino acids has been described at the brush border membrane of the
intestinal cell line Caco-2.73 Recent studies have shown that PAT1 is responsible for this
transport activity.72

4.7 Heterodimeric amino acid transporters


A growing number of amino acid transporters consist of two subunits, a light chain and
a heavy chain. These transporters are called heterodimeric amino acid transporters.13,14
This group includes systems l, y+L, b0,+, x-c, asc, and ag (Table 4.3). Two different heavy
chains, known as rBAT and 4F2hc, have been identified thus far. These heavy chains are
glycosylated, and they interact with the corresponding light chains and direct their traf-
ficking to the plasma membrane. The heavy chains themselves do not exhibit transport
activity but may influence the transport function of the heterodimer.74 rBAT interacts with
the light chain b0,+AT to constitute the Na+-independent amino acid transport system b0,+.
This transport system is expressed mostly in the intestine and kidney and is responsible
for the transport of neutral amino acids and cationic amino acids across the brush border
membrane of the absorptive cells in these tissues. Cystine is also a substrate for this system.
4F2hc interacts with a variety of light chains to constitute different amino acid transport
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74 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

systems. LAT1 and LAT2 are the light chains that interact with 4F2hc and form the two
known subtypes of amino acid transport system l. System l mediates the Na+-independent
transport of neutral amino acids. y+LAT1 and y+LAT2 are the light chains that interact
with 4F2hc to form the two known subtypes of amino acid transport system y+L. This
system mediates the transport of cationic amino acids in a Na+-independent manner and
the transport of neutral amino acids in a Na+-dependent manner. System x-c consists of
4F2hc and the light chain xCT. This transport system is Na+ independent and mediates
the cellular uptake of cystine coupled to the efflux of glutamate. System asc consists of
two subtypes. One of the subtypes consists of 4F2hc and the light chain Asc1. This system
mediates the Na+-independent transport of small neutral amino acids. The other subtype
consists of the light chain Asc2 and a hitherto unidentified heavy chain.75 Similarly, the
light chain of system ag has been identified (AGT1), but the nature of the heavy chain is
not yet known.76 The substrate specificity and transport properties of this newly identified
system ag do not conform to the functional features of any of the previously characterized
amino acid transport systems. At the structural level, the light chains of all of the known
heterodimeric amino acid transporters exhibit significant similarity. Since these light chains
also show sequence homology to the cationic amino acid transporters, they are considered
members of the SLC7 gene family. The two known heavy chains, rBAT and 4F2hc, are
structurally related to each other, but do not show any sequence homology to the light
chains. Therefore, the heavy chains are grouped under a separate solute carrier gene family
(SLC3). It is clear that there are additional heavy chains yet to be identified at the molecular
level. An interesting feature of these heterodimeric amino acid transporters is that they
all function as obligatory exchangers. They mediate the entry of amino acids into cells by
a process that is coupled to the exit of amino acids from the cells. This can be either homo-
exchange or hetero-exchange. Nonetheless, some of these transporters can facilitate the
vectorial transport of certain amino acids in polarized cells when functionally coupled to
other amino acid transporters.5,11,13
In terms of regulation, system x-c has received considerable attention in recent years.
The activity of system x-c is up-regulated by oxidative stress induced by nitric oxide,77
oxidized low-density lipoproteins,78 oxygen,79 and other oxidants.80 This regulation is
related to the role of system x-c in the influx of cystine into the cells where it is reduced
to cysteine and used for the synthesis of the antioxidant glutathione.

4.8 Conclusions
Most of the amino acid transport systems that are known to exist in mammalian cells based
on functional studies have now been cloned and characterized at the molecular level. The
information on the identity and structure of the genes coding for these transport systems
will undoubtedly help the investigators in the area of amino acid transport to generate
useful tools such as cDNAs and antibodies to usher this area of research in newer directions.
Detailed investigations of the various isoforms of different amino acid transport systems
in mammalian cells in terms of the regulatory aspects of their expression and function at
the gene and protein levels should now be feasible. There are, however, a few of the
classically identified amino acid transport systems that have not yet been cloned. This
includes the transport systems IMINO (a Na+-coupled transport system for imino acids
and glycine), X-A (a Na+-coupled transport system selectively for aspartate), and x-G (a Na+-
independent transport system selectively for glutamate). There is a very high likelihood
that these transport systems will be cloned and characterized at the molecular level in the
coming years.
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Chapter four: Cellular uptake of amino acids: systems and regulation 75

Acknowledgments
This work is supported by the National Institutes of Health grant GM65344.

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Part II

Physiology
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section A

Metabolism
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chapter five

Amino acid metabolism


and gluconeogenesis
Xavier M. Leverve
Université Joseph Fourier

Contents
Introduction....................................................................................................................................83
5.1 Gluconeogenesis at the cellular level: liver has a predominant but
nonexclusive role..................................................................................................................84
5.1.1 From amino acids to oxaloacetate ........................................................................86
5.1.2 From oxaloacetate to glucose ................................................................................86
5.1.3 Hormonal control of gluconeogenesis .................................................................87
5.2 Gluconeogenesis: interorgan cooperation ........................................................................88
5.3 Gluconeogenesis and pathological states.........................................................................90
5.4 Gluconeogenesis or glucose recycling? ............................................................................90
5.4.1 De novo gluconeogenesis ........................................................................................91
5.4.2 Glucose recycling: the futile cycles.......................................................................91
5.5 Conclusion .............................................................................................................................91
References .......................................................................................................................................92

Introduction
In terms of mass, glucose synthesis is probably the most important biosynthetic process
in living systems. It is a general feature of plants, microorganisms, and animals, and the
ability of heterotrophic cells to synthesize glucose or glycogen from lactate, pyruvate, and
also from nearly all amino acids present in proteins is one of the most fundamental
biosynthetic pathways of cellular metabolism. Classically, in highly complex organisms
such as mammals, and therefore in man, this function of glucose synthesis is mainly
devoted to the liver. While the kidney has also long been recognized as a gluconeogenic
organ, it has only recently been described that the gut is a gluconeogenic organ as well.
In fact, many other tissues, including skeletal muscle, are enzymatically equipped with
phosphoenolpyruvate carboxykinase, allowing glucose 6-phosphate synthesis. Hence,
these organs may also be considered as involved in the gluconeogenic pathway in a broad
sense, even if not able to release substantial amounts of glucose. In man, the main

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84 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 5.1 Origin of Hepatic Glucose Production in Postabsorptive


Healthy Man
Hepatic glucose output 100%
Glycogenolysis 75%
Gluconeogenesis 25%
Lactate 16%
Amino acids (mainly alanine) 6%
Glycerol 2%
Pyruvate 1%

Note: Liver glucose production from the different gluconeogenic substrates is


expressed as the percentage of total glucose production (10 to
12 mmol/kg/min).
Source: Data are from Newsholme, E.A. and Leech, A.R., Biochemistry for Medical
Sciences, John Wiley & Sons, Chichester, U.K., 1990; Windmueller, H.G. and
Spaeth, A.E., J. Biol. Chem., 255, 107–112, 1980; Fevrannini, E. et al., Diabetes, 34,
580–588, 1985. With permission.

substrates for gluconeogenesis are lactate, pyruvate, amino acids, and, to a lesser extent,
glycerol (Table 5.1; see Newsholme and Leech1 for a review). Since gluconeogenesis from
lactate is predominantly related to a recycling of carbons (Cori’s glucose–lactate cycle),
the net synthesis of glucose is essentially provided from amino acid sources and to a minor
extent from glycerol. Hence, in the absence of exogenous intakes of carbohydrates, the
oxidized glucose is replaced by newly synthesized molecules coming from the muscle
mass, and amino acids play a major role in glucose homeostasis.
Glucose is a major fuel in man, although its storage is limited (the glycogen storage is
consumed within 12 to 24 h) when compared to the lipid stores permitting weeks or months
of starvation.1 Hence, when exogenous carbohydrates are not sufficient, two mechanisms
permit the maintenance of glucose homeostasis: a decrease in glucose oxidation and an
enhancement of the synthesis of new glucose molecules from either liver glycogen or
gluconeogenic precursors2–4 (Table 5.2). Interestingly, the human body is able to self-satisfy
entirely its need of glucose through endogenous synthesis, conversely to lipids since essen-
tial fatty acid cannot be synthesized. If the relationships between amino acids and glucose
metabolism are predominantly considered through the role of amino acids as net gluco-
neogenic substrates, glucose and other carbohydrates are also precursors for nonessential
amino acid synthesis by transamination. Gluconeogenesis connects carbohydrate, lipid,
and amino acid metabolism in such a manner that it is not possible to study amino acid
gluconeogenesis without considering also lactate, glycerol, and lipid metabolism.

5.1 Gluconeogenesis at the cellular level: liver has a predominant


but nonexclusive role
It is classically believed that complete gluconeogenesis, i.e., glucose release, occurs only
in the liver and kidney,5,6 since only these tissues express the glucose-6-phosphatase gene.
However, it was recently shown that this gene is also expressed in the small intestine in
rats and humans.7–10 In fasting and diabetes the small intestine may contribute up to 20
to 25% of whole-body endogenous glucose production. In this organ, glutamine appears
to be the main gluconeogenic substrate, and therefore, glucose synthesis requires phos-
phoenolpyruvate carboxykinase (PEPCK), which is strongly induced in this tissue in some
conditions, such as long-term fasting and insulinopenia. Interestingly, glycerokinase is
also expressed in the small intestine, allowing the use of glycerol as a gluconeogenic
substrate together with glutamine. Alanine is substantially released by the gut, and since
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Chapter five: Amino acid metabolism and gluconeogenesis 85

Table 5.2 Glucose, Lactate, Alanine, and Glutamine Metabolism in Healthy Man at Basal
Postabsorptive Steady State
Total hepatic glucose output = total glucose uptake @ 10–12 mmol/kg/min
Glucose oxidation @ 5–7 mmol/kg/min
Brain glucose oxidation @ 4–5 mmol/kg/min
Glucose recycling via glycolysis @ 5.5 mmol/kg/min
Splanchnic @ 2.8 mmol/kg/min
Nonsplanchnic @ 2.4 mmol/kg/min
Muscle @ 2 mmol/kg/min
Blood cells, renal medulla @ 0.4 mmol/kg/min
Lactate turnover @ 4–5.5 mmol/kg/min
Alanine turnover @ 4–5 mmol/kg/min
Glutamine turnover @ 5–6 mmol/kg/min

Note: Averaged values of glucose metabolism in postabsorptive healthy man expressed as micromole
per kilogram of body mass per minute (mmol/kg/min). Although glucose oxidation concerns
about half of total glucose turnover, brain glucose oxidation accounts almost entirely for the
total glucose oxidation. The remaining half of glucose output is metabolized via recycling
pathways (lactate and alanine) divided into splanchnic and nonsplanchnic routes. The fluxes of
glucose oxidation, glucose recycling, lactate, alanine, and glutamine are of the same order of
magnitude: about 5 mmol/kg of body mass per hour.
Source: Data are from Cersosimo, E. et al., Metabolism, 49, 676–683, 2000; Cersosimo, E. et al., Diabetes,
49, 1186–1193, 2000; Mithieux, G., Curr. Opin. Clin. Nutr. Metab. Care, 4, 267–271, 2001; Pilkis, S.J. and
Claus, T.H., Annu. Rev. Nutr., 11, 465–515, 1991; Pilkis, S.J. and Granner, D.K., Annu. Rev. Physiol., 54,
885–909, 1992; Ferre, P. et al., Reprod. Nutr. Dev., 26, 619–631, 1986; Girard, J. et al., Reprod. Nutr. Dev.,
25, 303–319, 1985; Wahren, J. et al., J. Clin. Invest., 51, 1870–1878, 1972; Felig, P., Annu. Rev. Biochem.,
44, 933–955, 1975; Dechelotte, P. et al., Am. J. Physiol., 260, G677–G682, 1991.

this amino acid is a major precursor for liver gluconeogenesis, the gut appears to play an
important role in the gluconeogenesis from amino acids either directly (glutamine) or
indirectly (alanine).
Contrary to the classical view, PEPCK seems to be present in many tissues, including
the gut, as mentioned above, but also muscle and heart. Hence, all these organs are able
to achieve a net synthesis of phosphoenolpyruvate from oxaloacetate; therefore, they can
synthesize six-carbon intermediates, such as glucose 6-phosphate, from lactate. Interest-
ingly, this pathway is probably of major importance in muscle since it permits the restoring
of glycogen storage from lactate (and not from amino acids) in conditions where glycogen
is low and lactate is high, e.g., after intense muscular work.11
Theoretically, the lack of glucose-6-phosphatase prevents any glucose release from
muscle. In fact, in particular conditions of very active glycogenolysis, such a net release
of glucose can occur.12 Indeed, despite the absence of this enzyme, a very small amount
of nonphosphorylated glucose is released during hydrolysis of glycogen when this path-
way is highly activated. Renal gluconeogenesis is believed to be significant only after long-
term starvation. In this condition, this glucose formation is related to the need for an
increased proton excretion in urine, which is achieved when nitrogen is excreted predom-
inantly as ammonia from glutamine hydrolysis in the kidney, rather than urea synthesized
by the liver. Hence, in this particular condition, renal gluconeogenesis is mainly provided
by glutamine.1,13 Interestingly, a recent work has investigated the gluconeogenesis from
13C-lactate in patients during the anhepatic phase occurring in liver transplant surgery.14

This exceptional physiological condition of the complete absence of any liver function has
permitted the assessment of nonhepatic gluconeogenesis in a completely unequivocal
manner. In this particular condition, it was shown that as much as 50% of glucose synthesis
was achieved in the kidneys from labeled lactate.
The gluconeogenic pathway is classically described from pyruvate to glucose,
although several gluconeogenic pathways should actually be considered according to the
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86 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

precursor. Hence, gluconeogenesis from either pyruvate or lactate is different, since in one
case reducing equivalents must be exported from the mitochondrion to the matrix, whereas
in the latter NADH is provided directly in the cytosol. Moreover, pathways from alanine,
glycerol, or fructose are different.1 When amino acids are the gluconeogenic precursors,
oxaloacetate is a common intermediate, regardless of the amino acid substrate. Thus, the
amino acid gluconeogenic pathway can be divided into two parts: upstream and down-
stream of oxaloacetate.

5.1.1 From amino acids to oxaloacetate


As shown in Figure 5.1, amino acid enters the pathway at the site of pyruvate or of Krebs
cycle intermediates (2-oxoglutarate, succinyl-CoA, or fumarate) or directly via oxaloace-
tate. Entry of metabolites into the Krebs cycle via acetyl-CoA cannot lead to net oxaloac-
etate formation and therefore to glucose synthesis. Indeed, the Krebs cycle involves two
decarboxylative steps, while acetyl-CoA is a two-carbon metabolite. This is the case for
all substrates metabolized solely via acetyl-CoA, i.e., fatty acids, leucine, and lysine. Some
amino acids lead to both acetyl-CoA and pyruvate or citric acid cycle intermediates
(isoleucine, threonine, tryptophan, phenylalanine, or tyrosine); therefore, they are both
glucogenic and ketogenic. Among the different amino acids, the two most important for
gluconeogenesis are alanine and glutamine. In the liver, it is reported that alanine metab-
olism is largely controlled by its transport across the plasma membrane.15 However, in
experimental conditions of a very high rate of gluconeogenesis from alanine, it was
reported that the transamination step could be controlling.16 This point is of interest
because the cytosolic 2-oxoglutarate concentration is related to mitochondrial membrane
potential and then to cellular energy status. After transamination into pyruvate, alanine
is carboxylated into oxaloacetate by pyruvate carboxylase, a step also important for glu-
coneogenesis from lactate. Hence, the two major substrates for glucose synthesis, i.e.,
lactate and alanine, compete at the site of pyruvate carboxylase. Since the regulations of
alanine and lactate pathways have been studied separately so far, not much is known
about the reciprocal effect of lactate on alanine metabolism.

5.1.2 From oxaloacetate to glucose


This part of gluconeogenesis involves several steps shared with glycolysis (the near-
equilibrium steps), while three steps are far from equilibrium and are therefore irreversible.
These steps, catalyzed by pyruvate kinase, phosphofructokinase, and gluco (hexo) kinase,
need different reactions to reverse the flux; they are, respectively, pyruvate carboxylase
and PEPCK, fructose-1,6-biphosphatase, and glucose-6-phosphatase.1 For these three
steps, the presence of both forward and reverse enzymes creates a substrate cycling when
they are active simultaneously. This plays a major regulatory role, although it dissipates
some energy. The phosphoenolpyruvate (PEP)/pyruvate cycling is of crucial regulatory
importance in alanine, glutamine, and lactate gluconeogenesis, and the three main
enzymes, pyruvate carboxylase, pyruvate kinase, and PEPCK, share a large part of the
gluconeogenic flux control.17 Among the numerous factors involved in this regulation,
three should be emphasized: hormones (glucagon and insulin), acetyl-CoA concentration
(via free CoA to acetyl-CoA ratio), and cellular energy state (via phosphate potentials). It
should be emphasized that fatty acid oxidation increases acetyl-CoA, inhibits pyruvate
dehydrogenase, and activates pyruvate carboxylase in such a manner that the net result
is a simultaneous decrease in carbohydrate oxidation and an activation of gluconeogenesis.
Thus, fatty acid oxidation activates gluconeogenesis from lactate and alanine, as well as
from other amino acids entering the pathway at the site of pyruvate.
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Chapter five: Amino acid metabolism and gluconeogenesis 87

glucose

glucose-6-phosphate
alanine leucine
cysteine lysine
glycine phenylalanine
serine fructose-6-phosphate tyrosine
threonine
tryptophan fructose-1,6-disphosphate
acetoacetate

pyruvate phosphoenolpyruvate

isoleucine
leucine
acetylCoA threonine
aspartate oxaloacetate tryptophan
asparagine

malate citrate

phenylalanine
tyrosine fumarate isocitrate

CO 2 arginine
glutamate
succinate α-ketoglutarate glutamine
histidine
proline
succinyl-CoA

CO 2

isoleucine
methionine
valine

Figure 5.1 Shown are amino acids as gluconeogenic precursors and their relationships with Krebs
cycle intermediates. Amino acid metabolism is related to carbohydrate metabolism via the Krebs
cycle. Amino acids can be divided into three groups according to the site of their entry into the
pathway: acetyl-CoA, Krebs cycle intermediates, or pyruvate. (1) The entry via acetoacetate or acetyl-
CoA cannot lead to synthesis of net glucose precursors since the presence of two decarboxylative
steps at the cycle does not permit net synthesis of oxaloacetate from acetyl-CoA (a two-carbon
compound). Such a group of amino acids are precursors for ketone body synthesis The two amino
acids leucine and lysine, which enter the pathway at this site only, are not precursors for glucose
synthesis. (2) The majority of the gluconeogenic amino acids (12/18) enter the pathway at the site
of one of the Krebs cycle intermediates. (3) Six amino acids are metabolized via pyruvate, and among
them, alanine is the major gluconeogenic amino acid, sharing the gluconeogenic pathway with
lactate metabolism. The synthesis of glucose from oxaloacetate is mainly regulated via three substrate
cycles: the phosphoenolpyruvate/pyruvate, the fructose-1,6-bisphosphate/fructose-6-phosphate,
and the glucose 6-phosphate/glucose cycles.

5.1.3 Hormonal control of gluconeogenesis


The hormonal control of gluconeogenesis could be divided into transcriptional and post-
transcriptional regulation. The posttranscriptional regulation is achieved by protein phos-
phorylation mechanisms, which are related to either cAMP or calcium changes. Four
enzymes of the pathway are concerned: 6-phosphofructo-1-kinase, fructose-1,6-biphos-
phatase, fructose-2,6-biphosphatase, and pyruvate kinase. When phosphorylated, pyruvate
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88 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

kinase is inhibited, although fructose-2,6-biphosphatase is activated. Hence, the net result


is a decrease in fructose-2,6-biphosphate concentration and in the PEP/pyruvate recycling,
leading to an enhancement of gluconeogenesis. It can be noted that 6-phosphofructokinase
and fructose-1,6-biphosphatase18 also can be phosphorylated, although this does not affect
the enzyme activity,19 the meaning of such phosphorylation being unknown as yet.
The effects are mediated by four different hormones: glucagon, insulin, catechola-
mines, and cortisol. Glucagon is long known to stimulate gluconeogenesis from different
substrates by a rise in cAMP.20,21 It is a dominant hormone during fasting. Similarly,
b-adrenergic hormones increase cAMP, whereas a-adrenergic hormones act via intracel-
lular free calcium changes. The larger effect of the cAMP rise, as compared to that of the
calcium increase, may be due to the phosphorylation of both pyruvate kinase and fructose-
2,6-biphosphatase by cAMP, whereas calcium affects only fructose-2,6-biphosphatase. Cor-
tisol has no direct effect on phosphorylation, but it is known to amplify the effect of the
other hormones. Insulin is the dominant hormone during the fed state, but its effect is
located on muscle and adipose tissues, and the role of insulin on hepatic gluconeogenesis
is still a matter of controversy. Indeed, insulin addition is not responsible for an increased
glycogen deposition in the postprandial phase, and it seems that this hormone acts mainly
against the hyperglycemic effect of the other hormones by decreasing their effect on the
cAMP rise.22 The effect is probably due to an increased cAMP breakdown.23 It should be
added that posttranscriptional effects of hormones can also be mediated via changes in
energy metabolism, i.e., ATP-to-ADP ratio or transfer of reducing equivalents across the
mitochondrial membrane.24
The transcriptional regulation is involved in prolonged fasted states or in diseases
such as diabetes or acute illness, although it has been shown in early sepsis that the
hormonal effect of a-adrenergic stimulation by the a-agonist phenylephrine on gluconeo-
genesis was partly abolished, whereas ureagenesis stimulation was unchanged.25 This
transcriptional regulation concerns a glucagon-related cAMP rise leading to an increase
in several gluconeogenic enzymes (glucose-6-phosphatase, PEPCK, and possibly fructose-
1,6-biphosphatase) and a decrease in glycolytic enzymes (glucokinase, 6-phosphofructo-
1-kinase, and pyruvate kinase).23,26,27 Insulin or food intake has an opposite effect.27 This
cAMP effect on gene expression is of great importance in glucose homeostasis of the
newborn, since after birth PEPCK mRNA must be built for the first time. The rise in
glucagon immediatly after the birth plays a crucial role in energy metabolism at birth.28–30
It is of interest to note that the negative regulation on gene expression appears to be
dominant. Hence, the inhibitory effect of glucagon (cAMP) on glycolytic enzyme tran-
scription is stronger than the positive effect of insulin on these enzymes, whereas the
negative effect of insulin on gluconeogenic enzyme synthesis is stronger than that (posi-
tive) of glucagon.27

5.2 Gluconeogenesis: interorgan cooperation


The physiological need for hepatic gluconeogenesis is related to the condition where there
is a lack of exogenous glucose, i.e., the fasting state. In this condition, the muscle amino
acid balance is negative, and alanine and glutamine represent two thirds of the total
released amino acids.31–33 Such high alanine production is correlated with the use of amino
acid by the liver for glucose synthesis (Table 5.2). However, the question arises about the
origin of alanine and glutamine, since the muscle protein content of these two amino acids
is about 10%, whereas the net muscle production is about 60%. Hence, it appears that
these amino acids are synthesized in the muscle cells and simply not released after
muscular protein degradation.32 Alanine is probably formed from glucose carbon skeleton
via pyruvate and from ammonia as the result of leucine or other branched-chain amino
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Chapter five: Amino acid metabolism and gluconeogenesis 89

acid catabolism.34–37 As mentioned above, fatty acid oxidation reduces pyruvate oxidation
and enhances its use for gluconeogenesis; the same result can be achieved in muscle by
leucine oxidation, since its metabolism leads also to acetyl-CoA formation. Although the
muscle is not a gluconeogenic organ stricto sensu, such a mechanism of inhibition of
pyruvate oxidation when leucine is oxidized may contribute to the increase in muscle
alanine formation, which in turn affects the liver gluconeogenesis rate. Because alanine,
coming from glucose in muscle cells, is directly taken up by the liver mainly to resynthesize
glucose, Felig38 proposed 20 years ago the concept of an alanine–glucose cycle by reference
to Cori’s cycle (see Section 5.4). Indeed, it has been shown in man that glucose infusion
involves a decrease in both glucose and urea production, whereas the rate of appearance
of labeled alanine in the plasma, as well as the glucose synthesis from alanine, was
enhanced.39
Glutamine is also synthesized in muscle cells, but the carbon skeleton is provided
from the catabolism of other amino acids such as branched-chain amino acids or aspartic
acid; ammonia also results, at least for one part, from the branched-chain amino acid
catabolism.33,35–37,40 Glutamine is probably also a major gluconeogenic amino acid in vivo
at the level of the gut itself and indirectly as a precursor for alanine synthesis. It is actively
taken up by the gut and converted into pyruvate, which is either oxidized or transaminated
to form alanine released in the portal blood and potentially taken up by the liver.41,42
Therefore, if we consider the complete carbon pathway, glutamine is clearly also a quan-
titative substrate for gluconeogenesis, mainly after a first conversion into alanine. The
quantitative role of the other gluconeogenic amino acids in the hepatic glucose synthesis
rate is probably not very high, although glycine can be actively converted to glucose. It
is possible that pyruvate used in muscle for alanine synthesis is not only derived from
glucose but also from other gluconeogenic amino acids. This fact is of importance, since
when derived from glucose, this pathway represents a simple carbon recycling, whereas
in the second case, i.e., from other amino acids, there is a net neoglucose formation from
protein (see Section 5.4). It is difficult to estimate the exact contribution of alanine to
gluconeogenesis in vivo. Indeed, some studies using stable isostopes have measured ala-
nine, glutamine, and glucose turnover in man (see Table 5.2), and it has been shown that
alanine turnover represents about one third of glucose turnover, whereas glutamine flux
is about 40%.33,35–37,40 It is difficult to assess precisely the percentage of alanine synthesized
from glutamine in gut and taken up by the liver, since it is not possible to obtain in humans
portal blood samples. Assuming that two thirds of glutamine are completely oxidized in
the gut,3,41–43 it can be estimated, in terms of carbon equivalents, that alanine plus glutamine
contribution to hepatic glucose synthesis in overnight fasted healthy subjects may reach
about 25% of the total glucose synthesis. In fasted dogs, it has been shown that after an
oral glucose load, the liver glycogen was restored by exogenous glucose for 50% and by
endogenous substrates (lactate and alanine) for the other 50%.44 Moreover, alanine or
lactate was mainly coming from the gut.
During exercise, there is an accelerated rate of muscular glucose uptake, which must
be linked to major changes in gluconeogenesis/glycolysis regulation in order to prevent
dramatic changes in blood glucose concentration. Glycogenolysis and gluconeogenesis
are both involved in the increase in hepatic glucose production, glycogenolysis being
predominant in short-term exercise and gluconeogenesis being the major pathway in
prolonged exercise or during recovery. When the gluconeogenic pathway is enhanced by
exercise, the use of alanine as a hepatic substrate for glucose is increased. This effect is
mainly due to the increase in hepatic fractional extraction but also to a more efficient
channeling of alanine metabolism into glucose.45 During the recovery phase, the glucose
formation from alanine is further enhanced, since in addition to the increased alanine
extraction by the liver, there is a rise in alanine supply to the liver.45
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90 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

5.3 Gluconeogenesis and pathological states


It has long been known that gluconeogenesis is enhanced in severe illness, injury, and
diabetes, whereas it is decreased in long-term starvation.46 In stressed patients, the occur-
rence of an increased gluconeogenesis together with hyperglycemia is known as insulin
resistance. The increased gluconeogenesis is not suppressible by infusion of high amounts
of glucose.47 The splanchnic glucose output is increased by a factor of 2,47–49 whereas the
glucose formation from the kidney is not enhanced as in prolonged starvation. Wolfe and
coworkers50 have shown that the large increase in glucose production observed in burn
patients (24 µmol/kg/min), compared to controls (12 µmol/kg/min), was related to an
increase in both recycling and nonrecycling glucose production. The glucose oxidized,
when expressed as a percentage of total glucose uptake, was significantly decreased. The
increased use of amino acids for gluconeogenesis has been recognized for a long time,
since stress and the hypercatabolic state are associated with an increase in urinary nitrogen
loss. In fact, it was shown that in burn patients the entire increment in glucose production
could be accounted for by the increase in gluconeogenic precursors uptake by the
liver.48,49,51 Simultaneously, with such increased splanchnic glucose output and turnover,
some data showed, in these patients, a low respiratory quotient, demonstrating a small
contribution of glucose oxidation to total body oxygen consumption. This suggests that
the nonoxidative pathways (glucose–alanine or glucose–lactate) are primarily involved in
the increased glucose turnover, even when muscle amino acid contribution to gluconeo-
genesis is enhanced.48

5.4 Gluconeogenesis or glucose recycling?


The term gluconeogenesis means new glucose synthesis from a nonglucose compound. Thus,
we call gluconeogenesis the glucose formation from either fructose, glycerol, lactate, pro-
pionate, or amino acids, although the source and the pathways are quite different. In fact,
physiologically there are two kinds of gluconeogenesis, depending on the origin of the
carbons and not on their metabolic fate.52,53 Indeed, glucose can be formed from precursors
belonging to biochemical families other than carbohydrates. For example, ruminants absorb
very small amounts of glucose, and conversely, to nonruminant animals (like humans),
gluconeogenesis is maximally stimulated in the fed state and depressed during the fasting
state. The nutrients are first metabolized by bacteria, and fatty acids containing an odd
number of carbons are powerful gluconeogenic substrates via propionate.54 This is an
example of a true gluconeogenesis de novo since glucose is made from odd fatty acids. This
situation is comparable to the gluconeogenesis from amino acids, where proteins from
muscle are used to make new glucose molecules. On the other hand, when there is carbon
recycling, for instance, in the Cori’s cycle between glucose and lactate, there is only a partial
glucose hydrolysis (from six to three carbon intermediates), and the resynthesis occurs from
the hydrolysis products in another tissue.52 In this example, there is no net glucose synthesis,
but only a recycling of carbons between glucose and lactate, which consumes energy but
does not lead to net glucose accumulation, since the hydrolysis of one glucose into two
lactates is needed to rebuild a new glucose from the two lactates. In view of this fact,
gluconeogenesis from alanine is either a pathway of net gluconeogenesis if carbons are
coming from some amino acids resulting from muscle proteolysis or only a pathway of
carbon recycling if these carbons are coming from muscle glucose hydrolysis.

5.4.1 De novo gluconeogenesis


Another approach may relate true de novo gluconeogenesis and the total amount of oxi-
dized glucose. Indeed, in postabsorptive steady state, i.e., when the pools of the main
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Chapter five: Amino acid metabolism and gluconeogenesis 91

carbohydrates are constant — glucose, lactate, pyruvate, glycogen content — the glucose,
which is fully oxidized as CO2 and H2O, must be replaced by a true neo-glucose. Hence,
the measurement of oxidized carbohydrates gives an estimation of the true glucose syn-
thesis. In this fasting situation, gluconeogenesis from muscle protein amino acids can
account almost entirely (glycerol excepted) for such a true glucose formation; i.e., it is
equal to the net glucose oxidation. Hence, in the absence of exogenous intakes and in a
steady-state situation, the net glucose oxidation is an estimation of the net muscle protein
amino acid amount used for glucose synthesis.

5.4.2 Glucose recycling: the futile cycles


When considering the recycling of glucose, the situation is opposite. There is neither net
glucose oxidation nor newly synthesized glucose molecules, but only waste of energy in
the form of heat with increased oxygen consumption and CO2 production (Figure 5.2).
Although in the previous situation the muscle mass was decreasing parallel to glucose
formation, in this situation the lipid mass is used parallel to glucose recycling (see Figure
5.2). Indeed, the major metabolic fuel for liver is fatty acid, and consequently, acetyl-CoA
rise inhibits carbohydrate oxidation. Hence, the ATP consumption for liver gluconeogen-
esis (from lactate or alanine) is provided by fatty acid oxidation (b-oxidation ATP),
whereas peripheral glucose hydrolysis gives glycolytic ATP. Hence, these metabolic con-
siderations lead to different approaches of the two main glucose recycling cycles: glu-
cose–lactate cycle and glucose–alanine cycle. On the one hand, in terms of yield, there is
a clear loss of energy efficacy during recycling since two ATP are produced from one
glucose hydrolyzed into pyruvate, whereas six ATP are needed to make one glucose from
either two pyruvate, two lactate, or two alanine. Thus, two thirds of oxygen consumption
for ATP synthesis is directly lost as heat. On the other hand, when considering the
qualitative aspects instead of the quantitative, it appears that glucose hydrolysis into
pyruvate and finally into lactate (in insulin resistance, anoxic, or stressed tissue) or to
alanine or glutamine (in muscle to bring ammonia to the liver as a nontoxic form) provides
glycolytic anaerobic (or “nonrobic”) ATP to cells, whereas these compounds were made
with fatty acid aerobic ATP in the liver. Hence, one can say that these two main glucose
cycles allow the transfer of ATP made aerobically in the liver from lipid stores to glycolytic
anaerobic ATP made from glucose in peripheral cells. This is achieved with a decrease
in efficacy, but when considering the mass of lipid stores compared to the size of carbo-
hydrate and amino acid stores, it might be an advantage in these fasted states or acute
illnesses.

5.5 Conclusion
Gluconeogenesis is the pathway allowing glucose formation; however, regarding the
multiple precursors, this pathway is actually multiple. It is very important to consider
that glucose metabolism is divided in two parts: oxidative and nonoxidative routes. This
means that in the former case new molecules of glucose must be built from amino acids
provided from protein degradation, while in the latter it is only a carbon recycling with
energy dissipation. Lactate and pyruvate correspond solely to recycling; alanine and
glutamine correspond to both recycling and new synthesis, although recycling is probably
predominant. Other amino acids and all essential amino acids are involved in the neo-
synthetic pathway.
Contrary to the classical view, the liver is not the unique organ in glucose synthesis,
the kidney plays a major role, and the gut is probably an important source of neo-glucose
as well.
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92 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

H 2 O + CO2

oxidation
recycling recycling
glucose
LIVER MUSCLE
glucose
glucose
glucose 2 ADP
2 ADP 6 ADP

PERIPHERIC β oxidation VALINE


or other AA
CELL 2 ATP
6 ADP 2 ATP
PYRUVATE PYRUVATE
PYRUVATE

LACTATE GLUTAMINE
ALANINE

LACTATE ALANINE

ALANINE

AMMONIA
GLUTAMINE GLUTAMINE

GUT

Figure 5.2 Gluconeogenesis: net glucose synthesis and substrate recycling. Liver glucose
production can be divided in either de novo synthesized glucose or substrate recycling.
Lactate and alanine are the main glucose precursors, although other gluconeogenic amino
acids and glycerol are also involved. Glucose recycling via lactate or alanine creates a
futile cycle between three- and six-carbon compounds. Although it dissipates directly, two
thirds of the energy (six ATP are needed to build one glucose from two lactate or two
alanine, whereas two ATP only are produced when splitting glucose into lactate or ala-
nine), the liver energy source is mainly coming from fatty acids. Hence, glucose recycling
provides glycolytic ATP to peripheric cells, whereas this ATP comes from lipid stores.
Glutamine plays a special role as glucose precursor; it is poorly involved per se, but since
it is converted into alanine in enterocytes, it is taken up by the liver. Moreover, in muscle,
glutamine is formed from an amino acid carbon skeleton (mainly branched chain), explain-
ing that such glucose synthesis is mainly a true de novo glucose synthesis rather than
recycling.

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glucidique du nouveau-né, Méd. Sci., 9, 297–306, 1993.
31. Wahren, J., Felig, P., Cerasi, E., and Luft, R., Splanchnic and peripheral glucose and amino
acid metabolism in diabetes mellitus, J. Clin. Invest., 51, 1870–1878, 1972.
32. Felig, P., Amino acid metabolism in man, Annu. Rev. Biochem., 44, 933–955, 1975.
33. Dechelotte, P., Darmaun, D., Rongier, M., Hecketsweiler, B., Rigal, O., and Desjeux, J.F.,
Absorption and metabolic effects of enterally administered glutamine in humans, Am. J.
Physiol., 260, G677–G682, 1991.
34. Chang, T.W. and Goldberg, A.L., The metabolic fates of amino acids and the formation of
glutamine in skeletal muscle, J. Biol. Chem., 253, 3685–3693, 1978.
35. Darmaun, D., Métabolisme de la glutamine in vivo chez l'homme: implications pour la
nutrition artificielle, Nutr. Clin. Métabol., 4, 203–214, 1990.
36. Darmaun, D. and Dechelotte, P., Role of leucine as a precursor of glutamine alpha-amino
nitrogen in vivo in humans, Am. J. Physiol., 260, E326–E329, 1991.
37. Darmaun, D., Role of nutrients in the regulation of in vivo protein metabolism in humans,
Acta Paediatr. Suppl., 88, 92–94, 1999.
38. Felig, P., The glucose-alanine cycle, Metabolism, 22, 179–207, 1973.
39. Royle, G.T., Molnar, J.A., Wolfe, M.H., Wolfe, R.R., and Burke, J.F., Urea, glucose and alanine
kinetics in man: effects of glucose infusion, Clin. Sci., 62, 553–556, 1982.
40. Darmaun, D., Role of glutamine depletion in severe illness, Diabetes Nutr. Metab., 13, 25–30,
2000.
41. Hanson, P.J. and Parsons, S., Metabolism and transport of glutamine and glucose in vascu-
larly perfused small intestine rat, Biochem. J., 166, 509–519, 1977.
42. Haymond, M.W. and Miles, J.M., Branched chain amino acids as a major source of alanine
nitrogen in man, Diabetes, 31, 86–89, 1982.
43. Windmueller, H.G. and Spaeth, A.E., Identification of ketone bodies and glutamine as the
major respiratory fuels in vivo for postabsorptive rat small intestine, J. Biol. Chem., 253, 69–76,
1978.
44. Mitrakou, A., Jones, R., Okuda, Y., Pena, J., Nurjhan, N., Field, J.B., and Gerich J.E., Pathway
and carbon sources for hepatic glycogen repletion in dogs, Am. J. Physiol., 260, E194–E202,
1991.
45. Wasserman, D.H., Williams, P.E., Lacy, D.B., Green, D.R., and Cherrington, A.D., Importance
of intrahepatic mechanisms to gluconeogenesis from alanine during exercise and recovery,
Am. J. Physiol., 254, E518–E525, 1988.
46. Streja, D.A., Steiner, G., Marliss, E.B., and Vranic, M., Turnover and recycling of glucose in
man during prolonged fasting, Metabolism, 26, 1089–1098, 1977.
47. Long, C.L., Kinney, J.M., and Geiger, J.W., Nonsuppressability of gluconeogenesis by glucose
in septic patients, Metabolism, 25, 193–201, 1976.
48. Gelfand, R.A., Glickman, M.G., Jacob, R., Sherwin, R.S., and DeFronzo, R.A., Removal of
infused amino acids by splanchnic and leg tissues in humans, Am. J. Physiol., 250, E407–E413,
1986.
49. Wilmore, D.W., Aulick, H.L., and Goodwin, C.W., Glucose metabolism following severe
surgery, Acta Chir. Scand., 498 (Suppl.), 43–47, 1979.
50. Wolfe, R.R., Durkot, M.J., Allsop, J.R., and Burke, J.F., Glucose metabolism in severely burned
patients, Metabolism, 28, 1031–1039, 1979.
51. Wilmore, D.W., Goodwin, C.W., Aulick, L.H., Powanda, M.C., Mason, A.D., Jr., and Pruitt,
B.A., Jr., Effect of injury and infection on visceral metabolism and circulation, Ann. Surg.,
192, 491–504, 1980.
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Chapter five: Amino acid metabolism and gluconeogenesis 95

52. Leverve, X.M., Lactic acidosis: a new insight? Minerva Anestesiol., 65, 205–209, 1999.
53. Leverve, X.M., Energy metabolism in critically ill patients: lactate is a major oxidizable
substrate, Curr. Opin. Clin. Nutr. Metab. Care, 2, 165–169, 1999.
54. Demigne, C., Yacoub, C., Morand, C., and Remesy, C., Interactions between propionate and
amino acid metabolism in isolated sheep hepatocytes, Br. J. Nutr., 65, 301–317, 1991.
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chapter six

Contribution of amino acids


to ketogenesis
Milan Holecˇek
Charles University School of Medicine

Contents
Abbreviations.................................................................................................................................98
Introduction....................................................................................................................................98
6.1 General pathways of metabolism of ketone bodies .......................................................98
6.1.1 Synthesis of KB ........................................................................................................98
6.1.2 Utilization of ketone bodies.................................................................................100
6.2 Ketogenesis from amino acids .........................................................................................100
6.2.1 Contribution of amino acids to the total production of ketone bodies ..........101
6.2.2 Ketogenesis from leucine .....................................................................................101
6.2.2.1 Skeletal muscle ........................................................................................101
6.2.2.2 Adipose tissue .........................................................................................102
6.2.2.3 Liver ..........................................................................................................102
6.2.2.4 Brain ..........................................................................................................103
6.3 Physiology and pathophysiology of ketogenesis from amino acids.........................104
6.3.1 Ketogenesis from amino acids in hyperketonemic states...............................104
6.3.1.1 Starvation .................................................................................................104
6.3.1.2 Diabetes mellitus.....................................................................................105
6.3.2 Ketogenesis from amino acids in stress illness ................................................106
6.4 Conclusions .........................................................................................................................106
Acknowledgments ......................................................................................................................107
References .....................................................................................................................................107

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98 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Abbreviations
BCAA branched-chain amino acids (valine, leucine, and isoleucine)
BCKA branched-chain keto acids
CoA coenzyme A
FFA free fatty acids
HMG-CoA b-hydroxy-b-methylglutaryl-CoA
KB ketone bodies
KIC a-ketoisocaproic acid
KIV a-ketoisovaleric acid
KMV a-keto-b-methylvaleric acid
TAG triacylglycerols

Introduction
Physiologically important ketone bodies (KB) are represented by acetoacetate and
b-hydroxybutyrate. Minute quantities of acetone generated in the body by spontaneous
decarboxylation of acetoacetic acid are not metabolized and are a byproduct released by
lungs and kidneys. KB are an important fuel for the brain, heart, skeletal muscle, intestine,
and kidney both in physiological and pathological conditions. Production and utilization
of KB is markedly activated in starvation, a high-fat diet, strenuous exercise, and uncon-
trolled diabetes, and during development. Apart from acting as an important energy
substrate, KB can provide acetyl-CoA for synthesis of lipids in several tissues and play a
regulatory role in the metabolism of other substrates. Hyperketonemia results in the
sparing of glucose oxidation by the brain due to cerebral utilization of KB and inhibits
lipolysis in adipose tissue. Several studies suggested an inhibitory effect of KB on protein
breakdown.1
Ketogenesis occurs mainly in the liver from long-chain fatty acids derived from adi-
pose tissue. Other potential endogenous precursors of KB are short-chain fatty acids (e.g.,
acetate and butyrate) formed by gut flora and ketogenic amino acids (phenylalanine,
tyrosine, tryptophan, isoleucine, leucine, and lysine). These compounds are believed to
make insignificant contributions. However, five of six ketogenic amino acids are indis-
pensable, and their activated catabolism in reactions of ketogenesis may significantly affect
protein metabolism and the whole status of the human or animal. Unfortunately, there is
little attention given to understanding the regulation and clinical importance of ketogen-
esis from amino acids, and the available data are scattered throughout a limited number
of original research papers.

6.1 General pathways of metabolism of ketone bodies


The blood concentration of KB is given by the difference in their release to the blood and
uptake by extrahepatic tissues. In overnight fasted humans and rats it is around 0.1 to
0.4 mmol/l. At this point, the pathways and factors controlling KB synthesis from fatty
acids and pathways of KB utilization will be briefly described (see also Figure 6.1). The
pathways in which ketogenic amino acids contribute to ketogenesis are discussed later.

6.1.1 Synthesis of KB
Ketogenesis from fatty acids in overnight fasted humans is about 0.2 to 0.4 mmol/min,2
occurs almost entirely in the liver, and is controlled at several sites.
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Chapter six: Contribution of amino acids to ketogenesis 99

ADIPOSE TISSUE LIVER BRAIN, MUSCLE, KIDNEY,


HEART
TAG FFA FFA ATP
1 CoA
2

Acyl-CoA ATP

Acetyl-CoA Citric acid cycle


Acetyl-CoA
3
CoA

Acetoacetyl-CoA 2 Acetyl-CoA
Acetyl-CoA
4 3
CoA CoA
HMG-CoA Acetoacetyl-CoA
5 Succinate
Acetyl-CoA 7
Succinyl-CoA

Acetoacetate Acetoacetate
NADH+H + NADH+H +
6 6
NAD + NAD +
β-Hydroxybutyrate β-Hydroxybutyrate

Figure 6.1 Pathways of ketogenesis from fatty acids and ketone bodies utilization. TAG, triacyl-
glycerols; 1, hormone sensitive lipase (E.C.3.1.1.3); 2, acyl-CoA synthase (E.C.6.2.1.3); 3, acetoacetyl-
CoA thiolase (E.C.2.3.1.9.); 4, HMG-CoA synthase (E.C.4.1.3.5.); 5, HMG-CoA lyase (E.C.4.1.3.4.); 6,
b-hydroxybutyrate dehydrogenase (E.C.1.1.1.30); 7, 3-oxoacid-CoA-transferase (E.C.2.8.3.5).

Lipolysis and release of free fatty acids (FFA) from white adipose tissue to the blood
is the first step. The enzyme regulating lipolysis is hormone-sensitive lipase, which can
be activated by glucagon, epinephrine, norepinephrine, ACTH, thyroid-stimulating hor-
mone, growth hormone, and glucocorticoids. Insulin antagonizes the effect of lipolytic
hormones.
Fatty acids taken up by the liver in a concentration-dependent manner, after conver-
sion to the acyl-CoA, either are reesterified or enter the mitochondria via the carnitine
shuttle to be oxidized (second control point). In the majority of catabolic situations,
b-oxidation in mitochondrial matrix predominates. The rate of b-oxidation is regulated
through levels of fatty acyl-CoA, carnitine, and malonyl-CoA. Malonyl-CoA acts as a
potent inhibitor of carnitine acyltransferase I, the enzyme catalyzing the initial step in
fatty acid oxidation. As malonyl-CoA is an intermediate in synthesis of fatty acids, b-oxi-
dation is inhibited when fatty acid synthesis is active.
After b-oxidation, the resultant acetyl-CoA may be either converted to KB or con-
densed with oxaloacetate and enter the citric acid cycle (third control point). Ketogenesis
from acetyl-CoA occurs in mitochondria when the rate of supply of acetyl-CoA outstrips
the capacity for its oxidation by the citric acid cycle (e.g., increased lipolysis) and all
enzymes of pathway for ketogenesis are available (only in the liver). At the first step, two
molecules of acetyl-CoA condense (catalyzed by thiolase) to form one molecule of ace-
toacetyl-CoA (acetoacetyl-CoA also arises directly during the course of b-oxidation). Then
the ketogenesis pathway involves the condensation of acetoacetyl-CoA with acetyl-CoA
to b-hydroxy-b-methylglutaryl-CoA (HMG-CoA) catalyzed by HMG-CoA synthase. It
should be noted that the activity of HMG-CoA synthase is located in two different com-
partments, cytosol and mitochondria,and the genes for mitochondrial and cytosolic HMG-
CoA synthase are differently regulated.3,4 Control of ketogenesis is exerted by transcrip-
tional regulation of mitochondrial HMG-CoA synthase. Fasting, cAMP, and fatty acids
increase its transcriptional rate, while refeeding and insulin repress it.5 The HMG-CoA
produced in cytosol is the starting point of the isoprenoid pathway with cholesterol as
the main end product. HMG-CoA produced in mitochondria is split by HMG-CoA lyase
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100 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

to acetoacetic acid and acetyl-CoA. Part of the acetoacetic acid is converted into b-hydrox-
ybutyric acid. The interconversion of acetoacetate and b-hydroxybutyrate is controlled by
the mitochondrial ratio of [NAD+] to [NADH]. The [NAD+]/[NADH] ratio may increase
as result of rapid b-oxidation.

6.1.2 Utilization of ketone bodies


Ketone body utilization occurs rapidly in most extrahepatic tissues, particularly in the
brain, heart, skeletal muscle, and kidneys. The main pathway involves activation of ace-
toacetate to acetoacetyl-CoA by 3-oxoacid-CoA-transferase, an enzyme that appears to be
present in sufficient quantities in all tissues except liver. Acetoacetyl-CoA is split to acetyl-
Co by thiolase. Acetyl-CoA is oxidized in the citric acid cycle (major fate) or used for
synthesis of lipids. Biosynthesis of lipids from KB has been demonstrated in the developing
brain, adipose tissue, and lactating mammary gland.
In the liver, in contrast to other tissues, all enzymes essential for synthesis of acetyl
CoA from KB, the reaction necessary for oxidation of KB, are not expressed. The alternative
possibility of utilization of KB in liver is synthesis of cholesterol in the cytosol. This
pathway is not very active, and most KB synthesized in the liver are released to the
bloodstream and utilized in other tissues.
It appears that the main factor influencing the rate of KB utilization in extrahepatic
tissues is their concentration in the blood. If the blood KB level rises, oxidation of KB
increases. The pathway of KB oxidation is saturated at a KB concentration of approxi-
mately 12 mmol/l.

6.2 Ketogenesis from amino acids


Ketogenic amino acids are those yielding either acetoacetate or one of its precursors, acetyl-
CoA or acetoacetyl-CoA (phenylalanine, tyrosine, tryptophan, isoleucine, leucine, and
lysine). Amino acids whose catabolism yields pyruvate or one of the intermediates of the
citric acid cycle are glucogenic. Some amino acids have both ketogenic and glucogenic
functions (phenylalanine, tyrosine, tryptophan, and isoleucine). The only amino acids that
are exclusively ketogenic are leucine and lysine, because they cannot function as carbon
sources for the net synthesis of glucose. The final step in catabolism of leucine is break-
down of HMG-CoA to acetoacetate and acetyl-CoA. This explains its strong ketogenic
effect. Several studies have demonstrated that ketogenesis from amino acids, particularly
from leucine and isoleucine, is not a specific feature of hepatic tissue and also occurs in
the brain, skeletal muscle, adipose tissue, kidneys, heart, and gut.
The first step in ketogenesis from amino acids is their transamination followed by
elimination of the a-amino group in the form of urea. Most of the aminotransferase activity
is located in the liver. However, branched-chain amino acid (BCAA) aminotransferase, the
enzyme regulating the initial step in catabolism of BCAA (valine, leucine, and isoleucine),
has the highest activity in skeletal muscle. Acute regulation of transamination occurs by
the concentration of substrate and therefore is dependent mainly on the net rate of protein
breakdown or protein intake by food. Chronic regulation is by changing the activities of
the enzymes concerned. This may be achieved by glucagon, glucocorticoids, or other
humoral factors. It is unique in the catabolism of lysine that neither of its amino groups
undergoes transamination as the first step in their catabolism.
After the a-amino group has been removed from the amino acid, the remaining carbon
skeleton is available for conversion to intermediates of energy metabolism. Control of the
breakdown of the C-skeleton of amino acids depends on the nature of a-ketoacid
produced.
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Chapter six: Contribution of amino acids to ketogenesis 101

6.2.1 Contribution of amino acids to the total production of ketone bodies


Studies trying to estimate the contribution of ketogenic amino acids to the total KB pro-
duction are rare. Thomas et al.6 demonstrated that in rats starved for 3 h, 4.4% of KB carbon
is derived from the metabolism of leucine. The specific radioactivity of blood KB was four-
to fivefold higher after injection of labeled leucine than after injection of lysine or phenyl-
alanine. A similar result was obtained by Kulaylat et al.,7 who measured the hepatic
conversion of leucine to KB in overnight fasted dogs using continuous infusion of
L-U-[14C]-leucine and by determination of the appearance of [14C]-KB across the liver.
Authors calculated that hepatic conversion of leucine to KB accounted for 3.5% of net
hepatic production of KB.
It may be deduced from these studies that during the postprandial state (i.e., within
12 h of eating), the contribution of all ketogenic amino acids to ketogenesis is around 10%.
Considering the importance of ketogenic amino acids, particularly of leucine, in protein
economy, these amounts should not be considered negligible.

6.2.2 Ketogenesis from leucine


This chapter will mainly concentrate on leucine, as leucine is undoubtedly the most
significant ketogenic amino acid6 and because limited information about the contribution
of others exists. In an effort to elucidate the pathways of ketogenesis from leucine, we
should consider specific features in the metabolism of BCAA. The first step in BCAA
catabolism is reversible transamination leading to the production of corresponding
branched-chain keto acids (BCKA). Leucine is converted to a-ketoisocaproic acid (KIC),
isoleucine to a-keto-b-methylvaleric acid (KMV), and valine to a-ketoisovaleric acid (KIV).
The BCKA formed in this reaction then undergo irreversible decarboxylation catalyzed
by BCKA dehydrogenase to form thioesters of coenzyme A, and then through a series of
reactions is converted to acetoacetate and acetyl-CoA (leucine), propionyl-CoA and acetyl-
CoA (isoleucine), and succinyl-CoA (valine). In view of the importance of succinyl-CoA
in combustion of KB (see Figure 6.1), we can speculate about mutual relationships in
catabolism of valine (yielding succinyl-CoA) and KB generated from leucine and isoleu-
cine. This speculation is supported by the observation of Meguid et al.,8 who have shown
that an excess of leucine increases oxidation of valine both in vivo in humans and in isolated
epitrochlearis muscles from rats. Similarly, Block and Harper9 demonstrated that the
depression in plasma isoleucine and valine concentrations in rats fed by a high leucine
meal was associated with 50% increase in whole-body valine oxidation.
The activity of the first enzyme in BCAA metabolism, BCAA aminotransferase, is high
in muscle, heart, and kidney and very low in liver. The rate of transamination depends
primarily on the activity of the enzyme, and concentrations of substrates and products of
transamination. BCKA dehydrogenase is a multienzyme complex located in mitochondria
regulated by reversible phosphorylation (inactivation) and dephosphorylation (activa-
tion). Its activity is highest in the liver, while it is low in muscle, adipose tissue, and the
brain.10 Several studies provide strong evidence that these differences in BCKA dehydro-
genase activity are the basis of interorgan cooperation in BCAA metabolism. Therefore,
several tissues are involved and cooperate in ketogenesis from leucine.

6.2.2.1 Skeletal muscle


Due to a high activity of BCAA aminotransferase, the skeletal muscle has a remarkable
capability to transaminate BCAA to their ketoanalogues and is considered the initial site
of BCAA catabolism. BCAA are considered as essential donors of nitrogen in synthesis of
glutamine and alanine. Increased availability of BCAA enhances glutamine synthetase
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102 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

activities and release of alanine and glutamine from muscle.11,12 It can be suggested that
increased protein breakdown and activated catabolism of BCAA in muscle in stress illness
such as sepsis, burn injury, and trauma enable the body to provide for its increased need
for glutamine and alanine.
As the BCKA dehydrogenase activity in skeletal muscle is low, a significant portion
of BCKA generated in BCAA aminotransferase reaction is released to the blood and then
metabolized in other tissues.10,13 Therefore, under physiological conditions, a limited
amount of BCAA is catabolized in muscle to KB. These are mostly oxidized to CO2 and
water, and only a smaller part is released to the blood.14,15 Nevertheless, these data clearly
demonstrate that skeletal muscle may be a peripheral site of ketogenesis from amino acids.
KIC released to the blood may be an important precursor of KB in other tissues, particu-
larly in the liver.

6.2.2.2 Adipose tissue


Adipose tissue is very active in utilization of BCAA, particularly in a well-nourished state.
The capability of adipose tissue to concentrate BCAA is considerably high, even higher
than that of skeletal muscle. Leucine concentration in intracellular water of adipose tissue
is fourfold, while in skeletal muscle it is only 25 to 50% greater than that in extracellular
fluid.16,17 Available evidence suggests that in adipose tissue the aminotransferase activity
exceeds maximal rates of leucine oxidation and that the BCKA dehydrogenase activity is
the rate-limiting step controlling leucine degradation.18 Therefore, KIC formed from leu-
cine may accumulate to such an extent that 15% leaks to the blood.19
Degradation of leucine in adipose tissue is stimulated by insulin20 and coupled to
production of alanine and glutamine, like in the muscle. Generated acetyl-CoA and aceto-
acetate can be oxidized for energy or used for the synthesis of long-chain fatty acids and
cholesterol.21 In fact, in fed animals, the products of leucine catabolism are utilized for
fatty acid synthesis, while in fasted animals they might play a role in the development of
ketoacidosis. The significant contribution of leucine to ketogenesis in adipose tissue also
clearly demonstrates an increased amount of acetoacetate released by epididymal adipose
tissue of mice after addition of leucine to the incubation medium.22

6.2.2.3 Liver
The main end product of catabolism of leucine in liver is acetoacetate, together with
smaller amounts of b-hydroxybutyrate and acetyl-CoA. From rates of 14CO2 production
from [1-14C]-KIC and [U-14C]-KIC by isolated hepatocytes, it has been shown that only
10% of flux of KIC through BCKA dehydrogenase was oxidized to CO2; the reminder was
released as acetoacetate.23
Low activity of hepatic BCAA aminotransferase and high activity of BCKA dehydro-
genase implicate the important role of BCKA, delivered mainly from skeletal muscle and
adipose tissue, in hepatic ketogenesis. The hypothesis that the main substrate for hepatic
ketogenesis from amino acids is BCKA, particularly KIC, delivered by the bloodstream is
supported by a number of studies. Livesey and Lund24 demonstrated that the liver can
extract a quantity of BCKA equivalent to that released by muscle. Spydevold and
Hokland 25 demonstrated using perfused liver of rat that KB were the main product
released when liver was provided with KIC (ketoleucine) or KMV (ketoisoleucine). Kulay-
lat et al.7 measured hepatic conversion of leucine to KB by continuous infusion of U-[14C]-
leucine and by determination of the appearance of [14C]-KB in dogs fasted for 3 days. The
results showed that the rate of production of [14C]-KB exceeded the rate of unidirectional
uptake of labeled leucine by the liver. This finding clearly indicates that some leucine
carbon converted to KB must have been derived from one of the metabolites of leucine,
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Chapter six: Contribution of amino acids to ketogenesis 103

most likely KIC, produced in extrahepatic tissues. The crucial position of the liver in
ketogenesis from KIC is also demonstrated by the observations of Krebs and Lund,26 who
showed that KIC supported a rate of ketogenesis in isolated hepatocytes from 48-h starved
rats almost 100-fold higher than leucine in diaphragm muscle.
As BCAA aminotransferase reaction is reversible and its direction is determined by a
number of factors, a portion of BCKA delivered to liver from extrahepatic sources can be
reaminated to BCAA. Abumrad et al.27 demonstrated that after infusion of KIC into the
gut of postabsorptive dogs, 59% of the absorbed KIC was taken up by the liver and one-
third of this was transaminated to leucine. It can be suggested that partition of BCKA
between oxidation and reamination is an important regulatory mechanism supporting
either ketogenesis or resynthesis of leucine from KIC in the liver.

6.2.2.4 Brain
Undoubtedly, the brain is the tissue for which KB synthesized mostly in the liver and
delivered to the brain by the bloodstream are the most important energy fuel when there
is a lack of glucose, primarily in starvation. During prolonged fasting, more than half of
the brain’s energy supply is derived from KB. Recent findings show that leucine is an
important substrate of ketogenesis in astrocytes. KB produced by astrocytes might be used
as substrates for neuronal oxidative metabolism in situations of enhanced synaptic activity
and hypoxia.28 There are no data demonstrating the net release of KB from the brain.
It can be summarized that several tissues are involved in ketogenesis from leucine
and isoleucine. The interrelationships among muscle, liver, and adipose tissue are sche-
matically proposed in Figure 6.2. The principal source of KB synthesized from leucine and
released to the bloodstream is the liver. The main precursor seems to be the KIC delivered
to the liver mainly from skeletal muscle and adipose tissue. KB synthesized in the liver
are released to the blood due to the lack of enzymes, which enable conversion of KB to
acetyl-CoA. On the contrary, most of the KB produced in muscle or adipose tissue are
utilized in their place of origin, and only a smaller portion is released to the blood.

ADIPOSE TISSUE MUSCLE

Leu Leu PROTEINS Leu

KIC
KIC LIPIDS KIC

KB H2O + CO2 H2O + CO2 KB

Leu Leu
KIC KIC
KB KB
LIVER

Leu PROTEINS

KIC

KB H2O + CO2

KB

Figure 6.2 The cooperativity of skeletal muscle, adipose tissue, and liver in ketogenesis from leucine.
KIC, a-ketoisocaproic acid (ketoleucine); KB, ketone bodies.
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104 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

6.3 Physiology and pathophysiology of ketogenesis from amino acids


As pathways of ketogenesis from fatty acids and amino acids are different and differently
regulated, it may be assumed that ketogenesis from amino acids has different physiological
importance than that from fatty acids. While ketogenesis from fatty acids is activated
mainly in starvation and inhibited in satiety, ketogenesis from leucine is stimulated by
food intake. Several studies indicate that adipose tissue of fed animals converts leucine
to acetoacetate at a higher rate than in fasted ones.18,22 Block et al.29 have demonstrated a
strong correlation between ambient leucine concentrations and the activation state of
skeletal muscle BCKA dehydrogenase in rats fed four levels of dietary protein.

6.3.1 Ketogenesis from amino acids in hyperketonemic states


Several studies clearly demonstrate that hyperketonemia may significantly affect the keto-
genesis not only from fatty acids but also from amino acids. Paul and Adibi30 demonstrated
an almost linear increase in the rate of leucine decarboxylation by gastrocnemius muscle
homogenate when acetoacetate in the incubation medium was varied from 1 to 20 mM,
while a decrease was observed in homogenates of liver. The inhibitory effect of KB on
degradation of leucine intermediates in rat liver mitochondria was also observed by
Landaas.31 In another study,15 KB inhibited alanine release and 14CO2 production from
[1-14C]-BCAA by rat hemidiaphragms. Inhibition of leucine oxidation was also observed
during infusion of lipids.32 Hyperketonemia also affects ketogenesis from amino acids by
changes in protein turnover and delivery of leucine into body fluids.33 This occurs
undoubtedly in starvation and diabetes mellitus, the conditions in which marked hyper-
ketonemia is a common finding.

6.3.1.1 Starvation
Response to starvation follows three phases. The initial one (about 3 days in man and
1 day in rats) is concerned with the maintenance of glucose production (gluconeogenic
phase). The second phase (3 days to 2 months in man and 1 to 5 days in rat) is one of
protein conservation in which metabolism switches away from glucose to fatty acid and
KB oxidation. The decisive amount of KB produced is undoubtedly from b-oxidation of
fatty acids and acetyl-CoA overproduction in the liver promoted by the lack of glucose,
rise in glucagon, and decrease in insulin levels. Rous et al.22 have also found release of
greater quantities of KB by adipose tissue of 24-h fasted mice than of fed mice. The third
phase (premortal) is characterized by accelerated protein breakdown because energy stores
are exhausted.
The results of studies evaluating the effect of starvation on ketogenesis from amino
acids are not uniform. Thomas et al.6 observed that in rats starving for 48 h, 2.3% of KB
carbon is derived from metabolism of leucine, while in animals starving only for 3 h, the
corresponding value is 4.4%. Considering that starvation for 2 days caused a fourfold
increase in total blood KB, the results indicate a net increase in conversion of leucine to
KB. The main source of KB derived from leucine is probably the liver. Kulaylat et al.7
demonstrated that contribution of leucine to hepatic ketogenesis in dogs increases from
3.5% in overnight fasted animals to 10% in those fasted for 3 days. However, the changes
in leucine or KIC oxidation in isolated perfused liver of rats starving for 3 days are not
significant.34
The quantity of KB formed from leucine during fasting in adipose tissue is inhibited.
The amount of actetoacetate and 14CO2 released by adipose tissue of fasted mice incubated
in buffer containing [1-14C]-leucine was lower than that of fed mice.22 In epididymal fat
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Chapter six: Contribution of amino acids to ketogenesis 105

pads of rats fasted for 44 to 48 h, both the transamination of leucine and its subsequent
decarboxylation decreased.18
The effect of starvation on leucine conversion to KB in skeletal muscle was examined
by Palmer et al.,35 who demonstrated after addition of leucine to hemidiaphragms of 40-h
starved rats a higher release of KB than that from hemidiaphragms of fed rats. The
increased rates of KB release in the starved state were not a consequence of increased
leucine catabolism, as the rates of 14CO2 production from [U-14C]-leucine and [1-14C]-
leucine were lower in hemidiaphragms from starved than from fed rats. These results
indicate that leucine-stimulated production of KB in skeletal muscle of starved rats is
predominantly due to incomplete oxidation of leucine. This hypothesis is in agreement
with a marked decrease in BCKA dehydrogenase activity, indicating decreased leucine
oxidation, in gastrocnemius muscle and the heart of rats starving for 2 and 4 days, while
an increase was observed on the sixth day of starvation.36 Wagenmakers et al.37 demon-
strated that starvation decreased the activity state of BCKA dehydrogenase in the quad-
riceps muscle, although actual activity did not change. This finding is in agreement with
our observation of the gradual increase in the amount of BCKA dehydrogenase in skeletal
muscle and heart during starvation, which indicates an increasing capacity of these tissues
to oxidize leucine.36 In another study, starvation for 4 days increased the rate of leucine
decarboxylation by homogenates of skeletal muscle but was without effect on the rate of
leucine decarboxylation by the kidney and liver homogenates.30
It is not easy to summarize these contradictory reports and provide a reasonable
conclusion. It is clear that increased production of KB due to fatty acid oxidation in the
protein conservation phase of starvation is not accompanied by a corresponding increase
in ketogenesis from amino acids. It appears that enhanced ketogenesis from leucine during
starvation is more related to decreased utilization of KB in liver and muscle than to
increased catabolism of leucine. Leucine oxidation is activated in the premortal phase of
starvation.

6.3.1.2 Diabetes mellitus


Several factors contribute to diabetic hyperketonemia. The lack of insulin and excess in
anti-insulin hormones cause the inability of muscle and adipose tissue to take up glucose
from the blood, stimulating lipolysis and massive efflux of free fatty acids from adipose
tissue to the liver. These hormonal changes also enhance the ketogenic capacity of the
liver, and a higher proportion of acetyl-CoA produced in b-oxidation is converted into
KB. The hyperketonemia is amplified by limitation in the ability of extrahepatic tissue to
remove KB from the blood.
Ketogenesis from amino acids, at least from leucine, contributes significantly to hyper-
ketonemia in diabetes. In dogs fasted for 3 days with selective insulin deficiency induced
by a peripheral intravenous infusion of somatostatin and intraportal glucagon, the con-
tribution of leucine carbon to hepatic production of KB increased from 10 to 15%.7 This
finding is in agreement with observations of elevated activity of BCKA dehydrogenase in
skeletal muscle,30,38,39 kidney,30 and liver40,41 of diabetic rats. Further studies showed that
diabetes induced by streptozotocin treatment increased BCKA dehydrogenase activity in
skeletal muscle by inhibiting specific kinase.42 The elevated rates of KIC oxidation in
hindquarters of diabetic rats were depressed by the addition of acetoacetate to the perfu-
sion medium.43
The observations obtained in animals are in good agreement with observations in
insulin-dependent diabetics. In type I (insulin-dependent) diabetics, increased turnover
and oxidation rates of leucine that decreased with insulin therapy were reported.44–46
However, in type II diabetes, the values of leucine oxidation were not different from those
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106 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

of nondiabetic controls.47 One possible explanation is the difference in plasma insulin


concentration and insulin resistance.

6.3.2 Ketogenesis from amino acids in stress illness


Despite the importance of KB as an alternative fuel, several studies demonstrated signif-
icant depression of KB levels in severe illness, particularly in sepsis.48,49 In addition, KB
levels failed to rise in trauma patients during carbohydrate restriction.49 The inhibition of
ketogenesis from fatty acids in catabolic states is probably related to elevated insulin levels
due to increased production of catabolic hormones (cortisol, glucagon, and catechola-
mines) and to the metabolic response mediated by cytokines, particularly tumor necrosis
factor-a (TNF-a).50,51
Several studies evaluating changes in whole-body leucine and protein metabolism in
metabolic stress states, e.g., trauma and sepsis, or after administration of glucocorticoids
or pro-inflammatory cytokines demonstrated an increased rate in whole-body oxidation
of leucine.52,53 However, the stimulatory effect of cytokines on whole-body leucine oxida-
tion was not observed by others.54
It appears that in stress illness most of the leucine is completely oxidized in skeletal
muscle. Enhanced rates of leucine oxidation and BCKA dehydrogenase activity in skeletal
muscle showed endotoxins, cytokines, glucocorticoids, and acidosis.29,55–57 Unfortunately,
there are no studies evaluating the proportions of complete (to CO2 and water) and
incomplete (to KB) oxidation of leucine in muscle in stress conditions.
Most studies devoted to metabolism of ketogenic amino acids in the liver indicate
decreased ketogenesis, particularly from leucine. Pailla et al.58 demonstrated using isolated
rat hepatocytes that both TNF-a and interleukin (IL)-6 inhibit KB synthesis from KIC. In
further experiments, they demonstrated that inhibitory action of the cytokines on ketoge-
nesis is not related to changes in nitric oxide production or protein synthesis.51 Using
isolated perfused rat liver, we demonstrated that administration of endotoxin or TNF-a
decreases the flux of KIC through the BCKA dehydrogenase.56,59 Similar results were also
observed when endotoxin or TNF-a was added in the perfusion medium.53 These changes,
which also indicate an increased capacity of hepatic tissue to reaminate BCKA to BCAA,
were confirmed using an isolated perfused rat liver technique by adding KIC (leucine
precursor) to the perfusion medium. In effluent of hepatic tissue of endotoxemic rats, a
significantly higher leucine concentration was detected than in controls.59As severe illness
is commonly associated with anorexia, the activated resynthesis of BCAA in hepatic tissue
should be considered an important adaptive response of the body that can resupply
essential BCAA and prevent the rapid development of negative nitrogen balance.60 Sig-
nificant decrease in flux of KIC through hepatic BCKA dehydrogenase and decreased
b-hydroxybutyrate production from KIC were also observed in rats with cirrhosis.60–62

6.4 Conclusions
There are apparent species differences in BCAA metabolism, and the results obtained
mostly from laboratory animals should be carefully examined if valid also for humans.
The most important difference between humans and rats in BCAA catabolism is the lower
activity of hepatic BCKA dehydrogenase in humans.63 However, considering that the
capability of the liver to utilize KB is low in both humans and animals and that the majority
of studies evaluating changes in whole-body leucine metabolism in humans or dogs are
in good agreement with results obtained using small laboratory animals, the described
pathways of ketogenesis from leucine should also be valid for humans.
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Chapter six: Contribution of amino acids to ketogenesis 107

The results of several laboratories indicate that there is a significant potential for
formation of KB from ketogenic amino acids, at least from leucine. The ketogenesis from
lysine, tyrosine, phenylalanine, and tryptophane occurs mainly in the liver, whereas keto-
genesis from isoleucine and leucine occurs partly in extrahepatic tissues and partly in the
liver. As the contribution of individual tissues to ketonemia is determined not only by the
rate of KB production, but also by the presence or absence of key enzymes of KB utilization,
the contribution of KB synthesized in extrahepatic tissues is low. The liver should be
considered the most important source of blood KB derived from amino acids because of
the high activity of BCKA dehydrogenase (the key enzyme in BCAA catabolism), delivery
of significant amounts of KIC from extrahepatic tissues, and incomplete set of enzymes
for oxidation of KB.
Increased ketogenesis from amino acids in starvation is caused by incomplete oxida-
tion of leucine in muscle and liver rather than by its increased catabolism. The exact
reasons for this increase in the ratio of leucine conversion to KB and leucine oxidation is
not well defined. In insulin-dependent diabetes, the higher rate of ketogenesis from leucine
is associated with its increased oxidation. In stress conditions, activated leucine utilization
in skeletal muscle is not coupled with increased KB release to the blood. Decreased
ketogenesis from leucine in liver is related to enhanced resynthesis of BCAA from BCKA.

Acknowledgments
The author gratefully acknowledges the support of the Grant Agency of the Czech Repub-
lic (grants 306/94/1873, 306/98/0046, and 305/01/0578), the Internal Grant Agency of
Charles University (grants 152/95 and 276/98C), and the Internal Grant Agency of Min-
istry of Health of the Czech Republic (grants 3772-3 and 6793-3).

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ˇ M., Šprongl, L., and Tilser,
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1382_C07.fm Page 111 Tuesday, October 7, 2003 6:18 PM

chapter seven

Ureagenesis and ammoniagenesis:


an update
Alfred J. Meijer
Academic Medical Center, Amsterdam

Contents
Introduction.................................................................................................................................. 111
7.1 Control mechanisms ..........................................................................................................112
7.1.1 Regulation of carbamoyl-phosphate synthase..................................................112
7.1.2 Transport of ornithine, citrulline, and aspartate across the
mitochondrial membrane.....................................................................................114
7.1.3 Urea synthesis and pH homeostasis ..................................................................115
7.1.4 The periportal/pericentral glutamine cycle......................................................115
7.1.5 Metabolite channeling and urea synthesis........................................................116
7.1.6 Interaction of fatty acids with amino acid metabolism ..................................117
7.1.7 Interaction of glucose with urea synthesis........................................................117
7.1.8 Cell hydration and urea synthesis......................................................................118
7.2 Conclusions .........................................................................................................................118
References .....................................................................................................................................119

Introduction
The synthesis of urea occurs by a complex pathway that is localized both in the mitochon-
dria and in the cytosol of the liver cell (Figure 7.1). Synthesis of citrulline from NH4+,
HCO3–, ATP, and ornithine, catalyzed by carbamoyl-phosphate synthase and ornithine
carbamoyltransferase, is a mitochondrial process, whereas synthesis of urea from ATP,
citrulline, and aspartate via argininosuccinate synthase, argininosuccinate lyase, and argi-
nase I is a cytosolic process. Because of this dual localization, the transport of ornithine
into the mitochondria and the efflux of citrulline, aspartate, and ATP from the mitochon-
dria are essential steps in the pathway. Moreover, N-acetylglutamate, the essential activator
of carbamoyl-phosphate synthase, is synthesized in the mitochondria, whereas the com-
pound is degraded in the cytosol, which makes mitochondrial N-acetylglutamate efflux
another essential step in ureagenesis.

0-8493-1382-1/04/$0.00+$1.50
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112 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

cytosol mitochondrion
arg
acetate
+
glu AGA ? AGA glu
acetylCoA

UREA orn orn


+ 2ATP
HCO3-
CP NH3

arg

fum 1
as

AMP ATP Pi
cit cit

asp asp
NH3
2

glu glu αOG


NAD(P)+ NAD(P)H + H+
3
amino acids amino acids

Figure 7.1 Urea synthesis. AGA, N-acetylglutamate; glu, glutamate; asp, aspartate; CP, carbamoyl
phosphate; orn, ornithine; cit, citrulline; as, argininosuccinate; aOG, a-oxoglutarate; 1, ornithine/cit-
rulline translocator; 2, glutamate/aspartate translocator; 3, glutamate translocator. The putative
translocator for N-acetylglutamate is indicated by the question mark.

The regulation of urea synthesis has been reviewed extensively in the past.1–3 In the
previous volume of this series we discussed some features of the short-term regulation of
urea synthesis4; in the present chapter, only recent developments in this field are high-
lighted. For the transcriptional regulation of the enzymes participating in urea synthesis,
refer to recent reviews,2,3 and for a discussion of disorders in urea synthesis, refer to
Brusilow and Horwich.2

7.1 Control mechanisms


7.1.1 Regulation of carbamoyl-phosphate synthase
We previously pointed out repeatedly1,4 that discussions on the factors controlling flux
through the ornithine cycle should take into account that neither amino acid degradation
nor carbamoyl-phosphate synthase is sensitive to inhibition by their products, ammonia
and carbamoyl-phosphate, respectively. Thus, at constant substrate supply, any change in
the activity of one of the enzymes of the ornithine cycle, any change in N-acetylglutamate
synthase, or any change in the mitochondrial transport steps will influence neither orni-
thine cycle flux nor amino acid degradation. Such changes will only affect the steady-state
concentration of ammonia and of carbamoyl phosphate in the mitochondrial matrix. This
statement is best illustrated by the accumulation of ammonia in severe deficiencies of the
enzymes of the ornithine cycle and by the production of orotic acid when the deficiency
is distal to that of carbamoyl-phosphate synthase.1,2
Because of the lack of product inhibition by ammonia of amino acid degradation, we
previously argued that in vivo flux through the ornithine cycle is entirely controlled by
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Chapter seven: Ureagenesis and ammoniagenesis: an update 113

the rate of amino acid degradation.1 The capacity of the enzymes of the ornithine cycle is
more than sufficient to handle the amount of ammonia formed by amino acid degradation
in vivo under normal conditions. Perhaps the best evidence supporting this statement is
the fact that urea synthesis rates were near normal in asymptomatic carriers with ornithine
carbamoyltransferase (OCT) deficiency with an enzyme activity below normal.5 Another
illustration can be found in a recent study from the same group with spf mutant mice, in
which the activity of OCT in the liver was only 5% of that of control mice: a relatively
small increase in OCT activity by adenovirus-mediated gene transfer was sufficient for
complete correction of the defect in urea synthesis in the mutant mice.6
In the regulation of carbamoyl-phosphate synthase activity, N-acetylglutamate is of
crucial importance. As we indicated in the past, the intramitochondrial synthesis of
N-acetylglutamate from glutamate and acetylCoA is part of a mechanism to buffer the
intramitochondrial (and thus the intrahepatic) concentration of ammonia.1,4,7 When the
amino acid supply to the liver increases, flux through carbamoyl-phosphate synthase
increases because of two reasons. First, there is an increase in portal ammonia (derived
from amino acid breakdown in enterocytes, glutamine in particular8,9) and also in the rate
of intrahepatic production of ammonia (formed either by deamidation of some amino
acids or by glutamate dehydrogenase). Second, there is an increase in synthesis of N-acetyl-
glutamate. Thus, not only is there an increase in substrate (i.e., ammonia) supply but
simultaneously there is an increase in the number of active, already existing carbamoyl-
phosphate synthase molecules. This results in a sigmoidal relationship between the con-
centration of ammonia and the rate of urea synthesis and allows large changes in ornithine
cycle flux at relatively constant ammonia concentrations.7 Such a mechanism makes perfect
sense, and it is surprising that in a recent review this mechanism was not considered and
a role of N-acetylglutamate in the short-term regulation of urea synthesis was even ques-
tioned.3 Perhaps some of the confusion is caused by the fact that intrahepatic N-acetyl-
glutamate levels do not necessarily reflect the intramitochondrial concentration of this
compound. It is also important to stress that the concentration of carbamoyl-phosphate
synthase in the mitochondrial matrix is extremely high and similar to that of N-acetyl-
glutamate in the mitochondrial matrix, so that deviations from normal Michaelis–Menten
kinetics may be expected.1
Recently, the gene of the mouse and human liver N-acetylglutamate synthase was
cloned.10,11 As expected, the gene is highly expressed in the liver and small intestine; in
the latter tissue, the enzyme plays an important role in the synthesis of citrulline from
glutamine, which, after entry into the bloodstream, is subsequently converted into arginine
by the kidneys.1–3,8 The protein contains a putative mitochondrial target sequence at its
aminoterminus,10 and its activity is stimulated by arginine,10,11 as predicted by Kawamoto
and coworkers.12 Even though the capacity of arginase in the liver is extremely high,
changes in its activity influence the cytosolic concentration of arginine and, assuming that
these changes are also reflected in the mitochondria,1 directly affect the synthesis of
N-acetylglutamate and thus the steady-state concentration of ammonia.
As indicated in the Introduction, degradation of N-acetylglutamate occurs in the
cytosol. Whether regulation of N-acetylglutamate levels can also occur at the level of its
degradation is not known, but a recent paper13 suggests that this may be the case: it was
shown that the increased rate of urea production in hypothyroid rats was accompanied
by a rise in N-acetylglutamate in the liver, which was caused not only by increased
synthesis but also by decreased degradation of the compound. Decreased degradation
may be caused by decreased activity of the cytosolic deacylase or by decreased transport
of N-acetylglutamate from the mitochondria. Only in the latter case, this will result in
increased intramitochondrial N-acetylglutamate, because N-acetylglutamate transport is
unidirectional, out of the mitochondria only.14
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114 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

7.1.2 Transport of ornithine, citrulline, and aspartate across the mitochondrial


membrane
Important progress has been made with the recent cloning of the genes encoding the
mitochondrial translocators for ornithine/citrulline, glutamate/aspartate, and glutamate.
The gene encoding the ornithine/citrulline translocator was cloned in 1999, and a
defect in this gene is the underlying cause for the so-called HHH (hyperornithinemia,
hyperammonemia, and homocitrullinemia) syndrome,15 as was postulated several years
ago.16–18 The transporter catalyzes an electroneutral exchange between cytosolic ornithine+
and mitochondrial citrulline plus one H+.19 This proton is produced in the ornithine car-
bamoyltransferase reaction,1 and its removal from the mitochondrial matrix by the orni-
thine/citrulline exchange presumably accounts for the well-known activation by ornithine
of flux through carbamoyl-phosphate synthase in intact but not broken mitochondria,
because acidification of the mitochondrial matrix would inhibit the production of HCO3–
by mitochondrial carbonic anhydrase.1 Such mitochondrial acidification is likely to occur
in HHH patients and presumably accounts for the morphological abnormalities of liver
mitochondria seen in this disorder.3
A second mitochondrial transport system that was cloned recently is the
glutamate/aspartate translocator.20,21 This transporter catalyzes the electrophoretic
exchange between glutamic acid (the protonated form) and aspartate; it is unidirectional,
out of the mitochondria, energy dependent, and driven by the mitochondrial proton
gradient.22
The hepatocyte contains both cytosolic and mitochondrial aspartate aminotransferase.
When the mitochondrial enzyme is used for aspartate synthesis, the efflux of aspartate
from the mitochondria becomes essential for urea synthesis.23 In principle, both transam-
ination pathways are available. However, when their relative contributions were studied
with 15NH4Cl (by measurement of the degree of labeling with 15N of the urea molecule),
they strongly depended on the experimental conditions.9,24–27
The protein responsible for aspartate transport in the liver is called citrin, and a
defect in this transporter is the underlying cause of adult-onset type II citrullinemia.20,21
The structure of citrin is similar to that of aralar1 encoded by another gene.28,29 While
citrin is found predominantly (albeit not exclusively) in the liver, aralar1 is found
mainly in the heart, skeletal muscle, brain, and pancreatic b-cells.20,28–31 This suggests
that citrin is mainly involved in liver-specific processes such as urea synthesis and
gluconeogenesis from lactate.21,31 On the other hand, aralar1 is involved in the operation
of the malate/aspartate shuttle responsible for the mitochondrial oxidation of glycolytic
NADH, which is of eminent importance for ATP production in muscle, brain, and
pancreatic b-cells.21,30 Interestingly, both citrin and aralar1 require Ca++ at the external
face of the mitochondrial inner membrane.21 This agrees entirely with the suggestion
made years ago that in hepatocytes transport of cytosolic-reducing equivalents to the
mitochondria via the malate–aspartate shuttle was stimulated by Ca++ at the level of
the glutamate/aspartate translocator.32
The third mitochondrial transport system, important for urea synthesis, which was
recently cloned, is the translocator for glutamate.33 This protein transports glutamate,
derived from cytosolic amino acid transaminations, to the mitochondria (Figure 7.1), in
association with a proton. There are two isoforms of this protein that differ in kinetic
properties: isoform 1 has a lower affinity for glutamate but a higher Vmax than isoform
2. Both forms are ubiquitously expressed in all tissues. Expression of the mRNA of
isoform 1 predominates in most tissues except in the brain, where both forms are equally
expressed.33
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Chapter seven: Ureagenesis and ammoniagenesis: an update 115

7.1.3 Urea synthesis and pH homeostasis


Atkinson and colleagues34,35 were the first to focus attention on the fact that synthesis of
urea consumes equal amounts of NH4+ and HCO3– (up to an amount of about 1 mol each
in man per day, assuming a daily protein intake of 100 g), and that its function is to remove
not only NH4+ but also bicarbonate. NH4+ and bicarbonate ions arise from the positively
charged amino groups and the negatively charged carboxylate groups during amino acid
oxidation. Because no more than 5% of the daily bicarbonate production can be removed
by urinary excretion, it was proposed that ureagenesis must inevitably play an important
role in the regulation of pH homeostasis.34,35 Although this issue has been extremely
controversial and the idea was challenged by many (cf. Meijer et al.,1 Brusilow and Hor-
wich,2 and Häussinger36 for a discussion of this issue), the chemistry of urea synthesis
(i.e., consumption of equal amounts of ammonia and bicarbonate ions) cannot be denied.
There is general agreement that urea synthesis decreases in metabolic acidosis in order to
save bicarbonate ions for neutralization of the excess of protons, and several mechanisms
contribute to this decrease (see Meijer et al.1 and Meijer4 for reviews). Under these condi-
tions, NH4+ ions that are not converted into urea are excreted into the urine, with glutamine
as the nontoxic carrier of ammonia in the circulation. The glutamine may be produced by
the pericentral hepatocytes37 but can also be produced elsewhere in the body.1 The con-
troversy, however, is about the prediction that metabolic alkalosis is expected when urea
synthesis is compromised, e.g., in patients with cirrhosis or with inborn errors of urea
synthesis. However, this is not always found2,38 (contrast with Häussinger et al.39). Accord-
ing to Shangraw and Jahoor,38 the rate of urea production in man in vivo is only a few
percent of the total bicarbonate flux, and a change in urea synthesis will have a negligible
effect on the acid–base balance. Unfortunately, however, the bicarbonate flux they referred
to was the rate of CO2 production in vivo, not the rate of HCO3– production.40 As indicated
above, HCO3– ions mainly arise from the negatively charged carboxylate groups of amino
acids during amino acid oxidation. Oxidation of (neutral) fat and carbohydrate produces
CO2, not HCO3–. Studies on the issue have so far never considered the fact that a decrease
in urea synthesis can only cause alkalosis by an increase in the concentration of HCO3– if
the rate of amino acid oxidation, and thus the rate of HCO3– production, is unchanged. If
amino acid oxidation in patients with cirrhosis or with an ornithine cycle enzyme defi-
ciency would decrease, metabolic alkalosis would be compensated for because less HCO3–
would be produced. This situation is analogous but opposite that in metabolic acidosis
where urea synthesis decreases, and proteolysis and amino acid oxidation in the muscle
increases,41 and the increased HCO3– production counteracts acidosis.1
A role for urea metabolism in pH control can also be found in nonmammalian cells.
For example, a high rate of urea synthesis in the liver (i.e., HCO3– consumption) allows
certain fish to survive alkaline environments of pH as high as 10.42–44 Oppositely, intra-
cellular degradation of urea by urease after H+-dependent membrane transport of urea
allows Helicobacter pylori to survive the acidic environment of the stomach.45

7.1.4 The periportal/pericentral glutamine cycle


The presence of periportal glutaminase and pericentral glutamine synthase results in
intercellular glutamine cycling.37,46 This is not a waste of energy: urea synthesis in peri-
portal hepatocytes has a low affinity for ammonia, and any ammonia escaping urea
synthesis will be scavenged by glutamine synthase in pericentral hepatocytes because this
enzyme has a very high affinity for ammonia in addition to a high capacity. Glutaminase
activity is strongly inhibited when the pH falls because the activation by ammonia, its
essential activator, is exquisitely sensitive to small pH changes within the physiological
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116 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

range.47 In acidosis, hepatic glutaminase flux decreases with relatively little change in
glutamine synthase flux.48 A possible source of glutamate for pericentral glutamine syn-
thesis is ornithine (in addition to plasma glutamate), because ornithine aminotransferase
colocalizes with the glutamine synthase-containing cells and is not present in the periportal
cells (see Meijer4 and Watford49 for literature), and because ornithine aminotransferase is
induced by high-protein feeding.50 Interestingly, pericentral cells contain some arginase II
(which is the kidney isoenzyme), but do not contain the other enzymes of the ornithine
cycle.51 It was suggested that this may help to supply the pericentral hepatocytes with
ornithine from circulating arginine. However, whether the ornithine supply is sufficient
to drive pericentral glutamine synthesis remains to be seen.49
The net balance of glutamine across the liver strongly depends on the metabolic
conditions. There is net consumption of glutamine after protein feeding and in uncon-
trolled diabetes, sepsis, and short-term starvation; there is a net release of glutamine from
the liver after long-term starvation and, as discussed above, in metabolic acidosis (see
Curthoys and Watford8 and Watford49 for reviews). However, it must be stressed again
that flux through periportal glutaminase and pericentral glutamine synthase may be
considerable, even though the net balance of glutamine across the liver is zero.8,37,46,49

7.1.5 Metabolite channeling and urea synthesis


Previously, kinetic evidence obtained with intact and permeabilized mitochondria and
with permeabilized hepatocytes strongly supported the possibility of channeling of metab-
olites between the enzymes of the ornithine cycle, even across the mitochondrial inner
membrane, and the concept was coined that the ornithine cycle is a metabolon spanning
the mitochondrial membrane (reviewed by Watford52 and Meijer4). In agreement with this
concept, Cohen and colleagues53,54 provided immunocytochemical evidence that the cyto-
plasmic enzymes argininosuccinate synthase and argininosuccinate lyase in hepatocytes
are located in the immediate vicinity of the cytoplasmic surface of the mitochondrial outer
membrane. However, until now, evidence directly supporting the occurrence of channeling
of ornithine cycle intermediates in hepatocytes, in perfused liver, or in vivo has not yet
been provided.
On the basis of experiments with isolated liver mitochondria, incubated with high
concentrations of glutamine (when flux through glutaminase was very high), we previ-
ously concluded that ammonia was directly channeled from glutaminase to carbamoyl-
phosphate synthase and did not escape the mitochondria.55 It was proposed that the
activation of glutaminase by ammonia in this way increased the affinity of carbamoyl-
phosphate synthase for ammonia.55 However, experiments by others carried out with
isolated rat hepatocytes and with rat liver perfused with [15N]glutamine, labeled in the
amide group [5-15N], did not support this conclusion.56,57 According to these data, 15NH3
derived from [5-15N]glutamine completely mixed with the ammonia in the incubation
medium, and the isotopic enrichment of [15N]citrulline was identical to that of [15N]NH3
in the medium. However, this conclusion must be considered with caution for two reasons.
First, the flux through glutaminase in the experiments of Meijer55 was at least 10-fold
higher than that in the experiments of Nissim and coworkers.56,57 Second, metabolite
channeling is never 100% tight,58 and after a certain period of time, depending on the
degree of channeling, complete equilibration of label with the extramitochondrial pool of
ammonia can be expected. The data in Nissim et al.56,57 would have been more convincing
if the degree of labeling of citrulline and of ammonia had been analyzed in the first few
minutes after [5-15N]glutamine addition rather than after 40 to 70 min.
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Chapter seven: Ureagenesis and ammoniagenesis: an update 117

7.1.6 Interaction of fatty acids with amino acid metabolism


Fatty acid oxidation provides both ATP for urea production and acetyl-CoA for the syn-
thesis of N-acetylglutamate.1 Indeed, defects in fatty acid oxidation are often accompanied
by hyperammonemia, in addition to hypoglycemia.1 It has now been shown that at least
in isolated liver mitochondria, high concentrations of long-chain fatty acids are also able
to inhibit carbamoyl-phosphate synthase in an irreversible manner by acylation of the
enzyme protein on cysteine residues.59 It was proposed that this mechanism reduces amino
acid utilization during long-term starvation and that it contributes to the nitrogen-sparing
effect in long-term starvation.59 Because the acylation of carbamoyl-phosphate synthase
was counteracted by N-acetylglutamate and MgATP,59 this may provide a mechanism to
prevent inactivation of the enzyme during short-term activation when amino acids are
needed for gluconeogenesis.
Another development in the interaction between fatty acid metabolism and amino
acid metabolism and urea synthesis has been the observation that activation of PPARa
(peroxisome proliferator-activated receptor a, a fatty acid-activated nuclear receptor
required for the expression of many genes involved in fatty acid oxidation) inhibits mRNA
expression of enzymes of amino acid catabolism and of urea synthesis. By contrast,
disruption of PPARa results in an increase in these messengers.60 It has been proposed
that PPARa activation (like acylation of carbamoyl-phosphate synthase, discussed above)
contributes to the nitrogen-sparing effect of long-term starvation when ketone bodies
replace glucose as a fuel for the brain.60
Interestingly, mice with juvenile visceral statosis (JVS) (caused by systemic carnitine
deficiency with accumulation of fat in the liver, hyperammonemia, and hypoglycemia)
also have a decreased expression of all the genes of the ornithine cycle.61 Although it is
tempting to conclude that PPARa activation may be part of the mechanism, this possibility
was considered to be less likely.61 The hyperammonemia is not caused by a fall in N-acetyl-
glutamate (because the carnitine deficiency is not complete) but rather by the diminished
activity of the enzymes of the ornithine cycle.61

7.1.7 Interaction of glucose with urea synthesis


The interaction between glucose and urea synthesis is another area of interest because
glucose has a nitrogen-sparing effect on metabolism; it reduces urea synthesis indepen-
dently of changes in plasma amino acid concentrations.62,63 Mechanisms underlying this
effect are direct inhibition by glucose of gluconeogenesis from amino acids63 and inhibition
of glucagon production.64 Conversely, in hypoglycemia, glucagon is the primary mediator
in the coordination of the increased fluxes through the ornithine cycle and through the
proteolytic pathway in muscle.65 Because glucagon does not directly affect muscle pro-
teolysis, it is likely that in hypoglycemia the increased catabolism of amino acids in the
liver results in decreased plasma amino acid concentrations and, in this way, accelerates
net protein breakdown in the muscle (cf. Chapter 16).65 Mechanisms by means of which
glucagon can activate ureagenesis in addition to its well-known stimulation of gluconeo-
genesis have been reviewed previously.1 These include increased amino acid transport
across the plasma membrane, stimulation of hepatic autophagic proteolysis (cf. Chapter
16), increased degradation of some amino acids (e.g., glutamine), stimulation of carbam-
oyl-phosphate synthase activity by increased N-acetylglutamate levels in the mitochon-
dria, and increased synthesis of enzymes involved in urea synthesis.1
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118 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

7.1.8 Cell hydration and urea synthesis


Changes in cell volume have profound effects on hepatic metabolism66 (cf. Chapters 16 to
18). In general, increases and decreases in cell volume exert anabolic and catabolic effects,
respectively. Experiments with the isolated perfused liver showed that the effects of cell
volume changes on urea synthesis are complex: cell swelling increased urea synthesis
from amino acids but decreased urea synthesis with ammonia as the substrate.66 Neither
the mechanism nor the physiological meaning of these opposing effects is entirely clear.
In a study with healthy men, Ivarsen et al.67 examined the effect of furosemide-induced
dehydration on the rate of synthesis of urea from infused alanine and found that moderate
dehydration down-regulated urea synthesis and its activation by glucagon. These data
are consistent with the effects of cell swelling on urea synthesis from amino acids in the
perfused liver (see above), but again, the mechanism and meaning of these effects are not
clear: dehydration of liver cells decreases protein synthesis and increases protein degra-
dation during an amino acid load,66 so that stimulation rather than down-regulation of
urea synthesis would be expected under these conditions.

7.2 Conclusions
Research on ureagenesis in the past decades has made it clear that flux through the
ornithine cycle (i.e., the rate of urea production) is primarily determined by substrate
supply, i.e., the rate of supply of ammonia from the intestine to the liver and the intrahe-
patic rate of amino acid degradation. Mechanisms involved in the control of the activity
of the ornithine cycle itself are essentially mechanisms of control of substrate concentra-
tion, i.e., mechanisms to keep the intrahepatic ammonia concentration constant. This is
logical because, as first pointed out by Krebs and coworkers,68 ammonia is not only toxic
for the brain but is also an essential metabolite in intermediary metabolism, so that its
concentration must be kept within certain limits.
Short-term regulation is primarily exerted at the level of carbamoyl-phosphate syn-
thase, in which N-acetylglutamate plays a crucial role. In addition, there is the long-term
regulation of urea synthesis via changes in the amount of the enzymes involved in the
synthesis of urea (not reviewed in this chapter). Mechanisms controlling the transcription
of these genes clearly form an important research area for the years to come.2,3 In connec-
tion to this, the mechanisms responsible for the peculiar pericentral localization of
glutamine synthase69 are another area of great interest.
The transport proteins responsible for the transfer of ornithine, citrulline, and aspartate
across the mitochondrial membrane also play important roles in urea synthesis. An intrigu-
ing question yet to be answered is the importance of metabolite channeling in the func-
tioning of the ornithine cycle and whether it does, indeed, enhance the efficiency of this
process. Is aspartate, transported out of the mitochondria, directly channeled into argin-
inosuccinate synthase? If so, how can this aspartate then be discriminated from the aspar-
tate engaged in the transfer of reducing equivalents across the mitochondrial membrane
or in the mitochondrial transfer of carbon during gluconeogenesis from lactate? It would
also be important to know how transport of N-acetylglutamate out of the mitochondria
is achieved. Is it by simple diffusion, or is it mediated by an as yet unknown translocator
or by one of the known translocators?
The relevance of regulation of urea synthesis by changes in cell volume is not entirely
clear. It is more likely that changes in flux through the ornithine cycle by changes in cell
volume are caused by a change in substrate supply, i.e., by an effect on protein synthesis
and degradation (cf. Chapter 16), either in the liver itself or in extrahepatic tissues (e.g.,
muscle).
Clearly, these are important issues that deserve to be explored in the near future.
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Chapter seven: Ureagenesis and ammoniagenesis: an update 119

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35. Atkinson, D.E. and Bourke, E., Metabolic aspects of the regulation of systemic pH, Am. J.
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36. Häussinger, D., Liver regulation of acid-base balance, Miner. Electrolyte Metab., 23, 249–252,
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37. Häussinger, D., Sies, H., and Gerok, W., Functional hepatocyte heterogeneity in ammonia
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38. Shangraw, R.E. and Jahoor, F., Effect of liver disease and transplantation on urea synthesis
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41. May, R.C., Hara, Y., Kelly, R.A., Block, K.P., Buse, M.G., and Mitch, W.E., Branched-chain
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E712–E718, 1987.
42. Randall, D.J., Wood, C.M., Perry, S.F., Bergman, H., Maloiy, G.M., Mommsen, T.P., and Wright,
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43. Lindley, T.E., Scheiderer, C.L., Walsh, P.J., Wood, C.M., Bergman, H.L., Bergman, A.L.,
Laurent, P., Wilson, P., and Anderson, P.M., Muscle as the primary site of urea cycle enzyme
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983S–987S, 2000.
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induces pericentral glutamate dehydrogenase and ornithine aminotransferase to provide
sufficient glutamate for pericentral detoxification of ammonia in rat liver lobules, Histochem.
Cell Biol., 111, 445–452, 1999.
51. O’Sullivan, D., Brosnan, J.T., and Brosnan, M.E., Hepatic zonation of the catabolism of
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52. Watford, M., Channeling in the urea cycle: a metabolon spanning two compartments, Trends
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53. Cohen, N.S. and Kuda, A., Argininosuccinate synthetase and argininosuccinate lyase are
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55. Meijer, A.J., Channeling of ammonia from glutaminase to carbamoyl-phosphate synthetase
in liver mitochondria, FEBS Lett., 191, 249–251, 1985.
56. Nissim, I., Yudkoff, M., and Brosnan, J.T., Regulation of [15N]urea synthesis from
[5-15N]glutamine: role of pH, hormones, and pyruvate, J. Biol. Chem., 271, 31234–31242, 1996.
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59. Corvi, M.M., Soltys, C.L., and Berthiaume, L.G., Regulation of mitochondrial carbamoyl-
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61. Saheki, T., Li, M.X., and Kobayashi, K., Antagonizing effect of AP-1 on glucocorticoid induc-
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64. Hamberg, O. and Vilstrup, H., Effects of glucose on hepatic conversion of aminonitrogen to
urea in patients with cirrhosis: relationship to glucagon, Hepatology, 19, 45–54, 1994.
65. Grofte, T., Wolthers, T., Jorgensen, J.O., Poulsen, P.L., Vilstrup, H., and Moller, N., Hepatic
amino- to urea-N clearance and forearm amino-N exchange during hypoglycemic and eug-
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313, 697–710, 1996.
67. Ivarsen, P., Greisen, J., and Vilstrup, H., Acute effects of moderate dehydration on the hepatic
conversion of amino nitrogen into urea nitrogen in healthy men, Clin. Sci. (Lond.), 101,
339–344, 2001.
68. Krebs, H.A., Hems, R., and Lund, P., Some regulatory mechanisms in the synthesis of urea
in the mammalian liver, Adv. Enzyme Regul., 11, 361–377, 1973.
69. Christoffels, V.M., Sassi, H., Ruijter, J.M., Moorman, A.F., Grange, T., and Lamers, W.H., A
mechanistic model for the development and maintenance of portocentral gradients in gene
expression in the liver, Hepatology, 29, 1180–1192, 1999.
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chapter eight

Metabolism of branched-chain
amino acids in man
Anton J.M. Wagenmakers
Maastricht University and University Hospital

Contents
Introduction..................................................................................................................................123
8.1 BCAA metabolism in the postabsorptive state .............................................................124
8.2 BCAA metabolism after ingestion of protein-containing meals ................................125
8.3 BCAA metabolism during one-leg knee-extension exercise: a model with
exceptionally high net protein degradation rates.........................................................126
8.4 Are BCAA a source for both the carbon and nitrogen of alanine and
glutamine? Studies with isotopic tracers .......................................................................128
8.5 The glucose–alanine cycle: a changing concept ............................................................128
8.6 The role of alanine aminotransferase and deamination of BCAA in TCA cycle
anaplerosis during exercise ..............................................................................................129
8.7 Lessons from patients with McArdle’s disease.............................................................131
8.8 Conclusion ...........................................................................................................................132
References ....................................................................................................................................132

Introduction
The branched-chain amino acids (BCAA) — leucine, valine, and isoleucine — are three of
the nine essential amino acids in mammals. Together, they make up 40% of the daily
requirement for essential amino acids in man. They serve among others as building blocks
for tissue proteins, and as carbon and nitrogen precursors for the synthesis of the most
abundant nonessential amino acids in muscle and plasma (glutamine, alanine, and
glutamate); they play an important role in the synthesis of tricarboxylic acid (TCA) cycle
intermediates in contracting skeletal muscle during workload transitions; they are oxi-
dized to acetyl-CoA and CO2 ; and they (especially leucine) are important signal molecules
that exert control on insulin production (pancreas) and on the translation initiation phase
of protein synthesis (liver and skeletal muscle). As such, a thorough understanding of the
metabolism of the BCAA is essential for a proper understanding of the cellular control
mechanisms of energy production, fuel selection, and protein metabolism (maintenance

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124 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

of muscle mass), both in healthy subjects and in patients with cachexia due to nutritional
and metabolic diseases.
The aim of this chapter is to present an update of the literature that has appeared
since the previous publication of this chapter in 1995.1 Few new publications have
appeared on the following topics: (1) oxidative pathways of BCAA and tissue distribution
of enzyme activities and (2) effect of diet, starvation, exercise, and hormones on the activity
of the enzymes catalyzing steps 1 to 3 in the BCAA degradative pathway. One exception
is the effect of cytokines on enzyme activities. For details, refer to Chapter 6, as stress and
trauma metabolism are out of the scope of this chapter. The minimal progress in current
knowledge on these topics is disappointing in light of the earlier conclusion1 that we need
to extend this knowledge for a proper understanding of, first, the physiological basis of
the interorgan collaboration in BCAA metabolism and of, second, the complex role that
these compounds play in the regulation of energy and protein metabolism at the cellular
and whole-body level. Therefore, we advise graduate students and others with a devel-
oping interest in BCAA metabolism to first read Chapter 5 of the 1995 book.1 Further
useful readings are the four books on BCAA metabolism that have been published so
far,2–5 and for an overview of the important early in vivo animal studies (e.g., on BCAA
antagonism), refer to the review of Harper and colleagues.6 In this chapter we will focus
on a few selected new aspects and physiological concepts of BCAA metabolism that have
been further developed or emerged since 1995.1 The focus of this review is on new studies
in man.

8.1 BCAA metabolism in the postabsorptive state


Most of the essential amino acids are degraded by the liver, but the BCAA are primarily
oxidized in skeletal muscle and probably also in adipose tissue.1 This conclusion was
originally based on data from rat muscles incubated in vitro.7,8
These muscles are in net protein breakdown (protein synthesis < protein degradation),
especially when insulin is omitted from the incubation or perfusion medium. This implies
that all the amino acids are released from the existing muscle proteins and that those
amino acids that are not metabolized in muscle will be released in proportion to their
relative occurrence in muscle protein, while a discrepancy will be found when amino acids
are transaminated, oxidized, or synthesized. Such comparisons revealed that rat skeletal
muscle can oxidize only six amino acids: the three BCAA, glutamate, aspartate, and
asparagine (Figure 8.1).7–9 Glutamine and alanine were released in excess of the relative
occurrence in muscle protein. This suggests that de novo synthesis of these amino acids
occurs.7–9 In 1972 Ruderman and Lund7 were the first to observe that addition of BCAA
to the perfusion medium of rat hindquarters increased the release of alanine and
glutamine. The relationship between the metabolism of BCAA on the one hand and the
release of alanine and glutamine on the other has since been the subject of many studies.
Most of this relationship today has been firmly established.9 In the BCAA aminotransferase
reaction the amino group is donated to a-ketoglutarate to form glutamate and a branched-
chain a-keto acid (Figure 8.1). In the reaction catalyzed by glutamine synthase, glutamate
reacts with ammonia to form glutamine. Alternatively, glutamate may donate the amino
group to pyruvate to form alanine and regenerate a-ketoglutarate. These reactions provide
a mechanism for the elimination of amino groups from muscle in the form of the nontoxic
nitrogen carriers alanine and glutamine (Figure 8.1).
Muscle amino acid metabolism has also been investigated in man in vivo by measuring
the exchange of amino acids across a forearm or a leg (arteriovenous difference multiplied
by blood flow gives the net exchange of amino acids).10–15 As muscle is the largest and
most active tissue in the limbs, the assumption that limb exchange primarily reflects
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Chapter eight: Metabolism of branched-chain amino acids in man 125

Figure 8.1 Schematic presentation of the interactions of amino acids with the TCA cycle. The trans-
amination products of leucine, isoleucine, and valine are a-ketoiscocaproic acid (a-KIC), a-oxo-
methylvaleric acid (a-KMV), and a-ketoisovaleric acid (a-KIV). Glucose 6-P, glucose-6-phosphate.
(This figure is reproduced, with permission, from Van Hall et al., 1999, Clin. Sci., 97, 557, © the
Biochemical Society and the Medical Research Society (UK).)

muscle metabolism seems reasonable. After overnight fasting there is net breakdown of
muscle proteins as protein synthesis is slightly lower than protein degradation.16 This
again (as explained for the incubated rat muscles) implies that those amino acids that are
not metabolized in muscle will be released in proportion to their relative occurrence in
muscle protein, while a discrepancy will be found when amino acids are transaminated,
oxidized, or synthesized. Human limbs at rest after overnight fasting released much more
glutamine (48% of total amino acid release) and alanine (25%)15 than would be anticipated
from the relative occurrence in muscle protein (glutamine, 8%; alanine, 9%).15 This implies
that glutamine with two N-atoms per molecule is dominant for the amino acid N-release
from human muscle. The BCAA (19% relative occurrence in muscle protein), glutamate
(7%), and aspartate and asparagine (together, 9%), on the other hand, are not released or
in lower amounts than their relative occurrence. Glutamate, in fact, is always taken up
from the circulation by human skeletal muscle.10–15 This implies that in human skeletal
muscle after overnight fasting the BCAA, glutamate, aspartate, and asparagine originating
from net breakdown of muscle proteins and glutamate taken up from the circulation are
metabolized and used for de novo synthesis of glutamine and alanine (Figure 8.1). All other
amino acids are released in proportion to their relative occurrence in muscle protein,
implying that little or no metabolism occurs.15

8.2 BCAA metabolism after ingestion of protein-containing meals


Several studies have investigated the fate of BCAA ingested orally either as a mixed meal
containing carbohydrate, fat, and protein or as a pure protein meal.12,19–22 Following inges-
tion of mixed protein-containing meals, small amounts of most amino acids are taken up
by muscle and most other tissues, as there is net protein deposition in the fed state (protein
synthesis > protein degradation), which compensates for the net losses in the overnight
fasting period.16,17 The high plasma insulin concentration that is seen in the postprandial
state, in combination with the increase in the intracellular concentration or availability
(arterial blood concentration) of the essential amino acids, has been shown to both
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126 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

stimulate protein synthesis (in liver and muscle at the initiation phase of mRNA trans-
lation18) and reduce protein degradation at the whole-body level and in skeletal muscle.16,17
An excessively large uptake of BCAA and glutamate is seen in muscle in the 4-h period
after ingestion of a protein meal12 and a mixed meal,19 and also after ingestion of a large
steak.20 Together, BCAA and glutamate then cover more than 90% of the muscle amino
acid uptake. The BCAA originate from dietary protein. After digestion of dietary protein,
most of the resulting BCAA escape from uptake and metabolism in gut and liver12 as a
consequence of the low BCAA aminotransferase activity in these tissues.1 In agreement
with this conclusion Hagenfeldt et al.21 found that 55% of a constant intravenous leucine
infusion (2.5 h at a rate of 300 mmol/min) in postabsorptive human subjects was taken
up by the peripheral tissues, 25% by the splanchnic region, and 10% by the brain. The
source of the glutamate is less clear. The diet only seems to deliver a minor proportion as
both a [15N]- and [13C]-glutamate tracer ingested orally were almost quantitatively removed
in the first pass through the splanchnic area (gut and liver).22,23 However, the splanchnic
area in man is producing significant amounts of glutamate after protein ingestion,12 after
overnight fasting,10 and after prolonged starvation.10 Equivalent amounts of glutamate are
taken up by muscle in all these conditions.10–15,19,20 After ingestion of a large steak, the
muscle release of glutamine more than doubles, while the alanine release is strongly
reduced to only 10% of the overnight fasted value.20 In the 4-h period after ingestion of a
mixed meal,19 the dominance of glutamine in carrying N out of skeletal muscle also was
much larger than after overnight fasting. Glutamine then accounted for 71% of the amino
acid release and 82% of the N-release from muscle.19 In summary, these data show that
after consumption of protein-containing meals, BCAA and glutamate are produced by the
splanchnic bed and taken up by muscle, and their carbon skeletons are primarily used
for de novo synthesis of glutamine.

8.3 BCAA metabolism during one-leg knee-extension exercise: a


model with exceptionally high net protein degradation rates
Van Hall et al.15 measured the exchange of amino acids across the leg muscles during 90
min of one-leg knee-extensor kicking exercise in six healthy trained human subjects. One-
leg knee-extensor exercise is primarily confined to the quadriceps muscle group (only
3 kg of active muscle, while there is 9 kg of muscle in a human leg). The maximal oxygen
consumption of the active muscle is about 800 ml/min/kg of muscle, which is two- to
threefold higher than during two-legged cycling on a normal cycle ergometer (which is
performed with a much larger muscle mass). This implies that the metabolic rate of the
active muscle during one-leg knee-extensor exercise is much greater than with whole-
body exercise. Such high metabolic rates have also been reached during electrical stim-
ulation of rat muscles, and in that case, the contractions lead to a reduction of the energy
charge and a decrease of the rate of muscle protein synthesis.24 A recent study25 has
disclosed the mechanism behind this observation. Increases in muscle AMP concentration
via activation of the AMP-activated protein kinase (AMPK) signaling cascade have been
shown to suppress protein synthesis in rat skeletal muscle through down-regulation of
the mammalian target of rapamycin (mTOR) signaling pathway. This is one of the impor-
tant pathways controlling the translation initiation phase of protein synthesis (see Chapter
16 for more details). The understanding of the AMPK signaling cascade recently has
received a lot of attention in exercise physiology. Activation of AMPK, among others,
leads to the phosphorylation of enzymes that exert control on glucose and fatty acid
transport and oxidation.26 AMPK is activated during exercise both by an increase of the
AMP concentration and by a reduction of the phosphocreatine/creatine ratio.26 These
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Chapter eight: Metabolism of branched-chain amino acids in man 127

Figure 8.2 The leg amino acid exchange observed during one-leg knee-extensor leg exercise — a
model with exceptionally high net protein degradation rates. For a detailed explanation of the
experimental conditions and methods used, see Van Hall et al.14,15

mechanisms seem to explain that one-legged knee-extensor exercise with its high meta-
bolic rate in comparison to the rest situation (and in comparison to normal dynamic
exercise such as running or cycling) leads to a large net protein degradation rate (protein
degradation >>> protein synthesis) (Figure 8.2).15 The conclusion that net protein degra-
dation was increased was based on the 10-fold increase in the release of amino acids that
are not metabolized (nmAA), transaminated, and oxidized, in human muscle.15 These
amino acids are only used for protein synthesis and produced by protein degradation,
and therefore, a net release indicates that protein degradation exceeds protein synthesis.15
As one-leg knee-extensor exercise appears to lead to the largest physiological imbal-
ance between muscle protein synthesis and protein degradation that has ever been
observed in the scientific literature in any other physiological condition, the amino acid
exchange data obtained in this model are uniquely suited to investigate the metabolism
of the large amounts of BCAA and glutamate that are released from muscle protein. It is
clear that BCAA and glutamate are vividly metabolized by human skeletal muscle, as
they, despite the high production rate by net protein degradation during one-leg exercise,
were taken up in substantial amounts (≥10-fold the resting uptake) from the circulation
(Figure 8.2). Data on aspartate were not obtained in these human experiments, but in
incubated rat muscles aspartate is rapidly transaminated to oxaloacetate and not released
to the medium.27 During one-leg knee-extensor exercise the alanine is released in an
amount about equal to its relative occurrence,15 and the amount released is not higher than
that in the resting state (Figure 8.2). Glutamine, on the other hand, is released in amounts
that are much larger than its relative occurrence,15 and the amount released is ninefold
higher than that in the resting state (Figure 8.2). Together, these data indicate that the
carbon skeletons of the BCAA, glutamate, aspartate, asparagine, and other amino acids
that are partly oxidized in muscle15 are not used for the synthesis of alanine but are solely
used for conversion into tricarboxylic acid cycle intermediates and glutamine (see Section
8.4 and Figure 8.1).
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128 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

8.4 Are BCAA a source for both the carbon and nitrogen of alanine
and glutamine? Studies with isotopic tracers
The next question is whether the carbon and nitrogen atoms of the BCAA can be used for
complete synthesis of both glutamine and alanine (Figure 8.1). Studies with [15N]-leucine
have shown that the amino group of the BCAA is indeed incorporated in humans in vivo
in the a-amino nitrogen of alanine28 and glutamine.29As glutamate is central in all ami-
notransferase reactions in muscle (Figure 8.1), this implies that the amino group of all six
mentioned amino acids (BCAA, glutamate, aspartate, and asparagine) is interchangeable
and can be incorporated in the a-amino nitrogen of alanine and glutamine. The source of
ammonia in glutamine synthesis (incorporated in the amide nitrogen) was not known in
19951 and still is not known today. Two deamination reactions (purine nucleotide cycle
and glutamate dehydrogenase) theoretically may deaminate the six metabolized amino
acids as discussed before,1,9 but the experimental evidence is still lacking.
In vitro muscle incubations and perfusions with [U-14C]-amino acids and in vivo exper-
iments in rats with [1,2-13C]-leucine have led to the general consensus that the carbon
skeletons of the six metabolized amino acids (Figure 8.1) are used for de novo synthesis of
TCA cycle intermediates and glutamine.1 Carbon from the BCAA enters the TCA cycle
both as acetyl-CoA (leucine and isoleucine) and as succinyl-CoA (isoleucine and valine)
and is then via a-ketoglutarate converted to glutamate amd glutamine (Figure 8.1). No,
or very little, radioactivity was found in lactate, pyruvate, and alanine during incubation
of rat diaphragms30 and perfusion of rat hindquarters1 with [U-14C]-valine. This implies
that there is no active pathway in muscle for conversion of TCA cycle intermediates into
pyruvate. It also implies that the carbon skeleton of the six amino acids that are converted
to TCA cycle intermediates (Figure 8.1) cannot be used for complete oxidation (which is
only possible when carbon enters the TCA cycle as acetyl-CoA, as is the case for leucine
and for part of the isoleucine molecule) or for pyruvate and alanine synthesis. Therefore,
the only fate of these carbon skeletons is synthesis of TCA cycle intermediates and
glutamine (Figure 8.1). The question then is what are the sources of the carbon atoms of
alanine? The remaining sources are muscle glycogen and blood glucose converted by
glycolysis into pyruvate (Figure 8.1). In agreement with this conclusion, Chang and
Goldberg30 reported that over 97% of the carbons of the alanine, pyruvate, and lactate
released by incubated diaphragms were derived from exogenous glucose. In a recent
whole-body tracer study in man,31 42% of the alanine released by muscle was reported to
originate from blood glucose.

8.5 The glucose–alanine cycle: a changing concept


The conclusion of the previous section slightly changes the concept of the glucose–alanine
cycle as proposed by Felig and colleagues32 and as explained today in most textbooks.
According to the original formulation of the glucose–alanine cycle the pyruvate used for
alanine production in muscle was derived from either glycolysis of blood glucose or
pyruvate derived from metabolism of other muscle protein-derived amino acids. The
alanine is then released to the blood and converted to glucose via gluconeogenesis in the
liver. Carbon derived from muscle protein and amino acids in this way was suggested to
be a major source used to help maintain blood glucose concentrations after overnight
fasting and during prolonged starvation. The implication, however, of the above conclu-
sions is that all pyruvate is either derived from glycolysis of blood glucose or from
breakdown of muscle glycogen followed by glycolysis. In a recent tracer study in man,31
42% of the alanine released by muscle was reported to originate from blood glucose. This
suggests that more than half of the alanine released by muscle is formed from pyruvate
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Chapter eight: Metabolism of branched-chain amino acids in man 129

generated by muscle glycogen breakdown. This route in fact may provide a mechanism
to slowly mobilize the large muscle glycogen stores (350 to 800 g)9 during starvation, such
that these stores can be used to help maintain the blood glucose concentration and function
as fuel in tissues that critically depend on glucose availability such as brain, red blood
cells, and kidney cortex. The amino acids liberated during starvation by increased net
rates of protein degradation16,17 are not converted to alanine but to glutamine, which also
is a precursor for gluconeogenesis in the liver in the postabsorptive state (for references,
see Wagenmakers9). Glutamine also is a precursor for gluconeogenesis in the kidney, but
renal gluconeogenesis only starts to be significant (>10% of total glucose output) in man
after starvation for more than 60 h (for references, see Wagenmakers9). Protein-derived
amino acids metabolized in muscle thus still can help to maintain blood glucose concen-
tration during starvation, but by a different route than suggested in the original formu-
lation of the glucose–alanine cycle. Recent tracer studies in man indeed confirm that
glutamine is more important than alanine as a gluconeogenic precursor after overnight
starvation,33 and that glutamine is more important than alanine as a vehicle for transport
of protein-derived carbon and nitrogen from muscle through plasma to the sites of glu-
coneogenesis or further metabolism.31

8.6 The role of alanine aminotransferase and deamination of BCAA


in TCA cycle anaplerosis during exercise
During one- and two-legged cycling exercise at intensities between 40 and 70% of Wmax,
only two amino acids change substantially in concentration in the muscle free amino acid
pool, i.e., glutamate and alanine (for references, see Wagenmakers9,16 and van Hall et al.14).
Glutamate decreases by 50 to 70% within 10 min of exercise, while alanine at that point
in time is increased by 50 to 60%. The low concentration of glutamate is maintained when
exercise is continued for periods up to 90 min or until exhaustion, while alanine slowly
returns to resting levels. Substantial amounts of alanine, furthermore, are released into
circulation during the first 30 min of exercise.14 Alanine release is reduced again when
exercise is continued and the muscle glycogen stores are gradually emptied.14,15
The functionality of the rapid fall in muscle glutamate concentration during the first
minutes of moderate-intensity exercise is conversion of its carbon skeleton via the alanine
aminotransferase reaction (glutamate + pyruvate ´ a-ketoglutarate + alanine) into a-keto-
glutarate and other TCA cycle intermediates (Figure 8.1). The sum concentration of the
most abundant TCA cycle intermediates in skeletal muscle has been shown to increase
5- to 10-fold within 5 min of the start of exercise both in rat muscle and in human muscle
(for references, see Wagenmakers9 and Gibala et al.34). The increase, furthermore, is pro-
portional to the exercise intensity in human muscle.35 We have previously suggested9 that
the increase in concentration of TCA cycle intermediates during the first minutes of
exercise is needed to increase the flux in the TCA cycle and make ATP production by
aerobic substrate oxidation match the increased ATP demand when going from rest to
exercise (for details of the theoretical basis of this conclusion, see Wagenmakers9).
In skeletal muscle, the rate of aerobic energy production, and therefore the flux in the
TCA cycle, can increase more than 80-fold going from rest to exercise. The understanding
of the mechanisms that lead to this massive increase within minutes of the start of exercise
is one of the most interesting and most complex academic challenges of the biochemistry
of exercise of the last decades.
Theoretically, other reactions could make contributions to the synthesis of TCA cycle
intermediates during the first minutes of exercise. Glutamate dehydrogenase (glutamate
+ NAD+ ´ a-ketoglutarate + NH4+ + NADH) can be excluded as a major contributing
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130 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

reaction, as ammonia production rates were very low in the first 10 min of exercise, when
the large decrease in muscle glutamate concentration was observed.14 For the same reason,
purine nucleotide cycling, suggested by Aragón and Lowenstein36 to be the major anaple-
rotic mechanism during exercise, can be excluded. The alanine aminotransferase reaction
is a near-equilibrium reaction. At the start of exercise the rate of glycolysis and thus of
pyruvate formation is high, as indicated by a temporary increase of the muscle pyruvate
concentration (for references, see Wagenmakers9). The increase in muscle pyruvate con-
centration automatically forces the alanine aminotransferase reaction toward a new equi-
librium with production of a-ketoglutarate and alanine from pyruvate (continuously
supplied by glycolysis) and glutamate (falling in concentration).
Felig and Wahren11 have shown that the rate of release of alanine from muscle
depended on the exercise intensity and suggested a direct relation between the rate of
formation of pyruvate from glucose and alanine release. This led to the suggestion that
the glucose–alanine cycle also operated during exercise: glucose taken up by muscle from
the blood via glycolysis is converted to pyruvate and then via transamination to alanine
to subsequently serve as substrate for gluconeogenesis in the liver and help to maintain
blood glucose concentration during exercise. Here we propose that the alanine aminotrans-
ferase reaction primarily functions for de novo synthesis of a-ketoglutarate and TCA cycle
intermediates at the start of exercise. The augmented glycolysis during exercise thus
appears to serve a dual function. More pyruvate is generated (1) to function as a substrate
for pyruvate dehydrogenase and subsequent oxidation and (2) to force the alanine ami-
notransferase reaction toward production of a-ketoglutarate and TCA cycle intermediates,
and thus to increase TCA cycle activity and the capacity to oxidize acetyl-CoA derived
from pyruvate and fatty acid oxidation.
Another metabolic need for a high net flux through the alanine aminotransferase
reaction during exercise is the increase in the oxidation rate of BCAA that occurs, among
others, due to activation of the branched-chain a-keto acid dehydrogenase complex.9,37
This also leads to an increase in the rate of the BCAA aminotransferase reaction (leucine
+ a-ketoglutarate ´ a-ketoisocaproate (KIC) + glutamate). In case of leucine transamina-
tion and oxidation, acetyl-CoA is the end product, and therefore, leucine transamination
would lead to a net loss of TCA cycle intermediates when the glutamate would not be
converted back to a-ketoglutarate via the alanine aminotransferase reaction. Previously,
we suggested that leucine transamination via its draining effect on the TCA cycle may
play a role in the gradual decrease in TCA cycle intermediates and the development of
fatigue in muscles with a low glycogen content and, therefore, low capacity for alanine
production.9,16
An alternative mechanism that may generate TCA cycle intermediates during more
prolonged exercise leading to glycogen depletion is increased deamination rates of amino
acids in muscle. A gradually increasing ammonia production has been observed during
prolonged one-leg knee-extension exercise by van Hall et al. (Figure 8.2).14 As there was
no net breakdown of adenine nucleotides to IMP in that study (ATP Æ ADP Æ AMP Æ
IMP + NH4+), the conclusion was drawn that the ammonia production must have origi-
nated from net deamination of amino acids either via purine nucleotide cycling or via
glutamate dehydrogenase (source is unknown today). Deamination of amino acids in
contrast to transamination does not use a-ketoglutarate as an amino group acceptor
(Figure 8.1). As TCA cycle intermediates are formed in the degradation routes of valine,
isoleucine, aspartate, and glutamate (Figure 8.1), deamination will lead to net synthesis
of TCA cycle intermediates. Deamination of leucine leads to acetyl-CoA formation and,
therefore, does not influence the TCA cycle intermediate concentration. As explained
above (Section 8.3), one-leg knee-extensor exercise is an exercise model with exceptionally
high net rates of protein degradation. The amino acid exchange observed under these
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Chapter eight: Metabolism of branched-chain amino acids in man 131

conditions (Figure 8.2) indicated that BCAA and glutamate released by the net breakdown
of muscle protein and taken up from the circulation were either deaminated (thus leading
to synthesis of TCA cycle intermediates) or used for the synthesis of vast amounts of
glutamine (Figure 8.2). Removal of amino groups from muscle in the form of glutamine
provides another mechanism for net synthesis of TCA cycle intermediates,15 as is illus-
trated by the following net reactions (see Figure 8.1 for the complete metabolic pathways):

2 glutamate Æ glutamine + a-ketoglutarate

Valine + isoleucine Æ succinyl-CoA + glutamine

Aspartate + isoleucine Æ oxaloacetate + glutamine

Valine + leucine Æ 2 acetyl-CoA + glutamine

Therefore, BCAA and glutamate released by the net breakdown of muscle protein and
taken up from the circulation seem to be used for net synthesis of TCA cycle intermediates
and glutamine during prolonged one-legged exercise. The extent to which these reactions
were accelerated was even larger when the exercise was performed by knee-extensor
muscles with a low glycogen content.15 It is far from clear whether dynamic whole-body
exercise as practiced by athletes during competition (cycling or running) leads to net
protein breakdown in muscle and helps to provide carbon skeletons for synthesis of TCA
cycle intermediates via the indicated reactions. Different stable isotope tracers used to
measure protein synthesis and degradation in laboratory conditions gave different
answers; different answers also were obtained for changes observed at the whole-body
level and muscle level (for references, see Wagenmakers9,16). Whole-body measurements
with L-[1-13C]-leucine suggest that there is increased whole-body net protein breakdown
during 1 to 6 h of cycling exercise at intensities of 30 to 50% VO2max, but with other tracers
(urea and [2H5-ring]-phenylalanine), no such increases have been observed.16 Carraro et
al.39 did not find an effect of cycling exercise at 40% VO2max on muscle protein synthesis
measured directly from the incorporation of [13C]-leucine. Furthermore, carbohydrate
ingestion as practiced by endurance athletes during competition reduces net protein
breakdown and amino acid oxidation and upgrades the relative importance of the alanine
aminotransferase reaction for TCA cycle anaplerosis.

8.7 Lessons from patients with McArdle’s disease


Patients with McArdle’s disease cannot substantially increase the glycolytic rate during
exercise due to the glycogen breakdown defect in muscle, and they therefore do not
increase muscle pyruvate. The arterial alanine and muscle alanine concentrations do not
increase in these patients during exercise.35,38 The muscle glutamate concentration is only
about 30% of normal and decreases only little during incremental exercise.35 This implies
that the anaplerotic capacity of the alanine aminotransferase reaction in these patients is
substantially reduced in comparison to that of healthy subjects. Sahlin and colleagues35
indeed observed that patients with McArdle’s disease could only marginally increase the
concentration of TCA cycle intermediates during incremental exercise. The maximal work
rate and oxygen consumption of these patients during cycling exercise is between 40 and
50% of the maximum predicted for their age and build. In ultraendurance exercise without
carbohydrate ingestion, healthy subjects have to reduce the work rate to about the same
level when the glycogen stores have been emptied, suggesting that muscle glycogen
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132 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

indeed is needed to maintain high work rates potentially by means of its ability to establish
and maintain high concentrations of TCA cycle intermediates.
An excessive release of ammonia and glutamine and an excessive net breakdown of
muscle protein (several-fold more than in one-leg knee-extensor exercise in healthy sub-
jects) also were observed during two-legged cycling in patients with McArdle’s disease,38
indicating that deamination of BCAA and glutamate and synthesis of glutamine and TCA
cycle intermediates from glutamate and BCAA also provide alternative mechanisms of
TCA cycle anaplerosis in this muscle disease with zero glycogen availability and low
pyruvate concentrations. The fact that high exercise intensities cannot be maintained by
these patients and in glycogen-depleted muscles seems to indicate that these alternative
anaplerotic reactions are not as effective as the alanine aminotransferase reaction and only
allow muscular work at 40 to 50% of Wmax.

8.8 Conclusion
Since 1995 little new information has been obtained on the activity of the enzymes involved
in BCAA degradation, their tissue distribution, and regulation by physiological stresses
and hormones. Maybe a disproportionate effort goes into the modern large-scale genomic
and proteomic techniques as the regulation of enzyme activities will always continue to
form the basis for our understanding of the interorgan collaboration in the handling of
metabolites and of short-term cellular control mechanisms. Today our understanding of
BCAA metabolism in man has primarily been extended by a number of studies investi-
gating the exchange of amino acids across tissue beds using arteriovenous cannulations
and muscle biopsies and with studies investigating the fate of the amino group and carbon
skeleton with stable isotope tracers. The most important new insights are that the main
six amino acids metabolized in human muscle are the BCAA, glutamate, aspartate, and
asparagine. Only leucine and part of isoleucine generate acetyl-CoA and can be oxidized.
The other carbon skeletons are used solely for de novo synthesis of TCA cycle intermediates
and glutamine. The carbon atoms of the alanine released by muscle originate from glyc-
olysis of blood glucose and from glycogen breakdown. During the first 30 min of endur-
ance exercise, the alanine aminotransferase reaction functions to establish and maintain
high concentrations of TCA cycle intermediates and counteract the drain of the leucine
transaminase reaction. Deamination of amino acids and glutamine synthesis present alter-
native anaplerotic mechanisms in glycogen-depleted muscles but only allow exercise at
40 to 50% of Wmax. One-leg knee-extensor exercise leads to an excessive rate of net muscle
protein degradation. The liberated amino acids are used for synthesis of TCA cycle inter-
mediates and glutamine. Interactions between the metabolism of the BCAA, glutamate,
and aspartate and the TCA cycle clearly play a central role in the energy metabolism of
the exercising muscle.

References
1. Wagenmakers, A.J.M., Metabolism of branched-chain amino acids, in Amino Acid Metabolism
and Therapy in Health and Nutritional Disease, Cynober L.A., Ed., CRC Press, Boca Raton, 1995,
chap. 5.
2. Walser, M. and Williamson, J.R., Metabolism and Clinical Implications of Branched Chain Amino
and Ketoacids, Elsevier/North-Holland, Amsterdam, 1981.
3. Adibi, S.A., Fekl, W., Langenbeck, U., and Schauder, P., Branched Chain Amino and Keto Acids
in Health and Disease, S. Karger, Basel, Switzerland, 1984.
4. Odessey, R., Problems and Potential of Branched-Chain Amino Acids in Physiology and Medicine,
Elsevier Science Publishers, Amsterdam, 1986.
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Chapter eight: Metabolism of branched-chain amino acids in man 133

5. Harris, R.A. and Sokatch, J.R., Branched chain amino acids, Methods Enzymol., 166, 1988.
6. Harper, A.E., Miller, R.H., and Block, K.P., Branched-chain amino acid metabolism, Annu.
Rev. Nutr., 4, 409, 1984.
7. Ruderman, N.B. and Lund, P., Amino acid metabolism in skeletal muscle: regulation of
glutamine and alanine release in the perfused rat hindquarter, Israel J. Med. Sci., 8, 295, 1972.
8. Goldberg, A.L. and Chang, T.W., Regulation and significance of amino acid metabolism in
skeletal muscle, Fed. Proc., 37, 2301, 1978.
9. Wagenmakers, A.J.M., Muscle amino acid metabolism at rest and during exercise: role in
human physiology and metabolism, Ex. Sport Sci. Rev., 26, 287, 1998.
10. Marliss, E.B., Aoki, T.T., Pozefsky, T., Most, A.S., and Cahill, G.F., Muscle and splanchnic
glutamine and glutamate metabolism in postabsorptive and starved man, J. Clin. Invest., 50,
814, 1971.
11. Felig, P. and Wahren, J., Amino acid metabolism in exercising man, J. Clin. Invest., 50, 2703,
1971.
12. Wahren, J., Felig, P., and Hagenfeldt, L., Effect of protein ingestion on splanchnic and leg
metabolism in normal man and patients with diabetes mellitus, J. Clin. Invest., 57, 987, 1976.
13. Eriksson, L.S., Broberg, S., Björkman, O., and Wahren, J., Ammonia metabolism during
exercise in man, Clin. Physiol., 5, 325, 1985.
14. van Hall, G., Saltin, B., van der Vusse, G.J., Söderlund, K., and Wagenmakers, A.J.M., Deam-
ination of amino acids as a source for ammonia production during prolonged exercise in
man, J. Physiol., 489, 251, 1995.
15. van Hall, G., Saltin, B., and Wagenmakers, A.J.M., Muscle protein degradation and amino
acid metabolism during prolonged knee-extensor exercise in humans, Clin. Sci., 97, 557, 1999.
16. Wagenmakers, A.J.M., Protein and amino acid metabolism in human muscle, Adv. Exp. Biol.
Med., 441, 307, 1998.
17. Wagenmakers, A.J.M., Tracers to investigate protein and amino acid metabolism in human
subjects, Proc. Nutr. Soc., 58, 987, 1999.
18. Kimball, S.R., Regulation of global and specific mRNA translation by amino acids, J. Nutr.,
132, 883, 2002.
19. Elia, M., Schlatmann, A., Goren, A., and Austin, S., Amino acid metabolism in muscle and
in the whole body of man before and after ingestion of a single mixed meal, Am. J. Clin.
Nutr., 49, 1203, 1989.
20. Elia, M. and Livesey, G., Effects of ingested steak and infused leucine on forearm metabolism
in man and the fate of amino acids in healthy subjects, Clin. Sci., 64, 517, 1983.
21. Hagenfeldt, L., Eriksson, S., and Wahren, J., Influence of leucine on arterial concentrations
and regional exchange of amino acids in healthy subjects, Clin. Sci., 59, 173, 1980.
22. Matthews, D.E., Marano, M.A., and Campbell, R.G., Splanchnic bed utilization of glutamine
and glutamic acid in humans, Am. J. Physiol., 264, E848, 1993.
23. Battezzati, A., Brillon, D.J., and Matthews, D.E., Oxidation of glutamic acid by the splanchnic
bed in humans, Am. J. Physiol., 269, E269, 1995.
24. Bylund-Fellenius, A.-C., Ojamaa, K.M., Flaim, K.E., Li, J.B., Wassner, S.J., and Jefferson, L.S.,
Protein synthesis versus energy state in contracting muscles of perfused rat hindlimb, Am.
J. Physiol., 246, E297, 1984.
25. Bolster D.R., Crozier S.J., Kimball, S.R., and Jefferson, L.S., AMP-activated protein kinase
suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian
target of rapamycin (mTOR) signaling, J. Biol. Chem., 277, 23977, 2002.
26. Hardie, D.G., Carling, D., and Carlson, M., The AMP-activated/SNF1 protein kinase sub-
family: metabolic sensors of the eukaryotic cell? Annu. Rev. Biochem., 67, 821, 1998.
27. Chang, T.W. and Goldberg, A.L., The metabolic fates of amino acids and the formation of
glutamine in skeletal muscle, J. Biol. Chem., 253, 3685, 1978.
28. Haymond, M.W. and Miles, J.M., Branched-chain amino acids as a major source of alanine
nitrogen in man, Diabetes, 31, 86, 1982.
29. Darmaun, D. and Déchelotte, P., Role of leucine as a precursor of glutamine a-amino nitrogen
in vivo in humans, Am. J. Physiol., 260, E326, 1991.
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30. Chang, T.W. and Goldberg, A.L., The origin of alanine produced in skeletal muscle, J. Biol.
Chem., 253, 3677, 1978.
31. Perriello, G., Jorde, R., Nurjhan, N., Stumvoll, N., Dailey, G., Jenssen, T.G., Bier, D.M., and
Gerich, J.E., Estimation of glucose-alanine-lactate-glutamine cycles in postabsorptive hu-
mans: role of skeletal muscle, Am. J. Physiol., 269, E443, 1995.
32. Felig, P., Pozefsky, T., Marliss, E., and Cahill, G.F., Alanine: a key role in gluconeogenesis,
Science, 167, 1003, 1970.
33. Nurjhan, N., Bucci, A., Perriello, G., Stumvoll, N., Dailey, G., Bier, D.M., Toft, I., Jenssen,
T.G., and Gerich, J.E., Glutamine: a major gluconeogenic precursor and vehicle for interorgan
carbon transport in man, J. Clin. Invest., 95, 272, 1995.
34. Gibala, M.J., Tarnapolski, M.A., and Graham, T.E., Tricarboxylic acid cycle intermediates in
human muscle at rest and during prolonged cycling, Am. J. Physiol., 272, E239, 1997.
35. Sahlin, K., Jorfeldt, L., Henriksson, K.G., Lewis, S.R., and Haller, R.G., Tricarboxylic acid
cycle intermediates during incremental exercise in healthy subjects and in patients with
McArdle’s disease, Clin. Sci., 88, 687, 1995.
36. Aragón, J.J. and Lowenstein, J.M., The purine nucleotide cycle: comparison of the levels of
citric acid cycle intermediates with the operation of the purine nucleotide cycle in rat skeletal
muscle during exercise and recovery from exercise, Eur. J. Biochem., 110, 371, 1980.
37. van Hall, G., MacLean, D.A., Saltin, B., and Wagenmakers, A.J.M., Mechanisms of activation
of muscle branched-chain a-keto acid dehydrogenase during exercise in man, J. Physiol., 494,
899, 1996.
38. Wagenmakers, A.J.M., Coakley, J.H, and Edwards, R.H.T., Metabolism of branched-chain
amino acids and ammonia during exercise: clues from McArdle’s disease, Int. J. Sports Med.,
11, 101, 1990.
39. Carraro, F., Stuart, C.A., Hartl, W.H., Rosenblatt, J., and Wolfe, R.R., Effect of exercise and
recovery on muscle protein synthesis in human subjects, Am. J. Physiol., 259, E470, 1990.
1382_C09.fm Page 135 Tuesday, October 7, 2003 6:23 PM

chapter nine

The glutamate crossway


Yasuo Wakabayashi
Kyoto Prefectural University of Medicine

Contents
Introduction..................................................................................................................................135
9.1 Enzymes and tissues involved.........................................................................................135
9.1.1 Distribution of enzymes in the body .................................................................135
9.1.2 Ontogenic development of enzymes..................................................................138
9.1.3 Nutritional and hormonal effects on enzymes.................................................140
9.1.4 Tissue-selective expression of enzymes .............................................................141
9.2 Pioneering experiments led to fundamental findings .................................................142
9.2.1 Nutritional studies with isotopes .......................................................................142
9.2.2 Perfusion studies of tissues..................................................................................143
9.3 Overall flow of metabolites and significance in human nutrition.............................144
9.3.1 Derivation from animal studies ..........................................................................144
9.3.2 Implication from human clinical studies...........................................................145
References .....................................................................................................................................147

Introduction
In this chapter, metabolism of glutamate (or glutamine) as a precursor for arginine and
proline is discussed. Interplay between organs, regulation, and nutritional, hormonal, and
molecular aspects will also be considered. For healthy humans and adult mammals, these
four amino acids are nutritionally dispensable.1 However, supplementation of arginine
and glutamine is essential for cultured cells.2 Thus, the dietary dispensability of arginine
and glutamine does not mean that individual cells constituting the body can form both
amino acids by themselves.

9.1 Enzymes and tissues involved


9.1.1 Distribution of enzymes in the body
Figure 9.1 and Table 9.1 summarize distribution and activity of enzymes involved in this
pathway on tissue basis. The enzymes can be categorized into three subgroups based upon

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136 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 9.1 Organs involved in synthesis and degradation of arginine and proline. Typical interorgan
relationships are shown. Citrulline synthesis from glutamate definitely requires the small intestine.
Conversions of citrulline to arginine, arginine to proline, or proline to glutamate are not exclusively
dominated by the kidney and liver. Enzymes denoted by Arabic numbers are (with abbreviations
in parenthesis) as follows: 1, phosphate-dependent glutaminase (GLNase); 2, pyrroline-5-carboxylate
synthase (PCS); 3, ornithine aminotransferase (OAT); 4, ornithine transcarbamylase (OTC); 5, argin-
inosuccinate synthetase (ASS); 6, argininosuccinate lyase (ASL); 7, chemically spontaneous and
reversible reaction, although the equilibrium strongly favors P5C formation; 8, pyrroline-5-carbox-
ylate reductase (PCR); 9, arginase (ARGase); 10, carbamyl-phosphate synthetase (CPS); 11, N-acetyl-
glutamate synthase (AGS); 12, proline oxidase (PROox); 13, pyrroline-5-carboxylate dehydrogenase
(PCDH). Abbreviations for specific amino acid intermediates: GSA, glutamic-g-semialdehyde; P5C,
pyrroline-5-carboxylate; Cit, citrulline; ASA, argininosuccinate; Orn, ornithine; AGA, N-acetyl-
glutamate.

the extent of ubiquity. The most ubiquitous enzymes are ornithine aminotransferase (OAT)
and pyrroline-5-carboxylate reductase (PCR). The second enzymes are moderately ubiq-
uitous, but there is some convergency. They are phosphate-dependent glutaminase
(GLNase), glutamine synthetase (GS), argininosuccinate synthetase (ASS), argininosucci-
nate lyase (ASL), pyrroline-5-carboxylate dehydrogenase (PCDH), and arginase (ARGase).
The third enzymes display extreme polarity. Several tissues are unable to detect activity.
They are pyrroline-5-carboxylate synthase (PCS), carbamoyl-phosphate synthetase (CPS),
ornithine transcarbamoylase (OTC), N-acetylglutamate synthase (AGS), and proline oxi-
dase (PROox). One will notice that the small intestine is the only tissue where all the
enzymes required to construct citrulline or ornithine from glutamate or glutamine are
assembled, particularly PCS, CPS, OTC, and AGS. On the contrary, enzymes from citrulline
to arginine, ASS and ASL, are widespread, though relative abundance is found in the liver
and kidney.3 This is in accord with Eagle’s observation that all the human cell lines studied
lived satisfactorily with citrulline in place of arginine.2 Enzymes for proline and arginine
Table 9.1 Distribution of Enzymes Involved in Arginine, Proline, Glutamine, and Glutamate Interconversion
Tissue GLNase GS PCS OAT CPS OTC ASS
Small intestine 19 252 117 10 100 68
Liver 5 244 0 188 284 4110 1386
Chapter nine:

Kidney 24 221 0 166 8 5 677


Brain 16 337 1 13 4 0 51
Thymus 6 22 8 0 2
Lung 18 0 15 0 2 22
Spleen 6 166 0 15 0 0 11
Muscle 1 10 0 13 0 0
Pancreas 12 40 3 2
Salivary gland 1 10 12
1382_C09.fm Page 137 Tuesday, October 7, 2003 6:23 PM

Mammary gland 3
References (95, 96) (97) (56) (31) (98) (98) (99)
The glutamate crossway

Tissue ASL AGS PCR PROox PCDH ARGase


Small intestine 6 31 28 0.0 0.4 129
Liver 308 327 33 5.3 33.5 2545
Kidney 41 0 24 2.0 14.5 57
Brain 10 0 15 0.4 0.4 3
Thymus 3 20 0.3 2
Lung 10 14 7 0.1 0.4 5
Spleen 14 7 19 0.0 0.4 1
Muscle 5 0 5 0.5 0.4 0
Pancreas 0 10 0.0 0.9 103
Salivary gland 8 57 0.0 0.4 170
Mammary gland 5 0.8 163
References (100) (101) (102) (102) (102) (103)

Note: Original data were rounded so that figures may expedite comparisons. This distribution profile is intended to reflect whether these
enzymes are distributed ubiquitously or limited to specific tissues. Activities are expressed as pmol/mg/min with PCS and AGS;
nmol/mg/min with GS; mmol/g/min with ARGase, GLNase, PCR, PROox, and PCDH; mmol/g/h with CPS, OTC, ASL, and OAT;
and nmol/mg/h with ASS. Abbreviations of enzymes are the same as in Figure 9.1 except that GS denotes glutamine synthetase.
Muscle represents skeletal muscle except that the heart muscle was reported with OAT, PROox, and ASL. The mammary gland
was taken from pregnant or lactating animals.
137
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138 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

degradation, PROox, PCDH, and ARGase, are sparse in many tissues. ARGase is a mixture
of isozymes, type I and type II. Type I is a cytosolic enzyme found almost exclusively in
the liver as a part of urea cycle enzymes, whereas type II is in the mitochondria and
distributed, more or less, in almost all extrahepatic tissues.4 Intracellular location of
enzymes is in either cytosol or mitochondria. PCS, PROox, and GLNase are bound to
mitochondrial inner membranes.5 GLNase has two isozymes, liver type and kidney type.
The liver type is found exclusively in the liver and supplies ammonia to the urea cycle,
and the kidney type is ubiquitous and essential for pH homeostasis in the kidneys. PCDH,
OAT, OTC, CPS, and AGS are in mitochondrial matrix. Human PCDH has been identified
as aldehyde dehydrogenase isozyme IV. Besides pyrroline-5-carboxylate (P5C), adipic and
glutaric semialdehyde are other substrates.6 OTC and CPS activities in the liver are much
higher than those in the small intestine on a tissue mass basis. However, concentrations
per mitochondria basis, as estimated by the electron microscope immunocytochemistry
technique, differ only by twofold. In addition, CPS and OTC mRNAs are enriched twice
as high in the liver as in the small intestine when an identical amount of mRNA or poly(A)-
rich RNA from the tissues was analyzed by dot blot or Northern hybridization or in vitro
translation.7 Therefore, enrichment of cells by the mitochondria and the population in
given tissues of the cells expressing enzyme activities explain the discrepancy. GS, ASS,
ASL, and PCR are in cytosol. Erythrocyte PCR exhibits vigorous conversion of P5C to
proline with concomitant oxidation of NADPH2 to NADP+. Formed NADP+ stimulates
the pentose phosphate pathway to produce a large amount of phosphoribosylpyrophos-
phate (PRPP). PRPP will be efficiently used for salvaging purine bases.8
An inefficient kinetic property of OAT for ornithine synthesis had been argued.
Strecker9 showed minor conversion from P5C to ornithine with partially purified rat liver
enzyme and obtained an equilibrium constant of 70, which suggested that OAT works
much easier toward formation of P5C but not ornithine. McGivan et al.10 showed that
liver mitochondria failed to synthesize ornithine from P5C, unless rotenone was incorpo-
rated in the system to prevent P5C from being consumed by PCDH and NADH dehydro-
genase. In the meantime, Herzfeld and Raper11 reported that a mitochondria fraction of
young rat small intestine was effective in converting P5C to ornithine, although the rate
was one sixth that of the reverse reaction. Nonetheless, they were unable to detect ornithine
production from P5C by similar fractions of submaxillary gland and liver under similar
conditions.11 Since purified OAT proteins from the liver, kidney, and small intestine are
identical by kinetic, electrophoretic, and immunological criteria,12 the small intestine is
unique in its high OAT and low PCDH content, which permits ornithine synthesis once
P5C is supplied by PCS. When [14C]glutamate was injected into mice fed an arginine-free
diet, [14C] was incorporated into tissue arginine and ornithine. However, administration
of gabaculine, a potent suicide inhibitor of OAT, reduced the incorporation to one-third
of control. This provides direct evidence for involvement of OAT in arginine and ornithine
synthesis in vivo from glutamate.13 In the meantime, Ross et al.14 demonstrated conversion
of [14C]glutamate to [14C]ornithine using cell-free extract of rat intestinal mucosa.

9.1.2 Ontogenic development of enzymes


Developmental patterns of relevant enzymes are twofold. Typically, synthetic enzymes
are premature, whereas degradative enzymes are gradual.
PCS activity in fetus small intestine immediately before birth is one fifth that of adult
rats.15 The activity on postnatal day 1 is 180 pmol/mg/min, a value similar to that of
adults and is triplicated to 540 pmol/mg/min at day 14 and reduced to an adult level at
day 24. Thus, PCS of small intestine is high during suckling period and reduces with the
beginning of weaning. Wu et al.16 showed that the glucocorticoid surge at this postnatal
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Chapter nine: The glutamate crossway 139

period is the trigger for the enhancement of PCS activity in the small intestine, and the
enhanced activity of PCS determines the high output of citrulline from this tissue.16
Development of GLNase in the small intestine is similar to the profile of PCS.17 The highest
peak is shown in the midst of the weaning period. OAT in small intestine develops with
a prominent activation by about 400% during the suckling period.11 The crucial role of
this peak for the intestinal supply of ornithine and citrulline in the normal development
was shown by a gene-targeted knockout technology. OAT-null mice developed hypoargin-
inemia, hypocitrullinemia, hypoornithinemia, and hyperammonemia during the early
postnatal period. Without arginine supplementation, the mice died immediately after
birth, but with daily supplementation of arginine, the mice survived. Interestingly, the
adult mice developed hyperornithinemia and retinal degeneration similar to human
Gyrate atrophy (i.e., OAT deficiency).18 Therefore, the role of OAT in the developing period
favors ornithine synthesis and that in adult degradation. Liver OAT stays low until day
18 and then increases to an adult level after weaning. Also, liver ARGase develops with
a similar profile.11 CPS in the small intestine expresses the highest activity at birth, and
then activity monotonously decreases to an adult level.19 OTC in the small intestine is
more than 150% of adult activity at the 2nd postnatal day and approaches adult activity
at the 18th day.11 The mRNA level of CPS in the small intestine is highest at the 21st
gestational day; after birth, it decreases to an adult level gradually.20 On the contrary, the
mRNA of liver CPS continues to increase gradually to an adult level after birth. Thus, the
mRNA profiles explain the developmental changes in CPS activities of both tissues. mRNA
of OTC in the small intestine is highest at the 21st gestational day and accords to its activity
profile, except that the activity immediately before birth is too low for the highest mRNA
level at this stage. Also, the prenatal activity of liver OTC seems to be lower for the
abundant mRNA for the enzyme.11,20
PCR activity in the small intestine has two peaks, highest at birth and day 24.11 GLNase
protein and mRNA have their peak at postnatal day 19, and then mRNA drops, while
protein remains almost at the adult level.21 Activity of AGS in the fetus intestine is 14
pmol/mg/min and expresses a sudden peak of 64 pmol/mg/min at the first postnatal
day. It drops to 20 pmol/mg/min at day 3 and then gradually increases to an adult level.15
The AGS profile of the liver is different. The activity in fetuses is only one twentieth that
of adults. It increases gradually after birth to an adult level for 10 weeks. Immunocy-
tochemical staining of ASS and ASL proteins in the kidneys revealed that both proteins
were detectable at the 15th gestational day and the density increased up to gestational
day 23. mRNAs for ASS and ASL were 2 and 7% of adult levels, respectively, at the 15th
day. The mRNAs increased toward delivery when 50% or more of an adult level was
expressed.22
The significant role of the small intestine in citrulline-to-arginine conversion in the
perinatal period was visualized by a study where spatiotemporal expression patterns of
mRNAs of OAT, CPS, OTC, ASS, and ASL, and proteins of CPS and ASS were investi-
gated.23 The mRNAs of ASS and ASL were found only in the upper villi; those of OAT,
CPS, and OTC were condensed in the crypts. ASS protein was located in the upper villi
up to the weaning period but was almost lost in the adult villi. CPS protein was found
over all layers. Other enzymological studies quantified the synthesis of arginine in the
postnatal intestines and established the significance of this phenomenon.24–26 Overexpres-
sion of ARGase I in the developing intestine, therefore, provokes a serious effect: the
promoter and enhancer element of the rat intestinal fatty acid-binding protein (FABPi)
gene fused to ARGase I structural elements of rat was constructed to generate ARGase I
overexpressing transgenic mice.27 The enterocytes of the mice expressed ectopic ARGase
I extensively, and the suckling mice became arginine deficient. Typical phenotypes were
hypoargininemia, about one third of controls, and retardation of hair, muscle growth, and
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140 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

development of the lymphoid tissues. Specifically, an impairment of transition from the


pro-B cells to pre-B cells in the bone marrow was elucidated. Daily injection of arginine
prevented these completely. This observation demonstrated that the synthesis of arginine
by the intestine of suckling mice is essential for normal development, and also that
extraintestinal conversion of citrulline to arginine is premature at early postnatal period.

9.1.3 Nutritional and hormonal effects on enzymes


The molecular mass of purified OTC from the liver and small intestine (38,000 Da), their
pI (isoelectric point) (7.3), the identical reactivity against antibody against the liver enzyme
as detected by Western hybridization, the identical size of mRNA as visualized on North-
ern hybridization using cDNA for the liver OTC, and the specific activities of the pure
preparations (928 to 966 mmol/min/mg) indicate that both proteins are the same gene
product.28 However, intestinal OTC activity decreased from 8.8 to 5.7 mmol/min/mg DNA
when 15% casein diet was changed to 60%, whereas that of the liver increased from 0.8
to 1.3 mmol/min/mg DNA. Moreover, mRNA abundance as probed by [32P]-labeled OTC
cDNA in the liver increased from 930 to 2300 cpm/100 mg RNA, whereas that in the small
intestine decreased from 370 to 210 by the protein surplus.29 A 70% casein diet compared
with 5% increased OAT activity by 10-fold in liver, whereas there was no change in the
small intestine and kidney.12 mRNA levels for CPS and OTC in the liver responded to
glucagon positively by 1.5 to 2.2 times, but those in the small intestine did not.20 A 50%
casein diet compared with 8% did not affect CPS activity in the small intestine at all, while
it augmented the liver CPS by threefold.19 Nonetheless, reduction in caloric intake without
change in protein intake enhanced intestinal CPS activity twofold, with mRNA level
unchanged, which suggested posttranscriptional activation of CPS in the intestine. At the
same time, the activity in the liver increased by five times with concomitant threefold
increases of mRNA and protein.30 The apparently opposite regulations on OTC, CPS, and
OAT in the liver and small intestine are consistent with the role of the organs for the
synthesis of metabolites and their breakdown (urea synthesis), respectively. OAT activity
in the female kidney is twice as high as that in the male. Ovariectomy erases the difference.
Estrogen enhances kidney OAT in both sexes.31 Lyons and Pitot32 investigated responses
of OAT activity and the relative rate of its synthesis in vivo as estimated by pulse-labeling
rats with [3H]leucine combined with immunoprecipitation of OAT proteins. Triiodothy-
ronine (T3) injection into rats increased activity and synthesis by twofold in the kidney
but not in the liver. Glucagon enhanced activity in the liver by 2-fold and its synthesis by
10-fold, but had no effect on the kidney enzyme. Thyroidectomy reduced activity and
synthesis by 50% only in the kidney. This treatment abolished the induction of kidney
OAT by estrogen. Adrenalectomy had no effect on the basal activity and synthesis in the
liver and kidney.
The kidney arginine synthetase (ASS + ASL) increased threefold in rats fed an 80%
casein diet compared with an 8% diet.33 mRNA abundance of ASS and ASL in the kidney
became 2.5 times as rich as control after starvation for 6 days and doubled after transition
from 27 to 60% casein. ASS was boosted by dibutyryl cAMP by 2.3-fold, but failed to
respond to dexamethasone. ASL did not respond to either compound.34 Interestingly,
activities of ASS and ASL of HeLa, KB, and L cell strains were multiplied up to 15-fold
when these cells were grown in arginine-deficient media.35 ARGase in the liver increases
its activity and mRNA abundance coordinately after starvation, a high-protein diet, and
dexamethasone treatment.36 Also, glucagon augments ARGase activity.
PCR in the developing small intestine is elevated up to fivefold by starvation and cortisol,
while liver PCR is not.37 PCS is regulated by the negative feedback control sensitive to
intramitochondrial ornithine.38 Lysine, arginine, proline, and citrulline are ineffective. Also,
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Chapter nine: The glutamate crossway 141

glucose is the crucial substrate to provide NADPH, via the pentose phosphate pathway, to
PCS as the mandatory reductant in the reaction.39 Intestinal GLNase does not respond to
acid–base changes, whereas kidney GLNase increased four times by NH4Cl-induced meta-
bolic acidosis.17 PROox activity in adrenalectomized rat livers is 50% of controls, and hydro-
cortisone restored normal activity.40 A 60% protein diet compared with 25% enhanced liver
activity by twofold, but had no effect to the kidney, brain, and heart activity.41
Skeletal muscle is the major tissue to synthesize and release glutamine into systemic
circulation.42 Administration of dexamethasone to rats at a dose that imitates corticosteroid
levels under severe stress (0.5 mg/kg/day) caused loss of muscular weight and increased
GS activity in the plantaris muscle by 40% with concomitant rise of its mRNA by five times.
This rise was accompanied by a 2.5 times enrichment of GS mRNA in total translatable
mRNA. A dose up to 5.0 mg resulted in, by far, prominent augmentation of activity and
mRNA content. However, such treatment did not affect heart GS.43 Inhibition and activation
of GS by exercise and neural denervation, respectively, are other regulatory factors.44

9.1.4 Tissue-selective expression of enzymes


The mechanism of tissue-selective expression of genes mostly depends on how regulatory
elements, including promoters of those genes, can find appropriate transcription factors
(TFs) in the particular cells. There are two types of TFs, ubiquitous and tissue-specific
ones. Many TFs are required for tissue-selective expression of OTC and CPS.
A recombinant gene, a 1.3-kb segment of the 5' flanking region of rat OTC gene fused
to rat OTC cDNA, was made to generate the transgenic mice. The transgene was expressed
only in liver and small intestine. However, the hepatic transgene expression was much
lower than the endogenous gene expression.45 The lower expression was normalized when
a 1.6-kb segment about 11 kb upstream from the transcription start site was appended to
the recombinant. This far upstream region contains two hepatocyte nuclear factor (HNF)-4
binding sites and two CCAAT/enhancer binding protein (C/EBP) sites. By a cotransfection
analysis in a nonhepatic cell line, the combination of HNF-4 and C/EBP, both of which
are liver-specific TFs, normalized the expression in the liver of the recombinant OTC
gene.46 Also, two more elements were found in the promoter region to which HNF-4 and
chicken ovalbumin upstream promoter-transcription factor (COUP-TF) can bind to acti-
vate and suppress, respectively, the promoter activity of OTC gene.47 HNF-4 is selectively
expressed in the liver and intestine, whereas chicken ovalbumin upstream promoter-
transcription factor (COUP-TF) is ubiquitous.48 Therefore, the repression of OTC promoter
by the competitive binding of COUP-TF against HNF-4 may be involved in empty expres-
sion of OTC in nonhepatic tissues.
The crucial role of HNF-4a was demonstrated by introducing liver-specific destruction
of HNF-4a in mice.49 The liver-specific HNF-4a-null mice survived, although they devel-
oped mild hyperammonemia and low plasma urea. Western blot showed that HNF-4a
and OTC were almost lost in the liver, whereas CPS, ASS, ASL, and ARGase were normally
expressed. The residual OTC activity was 5% of the control. However, CPS and OTC
proteins in the intestine remained intact. Therefore, intestinal OTC and CPS might com-
pensate, at least partly, the role of the liver urea cycle. A dual role in ammonia detoxifi-
cation and citrulline synthesis by the small intestine will be an intriguing matter to be
elucidated.
Interplay between promoter and far upstream elements was also revealed with the
CPS gene.50 Transgenic mice were analyzed for the expression of the reporter gene: the
first transgene was a 1.8-kb fragment immediately upstream from the transcription start
site of a rat CPS gene fused to a reporter gene (CAT), the second one was a 12-kb fragment
instead of 1.8-kb one, and the third one was a minimized promoter and 469-bp enhancer
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142 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

sequence at 6.3 kb upstream. No CAT activity was found in the livers of the transgenic
mouse of the first group. In the second group, CAT activity was high in the liver and
moderate in the intestine; there was no activity in the spleen and kidney. mRNA of CAT
was distributed in the liver at a periportal location, as was mRNA of endogenous CPS.
The mRNA was also found in the epithelium of the small intestine, and there was more
density in the crypt region, which was the same for the endogenous CPS expression. The
developmental changes of the CAT mRNA in the liver and intestine of the second trans-
gene were the same as those of the endogenous CPS. Dexamethasone treatment of the
transgenic mouse resulted in a severalfold increase of CAT mRNA in the liver but not
intestine, reproducing exactly the expression of endogenous CPS. The third transgene was
expressed only in the periportal area. Therefore, the far upstream enhancer, 469 bp, at –6.3
kb must determine the periportal location of CPS in the liver. Within this enhancer
sequence several regulatory elements like CRE, GRE, HNF-3, and HNF-4, or elements
with homology to such, are identified. The 469-bp enhancer is crucial for the hormone-
dependent expression of CPS gene in hepatocyte and requires a GAG box (which contains
a direct repeat of the nonamer, GAGGAGGGG) in the promoter region.51
ASS and ASL genes have sequences in the promoter proximal region for Sp1 or NF-Y,
this explains the widespread expressions of the enzymes.48 An octameric element in the
distal promoter region of ASS is similar to that found in the albumin gene, and therefore,
it may promote expression of ASS in the liver.
Observations on regulations of gene expression at posttranscriptional levels are accu-
mulating. A study of villus-crypt axis distribution of mRNA and protein of CPS in the
developing intestine of the rat and human found mosaicism in cellular expression of CPS
protein under uniform expression of CPS mRNA.52 The cells with mRNA but without
protein suggest cell-specific posttranscriptional processing of mRNA. Diversity in the
5¢-untranslated region (UTR) of mRNA transcripts brought an important effect in the
tissue-specific expression of ASS protein in endothelial cells.53 A strict colocalization of
ASS and ASL in a segregated subcellular compartment of endothelial cells, plasmalemmal
caveolae, was demonstrated and construed for the presence of tight channeling of citrulline
and arginine for nitric oxide synthase.54
Recently, an intriguing question as to the consistency of mRNA abundance in tissues
with the activity of the product was presented for PCS. Full-length human cDNA encoding
PCS was cloned and found to encode a bifunctional protein for g-glutamyl kinase and
g-glutamyl phosphate reductase activities.55 The mature peptides for PCS activity exist in
two isoforms, long and short ones. The short isoform is shorter by only two amino acids
interposed in the kinase domain of the long one, but this minimum difference confers the
ability of regulation by ornithine on the short isoform. Northern blot showed that most
human tissues had a single predominant mRNA transcript of 3.6 kb. A consensus sequence
for the feedback inhibition by proline in plants was conserved, although no inhibition by
proline of mammalian enzyme has been reported. The possibility that the short isozyme
works in the intestine for citrulline synthesis while the long isozyme works in the periph-
ery for proline synthesis is attractive. Probably, cell populations expressing activity in each
tissue and specific activities of both isozymes would help to understand the difference
between mRNA abundance and the activity profile.56

9.2 Pioneering experiments led to fundamental findings


9.2.1 Nutritional studies with isotopes
Womack and Rose57 fed rats with basal amino acid diets lacking glutamic acid, proline,
and arginine, and the rats gained 49 g on average. When they supplied additional
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Chapter nine: The glutamate crossway 143

glutamate, proline, or arginine, rats gained 58, 60, and 78 g, respectively. In another
experiment, rats gained 91 g on an arginine-supplemented basal diet, whereas the addition
of proline and glutamic acid separately and in combination to this basal diet promoted
rats to gain 97, 102, and 106 g, respectively. They concluded that proline and arginine can
be formed from glutamate, and these are interconvertible. Scull and Rose58 fed rats with
arginine-depleted casein hydrolysate diet, and found that two to three times as much
arginine accumulated in the carcass as estimated from dietary arginine intake.
In 1951, the first direct evidence that arginine and proline are formed from glutamate
was obtained.59,60 [14C]glutamate administered to rats by peritoneal injection or orally was
subsequently found as [14C]arginine and [14C]proline in the protein hydrolysate of carcass.
A similar feeding experiment by Stetten and Schoenheimer61 with proline that was doubly
labeled with deuterium and 15N showed that the carbon skeleton of proline was found as
deuterium in the arginine fraction of the body protein. Not only the amidine group of
arginine but also the a- and d-amino groups of ornithine hydrolyzed from arginine con-
tained 15N. Oxidation to glutamate was shown by detecting deuterium and 15N in the
glutamate fraction. Ornithine skeleton was also labeled with deuterium. Stetten62 labeled
a- or d-amino nitrogen of ornithine with 15N, fed it to rats, and found that about 6.5% of
the tissue arginine was derived from the dietary ornithine. a-Amino group contributed
more efficiently to proline and to a lesser extent to glutamic acid. d-Amino group, in
contrast, contributed much to glutamic acid. These results suggest that the initial step in
the ornithine catabolism is the breakdown of ornithine by preferential loss of the d-amino
nitrogen leading to glutamic g-semialdehyde.62 Clutton et al.63 demonstrated by a similar
feeding experiment using deuterated ornithine that 7.5% of arginine in carcass protein
was derived from the deuterated ornithine. Deuterated ornithine was also used by Roloff
et al.64 and found to be converted to glutamic acid and proline in vivo.
These studies altogether proved that glutamate, proline, ornithine, and arginine are
fully interchangeable in living animals.

9.2.2 Perfusion studies of tissues


Windmueller and Spaeth65 elaborated a novel recirculation circuit for studying the small
intestine. Pooled heparinized blood collected from donor rats was pumped into the supe-
rior mesenteric artery after oxygenation. Blood return was collected from the superior
mesenteric vein under venous pressure surveillance. Lymph drains were simultaneously
collected. In early experiments, [14C]glutamine was infused by a separate arterial perfusion
pump, and the venous outflow was serially collected and analyzed to detect single-passage
metabolism. Later, they refined the procedure to detect an even segmental metabolism of
intestine where vascular or luminary perfusion was achieved.66 First of all, 20 to 30% of
plasma glutamine was constantly cleared off from the perfusate in each transit through
the intestines of rats, dogs, cats, hamsters, and monkeys, whereas no other amino acids
were taken up. Carbon of trapped glutamine was recovered as CO2 (57%), citrulline (6%),
proline (5%), alanine (1%), lactate (8%), and citrate (5%) in the venous collections. Besides,
small amounts of aspartate, ornithine, and glutamate were found. Nitrogen of glutamine
was quantitatively distributed in citrulline (34%), alanine (33%), ammonia (23%), and
proline (10%). When glutamine or glutamate was administered intraluminally, 92% of
glutamine administered in the jejunal segments was recovered in the venous output. Of
this glutamine, 37% remained intact; the rest was distributed among similar metabolites
found in the vascular administration, with a similar ratio. With glutamate, 81% was
recovered in the venous blood. However, 97% of recovered glutamate had been metabo-
lized during its translocation, suggesting that the rate-limiting step is GLNase. Thus,
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144 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

glutamine was metabolized similarly by the mucosal cells, regardless of its entrance,
through luminal front or basolateral front.
In other experiments, rat livers were perfused with blood supplemented with graded
levels of ammonia and 21 amino acids to generate maximally 18.8 mmol/min of urea,
which is almost equal to that observed in rats fed a 30% protein diet.67 However, citrulline
release was 36.2 nmol/min, only equivalent to 13% of the rate of citrulline release by the
intestine. Therefore, unless unphysiologically high ornithine and ammonia are loaded
simultaneously, excess citrulline will not accumulate and flood into venous output. Also,
when [14C]citrulline added to the perfusate was recirculated about 40 passes through the
liver, still more than 90% citrulline remained unmetabolized. The residual radioactivity
was in urea. No radioactive arginine was released.
The kidneys extracted about 35% of the citrulline from blood in each pass; this much
of citrulline uptake was equivalent to 83% the rate of intestinal citrulline release. The
kidney released 75% of the citrulline as arginine into the systemic circulation.67 Levillain
et al.68 demonstrated the site of arginine formation along with the nephron. Under stere-
omicroscopic observation, pieces of glomerulus, proximal convoluted tubule, and others
were collected and reacted with [14C]citrulline, arginase, and urease. Arginine production
was counted as [14C]CO2. Arginine synthesis coexisted with the proximal tubules, with
decreasing intensity from the proximal convoluted to the proximal straight tubules, from
122 to 41 fmol/min/mm length. Arginine synthesis increased linearly up to 0.4 mM
citrulline. The proximal straight tubules retained substantial arginase, and this hydrolyzed
40 to 64% arginine formed to urea and ornithine, whereas no hydrolysis occurred in the
proximal convoluted tubules. Therefore, arginine to be returned into the renal vein must
be fabricated in the proximal convoluted tubules. Arginine formed in proximal straight
tubules may contribute to urine concentration after its hydrolysis by arginase to urea.
Localization of ASS and ASL to proximal tubules by immunohistochemical staining of
mouse kidney sections was reported.22 Dhanakoti et al.,69 using similarly prepared kidney
cortical tubules, showed that aspartate that is the second substrate in arginine synthesis
from citrulline can be substituted with glutamate or glutamine just as effectively. Also,
arginine synthesis was proportional to the citrulline concentration in the medium up to
0.5 mM, particularly at around 0.06 mM, a physiological value. Moreover, citrulline infu-
sion from saphenous vein raised plasma citrulline from 62 to 242 mM, while renal uptake
of citrulline increased from 61 to 224 nmol/min/100 g of body weight (BW) and renal
release of arginine from 79 to 265 nmol/min/100 g of BW. A parallel rise of plasma arginine
from 175 to 244 mM was observed. Linear regression of the relationship of citrulline uptake
and arginine output revealed stoichiometric unity. Thus, the plasma citrulline concentra-
tion is the primary factor to determine the rate of arginine production by the kidneys.
Exchange of stable isotopes between citrulline and arginine in the plasma from can-
nulated dogs revealed that the kidney’s contribution for arginine synthesis was about
60%, and the remaining 40% was by extrarenal tissues.70 Also in this species, substantial
amounts of citrulline were derived from the liver as well as the small intestine.

9.3 Overall flow of metabolites and significance in human nutrition


9.3.1 Derivation from animal studies
The significance of the endogenous arginine synthesis in growth and ammonia disposal
has been illustrated by the species differences in the response of animals put on arginine-
depleted status. The typical case is represented by rats and cats, as omnivores and carni-
vores, respectively. Postweaning rats fed on an arginine-deficient diet for 8 days gained
11 g, whereas those on an arginine-replete diet gained 33 g. Citrulline completely replaced
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Chapter nine: The glutamate crossway 145

arginine (35 g), although ornithine failed (13 g).71 Urinary excretion of orotic acid, as a
measure of depleted intermediates of the liver urea cycle, was multiplied 50-fold by
arginine depletion, but was normalized by citrulline or ornithine supplementation. On
the contrary, cats fed on an arginine-depleted diet could not maintain weight and lost 48 g
daily; plasma arginine dropped to 25% of normal.72 However, citrulline supplementation
supported cat’s growth completely, whereas ornithine did not. Plasma arginine was nor-
malized by citrulline feeding but not ornithine.73 Furthermore, some of the cats fed an
arginine-deficient diet acutely developed hyperammonemia and its clinical symptoms. In
such cats, the liver concentration of ornithine was significantly low compared with cats
without hyperammonemia. This suggests that OTC of the hyperammonemic liver was
not sufficiently saturated with endogenous ornithine under the highly catabolic dietary
conditions with arginine exhaustion.74 In fact, PCS activity of total cat intestine is remark-
ably low compared with rats: 16 nmol/min/kg of BW to 308 nmol/min/kg of BW.75
Deprivation of the intestinal citrullinogenesis has been attempted by two different
studies. Hoogenraad et al.76 used d-N-(phosphonacetyl)-L-ornithine (PALO) linked to
glycylglycine to deliver PALO through the cell membrane of intestinal mucosa and to
selectively inhibit OTC. PALO is a transition state analogue and a potent and specific
inhibitor of OTC. When rats were fed on a 30% casein diet treated with arginase to replace
arginine with ornithine, rats gained 15 g for 6 days. However, when rats were given
glyglyPALO in drinking water, the rats lost 15.5 g during the same interval. Interestingly,
supplementation of 1% of either citrulline or arginine in the diet resumed their weight
gain, 9.4 and 5.7 g, respectively. Furthermore, rats fed on normal rat chow, which we may
certainly presume had a sufficient dietary intake of arginine, lost weight when they
received glyglyPALO. Serum arginine reduced to one fourth by glyglyPALO but normal-
ized by addition of 1% citrulline. Serum ammonia was not elevated in any experiment,
which assured that this inhibitor had no effect on liver OTC. These results clearly indicate
that citrulline synthesis by the small intestine has primary importance to suffice arginine
demand for growth, and this fraction will not be fully replaced by dietary arginine.
Another experiment confirmed the indispensability of the small intestine for in vivo
synthesis of arginine.77 Eighty percent of the small intestines of rats was removed, and
the rats were fed by one of four experimental diets: complete (Complete), arginine-free (-
Arg), proline-free (-Pro), and arginine- and proline-free diet (-Arg,Pro). After 4 weeks, rats
fed -Arg or -Arg,Pro lost weight by 28 to 32 g, whereas rats fed Complete gained 80 g. -
Pro resulted in a gain of 58 g. Nitrogen balance was negative in rats fed -Arg and -Arg,Pro.
Urinary orotic acid was high in -Arg and -Arg,Pro but not -Pro. Excretion of nitrate, an
end product of nitric oxide, decreased significantly in -Arg and -Arg,Pro. Concentration
of arginine in the muscle decreased to one third and one fifth in -Arg and -Arg,Pro,
respectively. However, proline was not much affected by any of the diets. Plasma
glutamine increased by twofold in -Arg and -Arg,Pro. These results demonstrate that rats
cannot synthesize arginine from glutamate without the small intestine. Nevertheless,
proline can be synthesized from arginine in such tissues as the liver and kidney even in
the absence of the intestine.

9.3.2 Implication from human clinical studies


Is the kidney an indispensable organ in the conversion of citrulline to arginine? Tizianello
et al.78 showed that citrulline uptake and arginine release by kidneys from patients with
normal renal function were 6.9 and 7.0 mmol/min/1.73 m2, respectively, and those from
patients with renal insufficiency were 2.9 and 2.7 mmol/min/1.73 m2, respectively. Despite
the marked drop in the arginine release, the arterial concentration of arginine was not
lowered in renal insufficiency.78 However, citrulline was raised from 28.7 to 68.6 mM.
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146 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 9.2 Hyperammonemia in a short-bowel patient under essential amino acid supplementation.
On day 0, the patient with thrombosis of superior mesenteric artery had a massive resection of the
small intestine, with a remaining 40 cm left. The patient was maintained by TPN (total parenteral
nutrition) with essential amino acids and glucose to supply 3 g of nitrogen and 2200 kcal daily. Mild
hyperammonemia was noted on day 4. Plasma citrulline, ornithine, and arginine were low, and
orotic acid was high. On day 8, arginine infusion, 6 g daily, was introduced and the plasma ammonia
was corrected to a normal level. Withdrawal of arginine caused a relapse of hyperammonemia.

Studies with uremic animal models in which the kidneys had been resected drastically
reported similar changes. This indicates that accumulated citrulline can be converted to
arginine in other competent tissues, as suggested in Table 9.1.
The effect of an arginine-free diet (-Arg) on humans has been investigated.79 Although
-Arg transiently reduced postprandial plasma arginine, neither hyperammonemia nor
orotic aciduria developed. Castillo et al.,80 using stable isotopes, quantitated de novo a rate
of arginine synthesis in the human body and compared it between arginine-replete and
arginine-free diets for 6 days. They found that the rate (16 mmol/kg/h) did not change in
response to arginine availability. However, plasma arginine and ornithine were low and
citrulline was high on feeding of an arginine-free diet, compared to an arginine-replete
one.80 Therefore, the human intestine appears more efficient than that of rats in making
citrulline and ornithine.
However, strong lines of evidence can be found among clinical reports that suggest
that the small intestine is essential in arginine synthesis in the human body. One is the
observation that a short-bowel (SB) patient who had hypocitrullinemia developed
hypoargininemia, hyperammonemia, and orotic acidemia when the patient was main-
tained on essential amino acid hyperalimentation that lacked in arginine.81–83 Arginine
supplementation relieved the biochemical changes (Figure 9.2). On the contrary, when SB
patients were maintained on an oral therapeutic diet, which contains arginine, tailored
for them, they showed almost normal values for plasma arginine, ornithine, and proline,
whereas glutamine was high and citrulline was low. Neither hyperammonemia nor orotic
aciduria was detected.84 Heird et al.85 reported hyperammonemia in three infants receiving
both essential and nonessential amino acids. However, these infants had jejunal atresia or
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Chapter nine: The glutamate crossway 147

gastroschisis, suggesting severe impairment of intestinal citrullinogenesis. Hyperammone-


mia disappeared by arginine supplementation.85
Patients of urea cycle enzyme deficiencies, CPS, OTC, ASS, or ASL, developed hyper-
glutaminemia and hyperammonemia accompanied by hypoargininemia immediately after
supplementation of arginine, with which their basal diets on low-protein content were
fortified, was discontinued.86 Concomitant decrease in plasma and urinary urea suggested
that dietary arginine was a major origin of urea production in those patients. Therefore,
they lacked not only CPS, OTC, ASS, or ASL of the liver urea cycle, but also corresponding
extrahepatic enzymes that make up the arginine synthetic route. Moreover, the fact that
plasma ornithine did not change or somewhat increased signifies the integrity of OAT
and PCS of the small intestine in those patients. In addition, when patients of CPS, OTC,
or ASS deficiencies received liver transplantation in order to cure hyperammonemic intox-
ication, hypocitrullinemia or hypercitrullinemia persisted, although plasma ammonia and
orotic aciduria were normalized.87 This observation further emphasizes that extrahepatic
CPS and OTC are essential for the citrulline synthesis as precursors of releasable arginine,
and ASS in utilization of citrulline to form argininosuccinate.
In normal humans, plasma arginine increases promptly after oral citrulline load.88 The
major portion of circulating ornithine is a product of arginine by ARGase, because of the
low plasma ornithine levels in ARGase deficiency89 and because arginine ingestion elevates
plasma ornithine almost linearly but does not affect citrulline.90 The systemic release of
citrulline from the intestine evidently determines the plasma levels of citrulline. Crenn
et al.91 surveyed over 57 SB patients to find any correlations among residual length of
intestine, plasma citrulline levels, digestive absorption of proteins and lipids, differences
in surgical procedures, and radiographic findings of the remnant intestines. They found91
complete correlations between citrulline levels, and the lengths and surface areas of resid-
ual intestines. They proposed a cutoff value of 20 mmol/l citrulline to judge between
permanent and transient inability of residual functional enterocytes.
The first patients of PCS deficiency carried a missense mutation in the g-glutamyl-
kinase domain and showed progressive neurodegeneration, joint laxity, skin hyperelastic-
ity, and cataracts with low plasma arginine, citrulline, ornithine, and proline, and para-
doxical hyperammonemia (postprandial alleviation).92 These metabolic derangements
appear mainly to reflect the role of intestinal PCS (the short isoform of PCS) to supply
P5C end products into circulation. However, the peripheral symptoms may suggest the
importance of the long isoform of PCS in specific cells to play a vital role, not known yet.
In the meantime, the importance of ATP availability in the intestinal mitochondria for the
synthesis of citrulline (as substrates for CPS and PCS) was suggested in a clinical record
reporting patients whose mitochondrial oligomycin-sensitive ATPase activity was mark-
edly reduced.93
P5C itself is an aminoaldehyde and a reactive compound. P5C interacts with pyridoxal
phosphate (PLP), a coenzyme form of vitamin B6, to form adducts at physiological tem-
perature and pH.94 This reaction leads to deactivation of PLP and may have clinical
significance in provoking so far unexplained seizures in hyperprolinemia II (PCDH defi-
ciency) patients. The authors also note that PLP forms adducts with physiological inter-
mediates such as pyruvate, oxaloacetate, and acetoacetate. A survey of direct chemical
interactions between intermediates of different enzymological pathways and their effects,
and elucidation of roles of a given enzyme on tissue basis (the global role) and cell basis
(the role in a cellular microenvironment) in a human metabolism will open a new research
area from the glutamate crossway.
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de-activation by pyrroline-5-carboxylic acid: increased risk of vitamin B6 deficiency and
seizures in hyperprolinemia type II, J. Biol. Chem., 276, 15107–15116, 2001.
95. De Almeida, A.F., Curi, R., Newsholme, P., and Newsholme, E.A., Maximal activities of key
enzymes of glutaminolysis, glycolysis, Krebs cycle and pentose-phosphate pathway of sev-
eral tissues in mature and aged rats, Int. J. Biochem., 21, 937–940, 1989.
96. Ardawi, M.S.M. and Newsholme, E.A., Intracellular localization and properties of phos-
phate-dependent glutaminase in rat mesenteric lymph nodes, Biochem. J., 217, 289–296, 1984.
97. Chader, G. J., Hormonal effects on the neural retina. I. Glutamine synthetase development
in the retina and liver of the normal and triiodothyronine-treated rat, Arch. Biochem. Biophys.,
144, 657–662, 1971.
98. Jones, M.E., Anderson, A.D., Anderson, C., and Hodes, S., Citrulline synthesis in rat tissues,
Arch. Biochem. Biophys., 95, 499–507, 1961.
99. Ratner, S., A radiochemical assay for argininosuccinate synthetase with [U-14C]aspartate,
Anal. Biochem., 135, 479–488, 1983.
100. Ratner, S. and Murakami-Murofushi, K., A new radiochemical assay for argininosuccinase
with purified [14C]argininosuccinate, Anal. Biochem., 106, 134–147, 1980.
101. Wakabayashi, Y., Iwashima, A., Yamada, E., and Yamada, R., Enzymological evidence for the
indispensability of small intestine in the synthesis of arginine from glutamate. II. N-acetyl-
glutamate synthase, Arch. Biochem. Biophys., 291, 9–14, 1991.
102. Herzfeld, A., Mezl, V.A., and Knox, W.E., Enzymes metabolizing d1-pyrroline-5-carboxylate
in rat tissues, Biochem. J., 166, 95–103, 1977.
103. Herzfeld, A. and Raper, S.M., The heterogeneity of arginases in rat tissues, Biochem. J., 153,
469–478, 1976.
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chapter ten

Arginine metabolism in mammals


Guoyao Wu
Texas A&M University
Sidney M. Morris, Jr.
University of Pittsburgh School of Medicine

Contents
Introduction..................................................................................................................................153
10.1 Arginine synthesis .............................................................................................................155
10.1.1 Arginine as a semiessential amino acid for most mammals.........................155
10.1.2 The intestinal-renal axis for endogenous arginine synthesis ........................155
10.1.3 The citrulline–NO cycle .......................................................................................156
10.2 Arginine catabolism ..........................................................................................................156
10.2.1 Catabolism via arginases .....................................................................................157
10.2.2 Catabolism via arginine:glycine amidinotransferase......................................158
10.2.3 Catabolism via NOS .............................................................................................159
10.2.4 Catabolism via arginine decarboxylase.............................................................159
10.3 Metabolism of methylarginines.......................................................................................160
10.4 Concluding remarks and perspectives ..........................................................................160
Acknowledgments ......................................................................................................................161
References .....................................................................................................................................161

Introduction
L-Arginine (2-amino-5-guanidinovaleric acid) is an amino acid with remarkable metabolic
and regulatory versatility. In 1988, it was identified as the physiological precursor for the
synthesis of nitric oxide (NO) in animal cells (Table 10.1).1,2 The discovery of NO synthesis
has stimulated an enormous interest in arginine metabolism over the past decade. Thus,
much effort has been directed to explore nutritional or therapeutic roles of arginine to
treat many human diseases that are associated with a relative or absolute deficiency of
arginine or with a reduced bioavailability of NO3–5 (see Chapter 35). However, it should
be borne in mind that other aspects of arginine metabolism in addition to NO synthesis
play very important physiological roles. These include the synthesis of arginine itself, as
well as the catabolism of arginine to produce compounds such as proline, polyamines
(putrescine, spermidine, and spermine), creatine, agmatine, and glutamate (Figure 10.1).

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 153
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154 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 10.1 Enzymes Involved in the Synthesis and Catabolism of Arginine, Ornithine,
and Citrulline
Amino Acid Produced by: Used as Substrate by:
Arginine Argininosuccinate lyase Arginase
NOS
Arginine:glycine
amidinotransferase
Arginine decarboxylase
Ornithine Arginase Ornithine decarboxylase
Ornithine aminotransferase Ornithine aminotransferase
Arginine:glycine Ornithine carbamyltransferase
amidinotransferase
Citrulline Ornithine carbamyltransferase Argininosuccinate synthetase
NOS
Dimethylarginine
dimethylaminohydrolase

Note: Because the reaction catalyzed by ornithine aminotransferase can be reversible under physiologic
conditions, ornithine may be either a substrate or product of this reaction, depending on the specific
metabolic conditions.

Arginine

fumarate fumarate
aspartate aspartate

Citrulline-NO Urea Cycle


Cycle
-
NH3 + HCO3
1
NO + Citrulline Arginine Ornithine
2 4
glycine
urea
3 Proline
CO2
ornithine
7
Creatine Agmatine Ornithine P5C

5 6
Glutamate
urea CO2

Putrescine Polyamines

Figure 10.1 Overview of mammalian arginine metabolism. Not all enzymes depicted are expressed
in all cell types. For the sake of clarity, not all reactants or cofactors are shown. 1, NOS; 2, argin-
ine:glycine amidinotransferase; 3, arginine decarboxylase; 4, arginase; 5, agmatinase; 6, ornithine
decarboxylase; 7, ornithine aminotransferase; 8, arginine transporter; P5C, D1-pyrroline-5-carboxy-
late. The dashed line indicates that recycling of citrulline is variable and not quantitative.
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Chapter ten: Arginine metabolism in mammals 155

Therefore, the impact of arginine metabolism extends to virtually every cellular and organ
function in the body. The major objective of this chapter is to present an overview of
mammalian arginine metabolism and the biochemical basis for the clinical applications
of arginine. For more detailed coverage of arginine metabolism and its regulation, refer
to recent reviews.6–10

10.1 Arginine synthesis


10.1.1 Arginine as a semiessential amino acid for most mammals
Owing to its endogenous synthesis in most mammals (including humans, rats, and pigs),
arginine is classified as a nutritionally semiessential amino acid, depending on develop-
mental stage and metabolic state.3 On the basis of nitrogen balance, arginine is considered
as a nonessential amino acid for healthy adult mammals7 (see Chapter 27), with the
exception of strict carnivores.11 However, endogenous arginine synthesis may not be
sufficient to meet metabolic needs in growing infants3 and other neonates (e.g., piglets12
and young rats13), in adults under highly catabolic conditions (e.g., sepsis, burn injury,
and trauma),14,15 and in adults with massive small bowel resection16 (see Chapter 9). In
these conditions, arginine supplementation can become essential for health (see Chapter
35). Moreover, following liver transplantation, hepatic arginase is released into the circu-
lation and can result in a large decline in plasma arginine levels.17–20 This condition also
may require arginine supplementation for optimal recovery.

10.1.2 The intestinal-renal axis for endogenous arginine synthesis


Although arginine is formed in the liver via the urea cycle, there is no net synthesis of
arginine by this organ. Once formed, arginine is immediately hydrolyzed to produce
urea and ornithine. The latter is recycled in the urea cycle as the carrier molecule that
collects waste nitrogen atoms and bicarbonate (see Chapter 7). Instead, the majority of
whole-body arginine synthesis in adults is performed in a metabolic collaboration by
the small intestine and kidneys in what has been termed the intestinal-renal axis.7
Studies over the past 25 years have established that absorptive epithelial cells of the
small intestine (enterocytes) in mammals play the major role in whole-body synthesis of
citrulline, the precursor of arginine.7 Glutamine, glutamate, and proline in enteral diet, as
well as glutamine in arterial blood, are substrates for the intestinal synthesis of citrulline.21
Intestinal catabolism of glutamine via phosphate-dependent glutaminase, pyrroline-5-
carboxylate (P5C) synthase, and ornithine aminotransferase (OAT) yields ornithine, which
is converted to citrulline by carbamylphosphate synthase I and ornithine carbamyltrans-
ferase.7 All of these enzymes are located in mitochondria of enterocytes. Proline oxidase,
another mitochondrial enzyme, oxidizes proline to form P5C, which is converted into
citrulline via OAT, carbamylphosphate synthase I, and ornithine carbamyltransferase.7
The intestine-derived citrulline is released into the circulation and taken up by extrahepatic
tissues (primarily kidneys) for arginine synthesis. (Because it synthesizes N-acetyl-
glutamate, an essential allosteric activator of carbamylphosphate synthase-I,7 the activity
of N-acetylglutamate synthase may regulate the rate of intestinal synthesis of citrulline
and arginine. However, experiments are needed to fully test this hypothesis.)
Declines in plasma concentrations of citrulline have been used as a marker for reduced
small intestinal mass, intestinal injury, and rejection of small bowel transplants.22–24
Changes in plasma citrulline concentrations also can be reflective of changes in intestinal
citrulline synthesis as a function of diet or development.12,25 The crucial role of the small
intestine in endogenous arginine synthesis is dramatically illustrated by the arginine
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156 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

deficiencies, which can result from an inherited deficiency of P5C synthase in humans,26,27
the naturally very low activity of P5C synthase in the intestine of cats,28 a limited supply
of enteral proline in neonatal pigs,29 or resection of the small bowel.16 Dietary arginine is
essential in these circumstances.
With regard to arginine synthesis, enterocytes of neonates differ from those of adults
in two respects. First, unlike the case in adults, most of the citrulline synthesized in
enterocytes of neonates is converted locally to arginine because of relatively high argini-
nosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) activities, coupled with
relatively little intestinal arginase activity.30,31 As ASS expression declines and arginase
expression increases in enterocytes around the time of weaning, enterocytes release most
of the synthesized citrulline rather than converting it into arginine.32,33 Second, catabolism
of proline in neonatal enterocytes represents an important source of P5C for ornithine and
citrulline synthesis.34 Endogenous synthesis of arginine is of enormous nutritional and
physiological importance because arginine is remarkably deficient in the milk of most
mammals, including humans, cows, pigs, and rats,35,36 and because arginine requirements
by young mammals are particularly high.37 For example, we have estimated that endog-
enous synthesis of arginine must provide at least 60% of arginine for the 7-day-old sucking
piglet.3 Consistent with this notion is the finding that an inhibition of intestinal citrulline
synthesis for 12 h decreases plasma concentrations of citrulline and arginine by 52 and
76%, respectively, in neonatal pigs.25
In both neonatal and adult mammals, citrulline released by the small intestine is not
taken up by the liver but is utilized for arginine synthesis by ASS and ASL in extrahepatic
tissues, with the proximal tubules of the kidneys being the major site.7,38 In healthy adult
rats, rates of renal arginine synthesis are determined by the availability of citrulline rather
than by renal capacity for arginine synthesis.39 Because the uptake of arginine by the liver
also is low,40 intestinally derived citrulline or arginine is equally effective as a source of
arginine for the whole body.

10.1.3 The citrulline–NO cycle


Arginine also is formed from citrulline in a pathway known as the citrulline–NO cycle41,42
or the arginine–citrulline cycle43,44 (Figure 10.1). This pathway, which is comprised of the
enzymes ASS and ASL, does not represent de novo synthesis of arginine but is instead a
recycling pathway whereby citrulline produced by NO synthase (NOS) is converted back
into arginine. ASS and ASL are present at low levels in virtually all nonhepatic cells and
are induced when inducible NOS (iNOS) is expressed.41,42 However, the efficiency of this
recycling pathway is by no means quanititative, as evidenced by the net production of
citrulline by NO-producing cells.7 The quantitative contribution of this pathway to argi-
nine production in vivo is unknown.

10.2 Arginine catabolism


Arginine transport by the plasma membrane is the first step of arginine utilization by
animal cells. The most important mechanism for arginine uptake by many cell types is
system y+, a high-affinity, Na+-independent transporter of the basic amino acids — argi-
nine, lysine, and ornithine.40 However, other transporters, such as b0,+, B0,+, and y+L, also
transport arginine (see Chapter 4), and cells may express more than one type of arginine
transporter. Arginine transport can be highly regulated by a variety of stimuli.40,45 Once
inside the cell, arginine can serve as a substrate for only four enzymes in mammals in
addition to arginyl-tRNA synthetase: the NOS isozymes, the arginase isozymes,
arginine:glycine amidinotransferase, and arginine decarboxylase (Figure 10.1). Owing to
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Chapter ten: Arginine metabolism in mammals 157

ARGININE

6
2 3
1 ARGININE X Ornithine Putrescine Polyamines

urea CO2 P5C Proline


[NG-OH-Ar gini ne]

ORNT1 ORNT1 ?
NO +
Citrulline 4 5
ARGININE X Ornithine P5C Glutamate

αKG Glu
Cytosol urea Mitochondrion

Figure 10.2 Metabolic roles and subcellular localization of the arginases and related enzymes. Not
all enzymes depicted are expressed in all cell types. For purposes of clarity, not all reactants or
cofactors are shown. 1, NOS; 2, arginase I; 3, ornithine decarboxylase; 4, arginase II; 5, ornithine
aminotransferase; 6, arginine transporter; ORNT1, mitochondrial transporter of ornithine/citrulline
and of arginine; P5C, D1-pyrroline-5-carboxylate. NG-OH-arginine, an intermediate in NO synthesis,
is an endogenous inhibitor of the arginases. (Modified from Figure 10.5 of Morris, S.M., Jr., Regu-
lation of arginine availability and its impact on NO synthesis, in Nitric Oxide: Biology and Pathobiology,
Ignarro, L.J., Ed., Academic Press, San Diego, 2000, pp. 187–197. Reprinted by permission of Aca-
demic Press.)

their impact on multiple cellular processes, changes in activity or expression of the NOS
and arginase isozymes probably represent the most important regulated changes in argi-
nine metabolism (Figure 10.2).
Changes in plasma concentrations of arginine, ornithine, or citrulline are often used
as markers for activities of specific arginine metabolic enzymes.17–27 However, it is not
always appreciated that each of these amino acids can be produced or catabolized by
multiple enzymes. The enzymes involved in metabolism of these key amino acids are
therefore listed in Table 10.1 as an aid to investigators in this field.

10.2.1 Catabolism via arginases


There are two distinct isozymes of mammalian arginase (types I and II), which are encoded
by separate genes and differ in molecular and immunological properties, tissue distribu-
tion, subcellular localization, and regulation of expression.46 Both isozymes catalyze the
hydrolysis of arginine to urea and ornithine. In healthy adults, type I arginase, a cytosolic
enzyme, is highly expressed in periportal hepatocytes as a component of the urea cycle
and, to a limited extent, in a few other tissues and cells, such as brain,47 small intestine,48
and red blood cells (only in primates).49 However, a variety of cytokines and other inflam-
matory stimuli can induce expression of type I arginase in many cell types.41,50 Type II
arginase, a mitochondrial enzyme, is expressed at modest to low levels in many extra-
hepatic tissues. In healthy adults, the highest expression of type II arginase is found in
the kidney, prostate, testis, small intestine, and lactating mammary gland,51–53 but its
expression also can be induced by inflammatory stimuli such as bacterial lipopolysaccha-
ride and by cyclic AMP.51,54,55 Type II arginase appears to play a significant role in arginine
catabolism and homeostasis, as plasma concentrations of arginine are doubled in mice
deficient in this enzyme.56 These results indicate that rates of endogenous arginine
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158 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

synthesis do not decrease sufficiently, if at all, to compensate for the reduced arginase
activity to restore normal plasma levels of arginine. This notion is thus consistent with
the finding that arginine catabolism is more important than arginine synthesis in main-
taining arginine homeostasis in adults.57 Similarly, overexpression of arginase I in the small
intestine of transgenic mice resulted in markedly reduced plasma arginine concentra-
tions,58,59 again demonstrating that endogenous arginine synthesis cannot compensate for
large changes in arginine catabolism. These transgenic mice also exhibited decreased
growth of the suckling neonate and impaired development of skeletal muscle and lym-
phoid organs.58,59
The different subcellular localization of the arginase isozymes may provide a mech-
anism for regulating the subsequent metabolic fate of ornithine (Figure 10.2). Ornithine
can be utilized for the formation of proline, polyamines, glutamate, and glutamine.7
Formation of these products from arginine is of great nutritional and physiological impor-
tance for diverse functions such as lactation, growth, development, issue remodeling, and
responses to a wide range of hormones and signaling molecules.42,60–63 Much of the orni-
thine produced by arginase is converted to P5C by OAT, as indicated by the occurrence
of hyperornithinemia in adult mammals (e.g., humans and mice) when OAT is defi-
cient.64,65 Owing to the production of ornithine, arginase can be limiting for polyamine
synthesis in several cell types, including vascular smooth muscle cells,66,67 endothelial
cells,68,69 and activated macrophages.70 Arginase is present in calf serum70,71 and thus may
generate sufficient ornithine to support the proliferation of cultured cells that lack this
enzyme. Conversely, elevated arginase activity in some tumor cells may contribute to their
high proliferation rate.72 Remarkably, arginase-dependent production of polyamines also
may be essential for regeneration of injured neurons.73
The arginases can inhibit both inducible74–76 and constitutive68,77–79 NO synthesis in
mammalian cells by regulating the availability of the common substrate arginine. Inter-
estingly, recent work has indicated that high-level expression of arginase I in astrocytes
can apparently deplete arginine to such an extent that subsequent induction of iNOS
expression also is inhibited (R.R. Ratan, personal communication). It remains to be deter-
mined whether this observation will hold for other cell types. Depletion of arginine by
arginase can also have other consequences. For example, elevated arginase activity in
activated macrophages can deplete extracellular arginine sufficiently to result in reduced
expression of the zeta chain of the T cell receptor in cocultured T cells (A. Ochoa, personal
communication). This may contribute to immune cell dysfunction in patients with arginine
deficiency. In some cases, increased expression of the arginases in macrophages may be
beneficial to invading pathogens via effects on the synthesis of NO or polyamines.80–84
The precise roles of the individual arginase isozymes in most cell types in vivo remain
unknown, but investigations of this exciting area are increasing. The recent development
of genetic knockout mouse models of arginase deficiencies56,85 will facilitate efforts to
define the physiological roles of these important enzymes.

10.2.2 Catabolism via arginine:glycine amidinotransferase


Creatine synthesis represents another major use of arginine.7 This pathway is initiated by
arginine:glycine amidinotransferase, which transfers the guanidino group from arginine
to glycine to form guanidinoacetate and ornithine. This enzyme is present predominantly
in the renal tubules, pancreas, and, to a much lesser extent, liver and other organs. The
kidneys are the principal site of guanidinoacetate formation.86 Guanidinoacetate is methyl-
ated by guanidinoacetate N-methyltransferase, which is located primarily in the liver,
pancreas, and, to a much lesser extent, kidneys to form creatine. Circulating creatine is
actively taken up by skeletal muscle and nerve where it is phosphorylated and eventually
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Chapter ten: Arginine metabolism in mammals 159

undergoes nonenzymatic, irreversible dehydration to yield creatinine. The latter is


excreted by the kidneys. Thus, creatine homeostasis primarily involves the kidney, liver,
and skeletal muscle. Approximately 2.3 g of arginine per day is required to maintain
creatine homeostasis in adult humans.7
The methylation of guanidinoacetate to form creatine consumes more methyl groups
than all other methylation reactions combined.87 Thus, creatine synthesis from arginine
plays an important role in regulating the availability of the methyl group donor for other
methylation reactions, such as the synthesis of methionine from homocysteine, an inde-
pendent risk factor for cardiovascular disease.88 The importance of creatine biosynthesis
is graphically demonstrated by the finding that a deficiency of guanidinoacetate N-methyl-
transferase in humans causes a severe creatine deficiency and developmental abnormali-
ties in muscle and brain during early infancy.89 Furthermore, an arginine deficiency in
mice decreases creatine synthesis and results in abnormal neuromotor behavior.58

10.2.3 Catabolism via NOS


As this topic is covered in detail in Chapter 14, only a few points will be noted here. NO
is formed from L-arginine by three NOS isoforms: nNOS or NOS1 (originally identified
as constitutive in neuronal tissue), iNOS or NOS2 (originally identified as being inducible
by cytokines in activated macrophages and liver), and eNOS or NOS3 (originally identified
as constitutive in vascular endothelial cells).90 A quantitatively small amount of NO is
produced by nNOS or eNOS in animal cells,30,68,91–93 whereas much larger amounts of NO
are generated by iNOS in almost all cell types following its induction by inflammatory
cytokines and lipopolysaccharide.70,91,94–96
Because arginine is the only physiologic nitrogen-containing substrate for NOS, pro-
cesses that regulate arginine availability can play an important role in regulating NO
synthesis.42 In fact, a major conundrum in this field is known as the arginine paradox.97
This is based on the observation that cellular rates of NO synthesis are a function of
extracellular arginine concentration, despite the fact that intracellular levels of arginine
should be sufficient to fully saturate the NOS enzymes, based on their Km values for
arginine.98 Thus, recent studies have shown that arginine transporters91,95 and the arginases
(see Section 10.2.1) can play important roles in regulating NO synthesis. Because deficien-
cies or excesses of NO production can lead to numerous cellular and organ dysfunctions,99
elucidating the roles that dietary arginine and arginine metabolic enzymes play in regulat-
ing NO synthesis is important for developing strategies to maintain health and treat disease.
NOS activity also can have an impact on arginase activity via production of
NG-hydroxy-L-arginine (Figure 10.2), a potent endogenous inhibitor of the arginases.100,101
NG-hydroxy-L-arginine, a relatively stable intermediate in NO synthesis, can be released
from NOS and accumulate in sufficient concentration to inhibit arginase activity in cul-
tured cells.96,102 However, the impact of this compound on arginase activity in vivo remains
to be determined.

10.2.4 Catabolism via arginine decarboxylase


Arginine decarboxylase, a mitochondrial enzyme that produces agmatine, is the most
recent addition to the family of mammalian arginine metabolic enzymes. Relatively little
is known about this enzyme, which has not been purified or cloned from any vertebrate
species. However, arginine decarboxylase activity has been reported for rat aorta, brain,
liver, kidney, stomach, and intestine.103 Agmatine is of considerable interest because of its
effects as a cell signaling molecule.104–106 However, the physiologic roles of endogenously
synthesized agmatine have yet to be established.
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160 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Agmatine is hydrolyzed by agmatinase, also a mitochondrial enzyme,107 to produce


urea and putrescine, thus representing an alternate pathway for polyamine synthesis in
animal cells (Figure 10.1). The first cDNAs for vertebrate agmatinase were recently
cloned,108,109 allowing investigators to determine that agmatinase is expressed to varying
degrees in several human cell types, with highest expression in liver and kidney.108,109 Its
expression in human hepatocytes can be induced by hepatitis B virus,108 possibly suggest-
ing a role for agmatinase in the pathogenesis of viral hepatitis.

10.3 Metabolism of methylarginines


Because the methylarginines NG-monomethyl-L-arginine (NMMA) and NGNG-dimethyl-
L-arginine (asymmetrical dimethylarginine (ADMA)) are competitive inhibitors of all NOS
isozymes, there has been growing interest in mammalian metabolism of this family of
arginine derivatives.5,109–111 Free arginine is not a substrate for methylation; arginine resi-
dues in proteins are methylated by a family of protein arginine N-methyltransferases
(PRMT)112 to form NMMA, ADMA, or NGNNG-dimethyl-L-arginine (symmetrical dimethyl-
arginine (SDMA)). Methylation of arginine residues in proteins appears to play roles in
regulating intra- or intermolecular interactions of proteins, protein sorting, gene transcrip-
tion, and cell signaling.112 All PRMT identified to date can monomethylate arginine resi-
dues in proteins. However, further methylation of NMMA to form ADMA and SDMA is
catalyzed by type I PRMT and type II PRMT, respectively. All of the arginine methylation
reactions involve the modification of guanidino nitrogen atoms and require S-adenosyl-
methionine. When intracellular proteins are degraded by proteases and peptidases, free
methylarginines (NMMA, ADMA, and SDMA) are released.
Free NMMA and ADMA produced in the body are hydrolyzed by dimethylarginine
dimethylaminohydrolase (DDAH) to form citrulline and methylamines.113 Two DDAH
isozymes are expressed, and DDAH activity is widespread in mammalian tissues and
cells, including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, and
endothelial cells.114 Interestingly, the crystal structure of a Pseudomonas DDAH revealed
similarities to arginine:glycine amidinotransferase and arginine deiminase.115 Homo-
cysteine recently was shown to inhibit DDAH in a cell-free system, suggesting that this
may represent a mechanism whereby hyperhomocysteinemia results in elevated plasma
levels of ADMA, which in turn inhibits NO production in the cardiovascular system.116
Concentrations of free NMMA, ADMA, and SDMA in plasma are low in healthy
subjects (0.5–1 mM)117,118 but can be elevated in patients with various cardiovascular and
other disorders, such as diabetes, renal failure, hypercholesterolemia, atherosclerosis,
schizophrenia, and multiple sclerosis,110,111 suggesting a role for dimethylarginines in these
diseases. Interestingly, endogenous NMMA and ADMA have recently been shown to
inhibit neuronal NO production and prevent NO-mediated excitotoxic injury in cultured
neurons.119 Although SDMA itself is not an inhibitor of NOS, it is a competitive inhibitor
of arginine transport by cells, as are the other methylarginines.40 Therefore, elevated
concentrations of SDMA under certain pathological conditions may indirectly lead to
reduced NO synthesis.

10.4 Concluding remarks and perspectives


Recent studies have shown that arginine displays remarkable metabolic and regulatory
versatility in mammals. Therefore, the beneficial or deleterious effects of arginine
metabolism critically depend on the relative activities of specific arginine-catabolizing
enzymes; thus, precise regulation of these enzymes in vivo has important implications for
health and disease. Much of the information about the role of arginases in regulating NO,
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Chapter ten: Arginine metabolism in mammals 161

polyamine, proline, and glutamate synthesis have been generated from in vitro studies
and should be verified in vivo. In addition, the recent cloning of cDNA for mouse N-acetyl-
glutamate synthase120 will provide a powerful tool to define the regulatory role of this
enzyme in intestinal citrulline synthesis. Furthermore, future studies to fully explain the
arginine paradox97 will not only provide new knowledge about the regulation of mam-
malian NO synthesis but will also have important implications for the clinical application
of arginine.4 Finally, arginine and citrulline were recently found to be unusually abundant
in porcine allantoic fluid (e.g., 4 to 6 mM arginine on day 40 of gestation)121,122 and in
ovine allantoic fluid (e.g., 9.7 mM citrulline on day 60 of gestation)119; thus, the roles of
arginine or citrulline in conceptus development should be elucidated. We predict that
arginine or citrulline will provide an effective nutritional or pharmacotherapeutic treat-
ment for many human diseases. We also anticipate that exciting new roles for arginine
in regulating cell and tissue function in health and disease will be discovered in the
coming years.

Acknowledgments
Work in our laboratories is supported, in part, by grants from the U.S. Department of
Agriculture, American Heart Association, and Juvenile Diabetes Research Foundation; by
a Hatch project from the Texas Agricultural Experiment Station; by a Texas A&M Univer-
sity Faculty Fellowship (to G.W.); and by grants from the National Institutes of Health (to
S.M.M.). We thank Frances Mutscher for office support.

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macrophage effector molecule, Biochem. Biophys. Res. Commun., 157, 87–94, 1988.
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development, health and disease, Curr. Opin. Clin. Nutr. Metab. Care, 3, 59–66, 2000.
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Chapter ten: Arginine metabolism in mammals 167

114. Leiper, J.M., Maria, J.S., Chubb, A., MacAllister, R.J., Charles, I.G., Whitley, G.S.J., and
Vallance P., Identification of two human dimethylarginine dimethylaminohydrolases with
distinct tissue distributions and homology with microbial arginine deiminases, Biochem. J.,
343, 209–214, 1999.
115. Murray-Rust, J., Leiper, J., McAlister, M., Phelan, J., Tilley, S., Santa Maria, J., Vallance, P.,
and McDonald, N., Structural insights into the hydrolysis of cellular nitric oxide synthase
inhibitors by dimethylarginine dimethylaminohydrolase, Nat. Struct. Biol., 8, 679–683, 2001.
116. Stuhlinger, M.C., Tsao, P.S., Her, J.H., Kimoto, M., Balint, R.F., and Cooke, J.P., Homocysteine
impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine, Circulation,
104, 2569–2575, 2001.
117. Teerlink, T., Nijveldt, R.J., de Jong, S., and van Leeuwen, P.A.M., Determination of arginine,
asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other
biological samples by high-performance liquid chromatography, Anal. Biochem., 303, 131–137,
2002.
118. Vishwanathan, K., Tackett, R.L., Stewart, J.T., and Bartlett, M.G., Determination of arginine
and methylated arginines in human plasma by liquid chromatography-tandem mass spec-
trometry, J. Chromatogr. B, 748, 157–166, 2000.
119. Cardounel, A.J. and Zweier, J.L., Endogenous methyl-arginines regulate neuronal nitric oxide
synthase and prevent excitotoxic injury, J. Biol. Chem., 277, 33995–34002, 2002.
120. Caldovic, L., Morizono, H., Yu, X.L., Thompson, M., Shi, D.S., Gallegos, R., et al., Identifica-
tion, cloning and expression of the mouse N-acetylglutamate synthase gene, Biochem. J., 364,
825–831, 2002.
121. Wu, G., Bazer, F.W., Tuo, W., and Flynn, S.P., Unusual abundance of arginine and ornithine
in porcine allantoic fluid, Biol. Reprod., 54, 1261–1265, 1996.
122. Wu, G., Pond, W.G., Ott, T., and Bazer, F.W., Maternal dietary protein deficiency decreases
amino acid concentrations in fetal plasma and allantoic fluid of pigs, J. Nutr., 128, 894–902,
1998.
123. Kwon, H., Wu, G., Bazer, F.W., and Spencer T.E., Developmental changes of amino acids in
ovine uterine and fetal fluids during pregnancy, Biol. Reprod., 66 (Suppl. 1), 186–187, 2002.
1382_C10.fm Page 168 Tuesday, October 7, 2003 6:27 PM
1382_C11.fm Page 169 Tuesday, October 7, 2003 6:29 PM

chapter eleven

Glutamine metabolism
Rudolf Oehler
University of Vienna
Erich Roth
University of Vienna

Contents
Introduction..................................................................................................................................169
11.1 Biochemical reactions and their regulation...................................................................170
11.1.1 Gln synthesis .........................................................................................................170
11.1.1.1 Glutamine synthetase reaction ............................................................171
11.1.1.2 Gln transport through cell membranes..............................................171
11.1.1.3 Gln is a carrier of nitrogen and carbon..............................................172
11.1.2 Gln breakdown......................................................................................................173
11.1.2.1 Glutaminase reaction.............................................................................173
11.1.2.2 Gln utilization.........................................................................................173
11.1.2.3 Gln is a precursor of a variety of molecules .....................................174
11.1.3 Gln interorgan exchanges....................................................................................174
11.2 Effects of Gln starvation ...................................................................................................175
11.2.1 Energy metabolism ...............................................................................................176
11.2.2 Protein synthesis rate ...........................................................................................177
11.2.3 Cell protective mechanisms ................................................................................177
11.2.4 Apoptosis ...............................................................................................................178
11.2.5 Osmosignaling.......................................................................................................179
11.2.6 Immune response..................................................................................................179
Acknowledgments ......................................................................................................................180
References .....................................................................................................................................180

Introduction
The pioneer of biochemical research, Sir Hans Krebs, once stated that “most amino acids
have multiple functions, but glutamine appears to be the most versatile.” The present
chapter describes the special biochemical properties of glutamine (Gln), which are respon-
sible for the versatility of this amino acid. It explains how Gln is involved in intracellular
metabolism and in interorgan nitrogen transport. It delineates how Gln is used as energetic

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 169
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170 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

fuel by several cell types, which implies that these cells suffer from Gln starvation during
catabolic states. In addition, the chapter summarizes recent findings on the effect of Gln
starvation on function, stress response, and apoptosis of Gln-utilizing cells and gives an
overview of the current knowledge on the molecular mechanisms of Gln sensing.

11.1 Biochemical reactions and their regulation


11.1.1 Gln synthesis
Gln is a polar amino acid that is uncharged at pH 7.0. It contains five C-atoms and bears
a 5-carboxamide in its R-group, which can be readily deaminated for nitrogen release (see
Figure 11.1). The C5 structure makes Gln compatible with the C5-dicarboxylic acids of the
tricarboxylic acid (TCA) cycle and their metabolites. Therefore, Gln plays a central role in
nitrogen as well as carbohydrate metabolism.

alanine
pyruvate
lactate purines
GSH pyrimidines
GABA glucosamines

NH4+
ATP GLN- ADP+Pi
AA αKA synthetase
TCA- TA
cycle αKG Glu Gln
NH4+ Glutaminase H2O

NADH GLU-DH NAD+


NH4+ CO2
ATP
CO2 + H2O
ATP urea-
cycle

expressed in some tissues


expressed in all tissues

urea

Figure 11.1 Metabolic functions of Gln. Gln is formed by the combination of the TCA cycle inter-
mediate a-ketoglutarate (a-KG) with two nitrogens in a two-step reaction. In the first reaction a-
KG is converted to glutamate (Glu) by the activity of transaminases (TA). In this reaction the a-
amino group is enzymatically transferred from an amino acid (AA) to the a-carbon atom of a-KG,
resulting in the corresponding a-keto acid analog of the incoming amino acid and Glu. Glu is then
transformed into Gln by the combination with an ammonium ion catalyzed by the ubiquitous
expressed energy-dependent glutamine synthetase (GS). In Gln catabolism, Gln is converted to Glu
through hydrolysis catalyzed by the enzyme glutaminase, which is expressed only in Gln-utilizing
cells. Glu can then be transformed to a-KG by either transamination or glutamate dehydrogenase
(GLU-DH). The free ammonia resulting from the glutaminase reaction and from the GLU-DH
reaction is then either introduced into the urea cycle (in liver) or released from the tissue (in kidney).
As a-KG, the carbon backbone of Gln enters the TCA cycle. Once in the TCA cycle, the Gln-derived
carbons can either be oxidized to CO2 and water resulting in ATP formation or be introduced in the
synthesis of lactate, pyruvate, or alanine. Gln is also used in the synthesis of purines, pyrimidines
and glucosamines. Glu is a substrate for synthesis glutathione (GSH) and g-aminobutyrate (GABA).
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Chapter eleven: Glutamine metabolism 171

11.1.1.1 Glutamine synthetase reaction


Gln is formed from glutamate, which itself is readily convertible to a-ketoglutarate,
feeding the TCA cycle (Figure 11.1). For Gln formation, glutamate is enzymatically
combined with an ammonium ion (NH4+) by the action of glutamine synthetase.
Glutamine synthetase is an enzyme that can be found in numerous tissues. It promotes
the reaction

ATP + NH4+ + glutamate Æ ADP + Pi + Gln + H+

In the first step of this reaction, the enzyme-bound high-energy intermediate glutamyl
5-phosphate is formed, an acyl phosphate resulting from the phosphorylation of the
5-carbocyl group of glutamate by ATP. Then in the second step, the glutamyl 5-phos-
phate combines with an ammonium ion in the active site to form Gln and release
phosphate. This energy-dependent reaction is irreversible and tightly regulated. It was
found that the activity of glutamine synthetase is subject to regulation by over 40
metabolites.1 Some of these serve as substrates or allosteric effectors, including Gln,
glutamate, ammonia, ATP, and a-ketoglutarate. Thus, glutamine synthetase is regulated
by its substrates and products. The Gln formation rate in skeletal muscle increases in
response to enhanced Gln withdrawal from the plasma by Gln-utilizing cells under
stress conditions. In contrast, the Gln formation rate decreases when plasma Gln is
restored in patients by parenteral Gln administration (see Chapter 36 for details). This
indicates that glutamine synthetase activity in skeletal muscle is regulated to maintain
plasma Gln at a defined level. Substitution of a-ketoglutarate can also help to spare
muscle Gln pools in these patients (see Chapter 37 for details), demonstrating that
synthesis of Gln in skeletal muscle can keep pace with Gln consumption if ample
precursors are provided.

11.1.1.2 Gln transport through cell membranes


The Gln formed by glutamine synthetase is a water-soluble compound that can readily
pass through cell membranes, whereas the precursor glutamate, which bears a net charge,
needs facilitation by specific transport systems.2 However, Gln does not freely cross the
plasma membrane. The cytoplasmatic Gln level is far above its transmembrane equilib-
rium distribution, and the transport of Gln is highly regulated. Several Gln-transporting
systems have been described in the past few years that can be divided into Na+-dependent
and Na+-independent systems. The former utilizes the potential energy present in the
transmembrane Na+ electrochemical gradient, maintained largely by the Na+/K+-ATPase,
to drive the uptake of amino acids against their concentration gradients. Na+-dependent
Gln transporters include systems ASC, N, A, B0,+, and y+L, whereas Na+-independent
transporters include systems L and b0,+.3 All of these transporters mediate the movement
of several other amino acids, to varying degrees. System ASC was originally named for
three of its preferred substrates (alanine, serine, cysteine) to distinguish it from system A
activity. It takes up Gln with high affinity and transports a wide panel of other zwitterionic
amino acids such as serine, threonine, cysteine, alanine, and asparagine, as well as
bulky/branched-chain amino acids (leucine, valine, methionine) to a lesser degree. The
catalytic mechanism of ASC involves a Na+-dependent exchange of intracellular and
extracellular amino acids. This transporter can therefore mediate either Gln uptake or
release. Net Gln movement through this transporter differs from cell type to cell type. The
ASC system is expressed in the liver, kidney, skeletal muscle, intestinal epithelia, lung,
placenta, and pancreas. In contrast, system N has a rather narrow substrate specificity of
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172 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Gln, histidine, and asparagine only — all substrates containing nitrogen in their side chain.
It is expressed in liver and skeletal muscle, in which it represents a potentially rate-limiting
step in metabolism. Hepatic and skeletal muscle system N activities exhibit reciprocal
regulation during catabolic states in which system N-mediated Gln uptake is predictably
enhanced in the liver and decreased in skeletal muscle. Gln transport mediated by system
N is electrogenic, with a proposed two Na+ ions and amino acid transported inward,
coupled to the efflux of one H+. The more ubiquitously expressed system A was found to
mediate Gln uptake only after prolonged periods of amino acid starvation. These data
show that Gln uptake and exchange are mediated by a number of transporters that show
a tissue-specific expression pattern. In addition, because of the wide range of intracellular
substrate levels and transmembrane electrical potentials, the affinity of each system for
Gln varies significantly between the different cell types even at similar expression levels.
Today it is not yet clear how the different Gln transporter systems are regulated, and
further studies are needed to understand the influence of these systems on the interorgan
Gln flux.

11.1.1.3 Gln is a carrier of nitrogen and carbon


The interconversion of charged glutamate to uncharged Gln includes the toxic compound
ammonia. The formed Gln is nontoxic and, as described above, membrane permeable.
Thus, Gln formation is suited to detoxify ammonia and to remove it from the cell. In fact,
approximately one-third of all amino acid-derived nitrogen transported by blood is in the
form of Gln.4 Its concentration in blood (approximately 650 mmol/l of plasma) exceeds
that of all other amino acids by far. More than 60 to 80% of the plasma flux of Gln is
provided by de novo synthesis in the postabsorptive state, whereas the contribution by the
diet plays only a minor role.5 De novo synthesis is inhibited only at very high levels of
Gln uptake (125 mg/kg*h) leading to a doubling of the normal plasma concentration. Gln
serves as a vehicle for transportation of ammonia in a nontoxic form from peripheral
tissues to visceral organs where it can be excreted as ammonium by kidneys or converted
to urea by the liver. Gln is the major source of both nitrogen used in hepatic ureagenesis
and nitrogen excreted in urine. Perfusion of isolated rat livers with Gln was found to
stimulate urea output.6 This indicates that Gln regulates the ammonia detoxification pro-
cesses in the liver.
Ammonia derives from degradation of nitrogen-containing compounds. A main
source of ammonia is amino acid catabolism in the course of protein breakdown. The 20
amino acid catabolic pathways converge to form only five carbohydrate products (pyru-
vate, acetyl-CoA, a-ketoglutarate, fumarate, and oxaloacetate), all of which enter the TCA
cycle. Thus, the carbohydrate backbone of all amino acids can be converted to any TCA
cycle intermediate. The amino group of the degraded amino acids is commonly transferred
to a-ketocarboxylic acids by transamination. A number of aminotransferases transfer the
amino group to the TCA cycle intermediate a-ketoglutarate to form glutamate. a-Keto-
glutarate is also converted to glutamate by combination with ammonia via the mitochon-
drial glutamate dehydrogenase. Glutamate itself is then combined with a second nitrogen
in the course of Gln formation via glutamine synthetase (see Figure 11.1). Thus, Gln can
potentially incorporate nitrogen as well as the carbohydrate skeleton from all 20 amino
acids and should therefore be a suitable end product of protein breakdown. In fact, amino
acids, mainly branched-chain amino acids, are the major source of nitrogen and carbon
for Gln synthesis by skeletal muscle. Net synthesis of Gln also occurs in the lungs, adipose
tissue, brain, and, under certain conditions, liver. The release of Gln from these tissues
serves to transport nitrogen and carbon to Gln-utilizing cells.
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Chapter eleven: Glutamine metabolism 173

11.1.2 Gln breakdown


11.1.2.1 Glutaminase reaction
Gln catabolic pathway results in the same intermediates as seen in Gln synthesis, but with
other enzymes involved. Gln is converted to glutamate and ammonia by the mitochondrial
enzyme glutaminase in the nonreversible reaction

Gln + H2O Æ glutamate + NH4+

Glutaminase activity was found only in certain tissues, including liver, kidney, entero-
cytes, and immune cells. The liver has evolved an elegant mechanism for Gln metabolism
and concerted ammonia detoxification. In the periportal region, ammonia and glutamate
are being generated from incoming Gln by the hepatic isoform of the enzyme glutaminase,
while at the same time high incoming concentrations of ammonia from the splanchic bed
are utilized for urea synthesis (see Chapter 7 for more details). In the perivenous portion
of the liver, high levels of Gln synthetase convert glutamate and excess ammonia into
Gln, effectively reducing the ammonia concentrations in hepatic venous blood to nontoxic
levels. With this intraorgan Gln cycle, the liver is able to effectively detoxify incoming
blood from ammonia and control output of Gln. In contrast, the kidney counteracts
acidosis utilizing the kidney isoform of glutaminase to generate ammonia from Gln. The
kidney disposes of excess nitrogen by consuming Gln and excreting the ammonia
produced.

11.1.2.2 Gln utilization


In the healthy individual, the major site of Gln catabolism is the small intestine, where
Gln is the principal respiratory fuel of enterocytes. In addition, a high rate of Gln utilization
was also found in all lymphoid tissues examined, including lymph nodes, spleen, thymus,
and Peyer’s patches, in neutrophils, in monocytes, and in macrophages. The catabolism
of Gln in the intestine results in the production of CO2, alanine, pyruvate, and lactate from
the carbon skeleton and of ammonia and alanine from the carboxamide and amino groups.
Complete oxidation of Gln via glutamate, a-ketoglutarate, and the TCA cycle results in
27 ATP equivalents, which is one of the highest rates of all nonessential amino acids.
Under physiologic conditions, Gln oxidation can account for as much as one third of the
ATP production in cultured enterocytes, lymphocytes, and monocytes. The rate of Gln
utilization of human neutrophils was recently found to depend on the extracellular con-
centration of the other major energy substrate, glucose.7 Decreasing the extracellular
concentration of glucose from 5.5 to 1 or 0 mM increased the apparent Vmax of Gln
utilization by 70 and 134%, respectively. This suggests that Gln metabolism may be par-
ticularly important as a fuel for neutrophils in situations where the blood glucose concen-
tration is low. The rate of glucose utilization, however, is independent of the extracellular
Gln concentration. As shown in isolated human monocytes, reduction of extracellular Gln
below the physiological plasma concentration leads to a decreased intracellular ATP level
in the presence of 11 mM glucose.8 This suggests that although both glucose and Gln are
used by leukocytes for synthesis of common metabolic intermediates, glucose cannot
replace Gln completely. Not all utilized Gln is used for ATP production. For example,
HeLa and CHO cells each convert approximately 30% of catabolized Gln carbons to CO2
and 15% to lactate, while approximately 20% of carbons are incorporated into macromol-
ecules. The total amount of cellular energy derived from Gln depends on the extent of
oxidation and the rate of Gln utilization. These factors in turn depend largely on the
absolute amounts and relative proportions of Gln and glucose available, as well as the
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174 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

type and proliferative state of the cell. For example, lymphocytes derive a large portion
of their energy from Gln oxidation. This portion increases strongly after activation with
RNA or mitogenic antibodies directed against CD3, which stimulate lymphocytes to
proliferate. This proliferation is highly dependent on the Gln concentration in culture
medium.

11.1.2.3 Gln is a precursor of a variety of molecules


Gln is used as an amino donor in a variety of reactions. For example, it is directly involved
in purine nucleotide synthesis. The amide groups of three Gln molecules can contribute
to each purine molecule that is synthesized. Similarly, formation of guanosine monophos-
phate requires nitrogen from Gln. In addition, pyrimidine nucleotides are synthesized by
a sequence of reactions that involve Gln and aspartate. Purines and pyrimidines are
substrates for DNA synthesis. The increased DNA synthesis in mitogen-stimulated lym-
phocytes is therefore believed to be the reason for the increased Gln utilization by these
cells.
Gln is the donor of nitrogen for synthesis of amino sugars such as glucosamine
6-phosphate, galactosamine, and N-acetylgalactosamine. Glucosamine is further metabo-
lized to synthesize N-acetylglucosamine and sialic acids. Amino sugars are found in both
glycoproteins and proteoglycans. The Gln product glutamate is part of the tripeptide
glutathione (GSH), in which its 5-carboxyl group forms the peptide linkage to the 1-amino
group of cysteine (GSH synthesis and function are described further below). In the central
nervous system, glutamate is an important excitatory neurotransmitter, and it also serves
as the precursor for g-aminobutyric acid (GABA), an important inhibitory neurotransmit-
ter. Nerve endings and neurons may also use Gln for synthesis of glutamate (and hence
GABA) because neurons have high glutaminase activity. Some of the glutamate and GABA
released by neurons are taken up by glial cells, which in turn have high glutamine
synthetase activity used in the resynthesis of Gln.

11.1.3 Gln interorgan exchanges


All these data indicate an extensive exchange of Gln between Gln-producing and Gln-con-
suming tissues. This Gln interorgan exchange is summarized in Figure 11.2. Although the
Gln-forming enzyme glutamine synthase is expressed in most cell types, net synthesis of
Gln occurs predominantly in skeletal muscle and lung. Excess Gln is released into the
blood and transported to Gln-consuming tissues. The Gln-degrading enzyme glutaminase
can only be found in a small number of tissues, such as liver, kidney, enterocytes, and
immune cells. Liver and kidney use Gln for nitrogen extraction and produce urea and
ammonia, respectively. In contrast, gut enterocytes and leukocytes utilize Gln as a main
respiratory fuel for ATP production. Thus, Gln plays an important role in nitrogen as well
as carbohydrate metabolism. The plasma Gln level is determined by the balance of Gln
production and Gln consumption.
About 20 years ago we found a marked muscle Gln depletion in septic patients that
correlated with poor survival.9 Numerous subsequent studies showed that Gln levels in
skeletal muscle as well as in plasma are strongly reduced in sepsis and other catabolic
states, which is associated with a worse prognosis of the disease. During catabolic states,
a combination of increased Gln use by Gln-consuming cells and decreased nutrient uptake
creates a Gln demand that is met primarily by increased Gln efflux from lung and muscle
tissue. Although plasma Gln levels are usually maintained in injured patients, lung and
muscle stores can be rapidly depleted after severe injury or infection. Muscle is a major
producer of Gln during normal and catabolic states. In the normal postabsoptive 200-g
rat, Gln is released from the hindquarter at a rate of approximately 0.4 mmol/min. Given
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Chapter eleven: Glutamine metabolism 175

muscle
protein
break down
lung

plasma
(~ 0,6 mM)
urea
ATP
glucose
Gln
liver gut

NH3 ATP

kidney leukocytes

Figure 11.2 Overview of physiological Gln interorgan exchanges. The plasma Gln level is deter-
mined by the balance of Gln production and Gln consumption. Net synthesis of Gln occurs pre-
dominantly in skeletal muscle and lung, whereas net Gln uptake occurs mainly in liver, kidney, gut,
and leukocytes. Liver and kidney use Gln for nitrogen extraction and produce urea and ammonia,
respectively. In contrast, gut enterocytes and leukocytes utilize Gln as a main respiratory fuel for
ATP production.

that skeletal muscle Gln concentrations are approximately 6 to 8 mmol/l of intracellular


water, even a normal rate of release cannot be sustained for long without de novo synthesis.
During catabolic states the efflux of Gln from muscle increases markedly, and increased
net production rate must eventually compensate for this heightened release. The muscles
maintain a rapid efflux of Gln in part by increasing the rate of proteolysis while decreasing
the rate of protein synthesis. This increases the intracellular pool of amino acids for
production of Gln, which can be derived from all amino acids through their conversion
to glutamate, either directly or through a-ketoglutarate. In critical illness, muscle Gln
stores and Gln supplied by muscle proteolysis do not suffice to meet demand for Gln,
because of either the magnitude of Gln use or depletion of muscle mass. This leads to a
decrease in plasma Gln levels, and Gln-utilizing cells therefore suffer from Gln starvation
under these conditions.

11.2 Effects of Gln starvation


Gln is used as a substrate for ATP formation mainly in gut enterocytes and leukocytes.
Thus, these cells exhibit the highest susceptibility to Gln starvation under catabolic con-
ditions. Both cell types play important roles in the defense against microorganisms: while
enterocytes are essential for integrity of gut mucosa, which prevents translocation of
bacteria from the lumen into the abdominal cavity, leukocytes detect and eliminate invad-
ing microorganisms in the course of immune response. Both capabilities are essential
during critical illness. However, reduced availability of Gln under these conditions has
adverse effects on energy metabolism, protein synthesis, cell protective mechanisms, via-
bility, and function of these Gln-consuming cells (summarized in Figure 11.3).
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176 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

AMPK pathway catabolic pathways ↑


ATP↓ AMPK anabolic pathways ↓
protein synthesis ↓

? Cellular redox state TRX:ASK1 ↓ JNK


GSH↓ redox-sensitive
kinases NF-kB
AP-1
Osmo-signaling
Fas ↑
delayed Hsp70-induction apoptosis
cell volume ↓
Erk ↓
p38 ↓ autophagic-proteolysis ↑
Gln-depletion
Translation p70s6k
mTOR ↓ 4E-BP1 translation ↓
...

glutaminyl-tRNA synthetase
QRS:Gln:ASK1 complex ↓ JNK

Figure 11.3 Cellular effects of Gln starvation. Reduced availability of Gln affects a number of dif-
ferent molecular mechanisms. Gln starvation leads to a reduction of intracellular ATP. Cellular
energy depletion leads to activation of AMPK, which regulates enzyme activity and gene expression.
However, until now there has been no study on the effect of Gln starvation on AMPK activation.
Gln starvation leads to a reduced intracellular glutathione (GSH) level. A decrease in the GSH:GSSG
ratio affects the activity of redox-sensitive kinases. Gln starvation leads to a cell shrinkage, which
affects the osmo-sensitive pathways. In addition, reduced availability of amino acids reduces the
activity of mTOR, which is a central regulatory factor in the control of translation. The available
Gln is also detected by glutaminyl-tRNA synthetase, which acts as a regulatory cofactor in the Fas-
mediated apoptosis pathway. All these mechanisms result in a reduction of anabolic pathways and
an increase in catabolic pathways in order to spare resources. In addition, several of these mecha-
nisms lead to an increased susceptibility of the cell to apoptosis triggers.

11.2.1 Energy metabolism


A major effect of Gln starvation is certainly the reduction of intracellular ATP. ATP is
required, directly or indirectly, for all energy-consuming processes. Energy deprivation
of mammalian cells leads to the activation of the AMP-activated protein kinase (AMPK)
cascade via an increase in the AMP:ATP ratio.10 The AMPK functions as a metabolic
master switch that inhibits anabolic processes and stimulates catabolic processes, thereby
preserving ATP. For example, AMPK inhibits acetyl-CoA-carboxylase (ACC) by phos-
phorylation, activates phosphofructokinase (PFK), and reduces the expression of genes
involved in fatty acid synthesis. It is still unclear whether the AMPK pathway becomes
activated by Gln starvation. To our knowledge, there is only one study to date that
investigated the role of AMPK in a Gln-utilizing cell type. Marsin and coworkers11
showed that hypoxia stimulates glycolysis in activated monocytes by phosphorylation
of PFK by AMPK. Thus, AMPK is involved in the regulation of the energy metabolism
of monocytes, but further studies are needed to characterize the role of AMPK in
monocyte function and the effect of Gln starvation on the AMPK pathway. However,
the sustained ATP decrease in Gln-starving monocytes8 indicates that even if ATP-
preserving countermeasures are activated, they are not sufficient to restore the normal
energy status of these cells.
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Chapter eleven: Glutamine metabolism 177

11.2.2 Protein synthesis rate


Studies employing a variety of cell types and different tissues demonstrate that cells
respond to reduction of amino acid availability by a decrease of overall protein synthesis
rate. This control occurs predominantly at the level of initiation of translation of mRNA
into proteins (see Chapter 16 for more details). Translation initiation is regulated by over
a dozen proteins referred to as eukaryotic initiation factors (eIFs). The phosphorylation
status of these factors determines the efficiency of unwinding of structured mRNA and
the binding of mRNA to the ribosomal complex. It has been shown that slight changes in
the phosphorylation patterns of eIFs can lead to preferential translation of mRNAs con-
taining highly structured 5'-untranslated regions or favor the binding of mRNAs contain-
ing a 5'-terminal oligopyrimidine (TOP) tract.12 Thus, the response of translation initiation
to a change in amino acid availability can be general, affecting the translation of most, if
not all, mRNAs, but it can also be specific, affecting the translation of a single subset of
mRNAs. A major regulating factor of eIF phosphorylation status is the mammalian target
of rapamycin (mTOR).13 The activity and phosphorylation state of mTOR and downstream
effectors are decreased in response to amino acid limitation and stimulated upon their
readdition. For example, administration of leucine promoted activation of mTOR in the
liver of food-deprived rats14 and in Xenopus laevis oocytes.15 However, the nutrient metab-
olites and signaling molecules that act upstream of mTOR are unknown. mTOR was
recently found to be down-regulated by AMPK activation in rat skeletal muscle cells,
which suggests a direct connection to energy metabolism.16 In addition, a recent study in
yeast showed that Gln starvation caused nuclear localization and activation of TOR-
inhibited transcription factors GLN3, RTG1, and RTG3, all of which mediate Gln synthe-
sis.17 These findings suggest that the TOR pathway senses Gln concentrations.

11.2.3 Cell protective mechanisms


In the course of inflammation cells are confronted with a number of cytotoxic mediators,
including endotoxin, cytokines, and reactive oxygen species (ROS). Cells express a group
of proteins that are essential to cellular survival under such stressful conditions, the heat
shock proteins. The 70-kDa heat shock protein (Hsp70) has attracted the most interest
because it is highly inducible in leukocytes.18,19 Preemptive Hsp70 induction protects cells
against cytotoxic mediators and reduces organ dysfunction and mortality in animal mod-
els of sepsis.20 Under conditions with low plasma Gln, such as polytrauma, severe sepsis,
and acute respiratory distress syndrome, Hsp70 expression was found to be impaired in
granulocytes,21 monocytes,22 lymphocytes,23 and intestinal epithelial cells.24,25 We have
shown in vitro in isolated lymphocytes that Hsp70 induction depends on the availability
of Gln in a dose-dependent manner.26 While Hsp70 can be induced at the physiological
Gln concentration to nearly maximum levels, it shows a reduced inducibility (–47%) at
pathological Gln levels (0.2 mM). This indicates that Gln-starving cells are unable to
express normal amounts of Hsp70. Since plasma Gln depletion occurs during systemic
inflammation, the impaired Hsp70 expression under these conditions is likely to have
deleterious effects on the survival and function of leukocytes and may contribute to the
immunosuppression observed in these patients.
Many researchers have found that Hsp70 expression can be induced in a variety of
cell types and healthy animals by administration of high-dose Gln (5 to 20 mM or 5 mg/kg,
respectively).27,28 The elevated Hsp70 level protects cells and animals from subsequent
stress situations such as heat shock,25 sepsis,28 and cold ischemia-reperfusion injury.29 The
increase in Hsp70 is probably induced by a stress response of the cell to the nonphysio-
logically high Gln concentration rather than being related to a starvation effect.
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178 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

While Hsp70 protects cells against oxidative stress by repair or removal of damaged
proteins, the second major protection factor of mammalian cells, the antioxidant glu-
tathione (GSH), reacts directly with ROS in order to prevent oxidative damage. Gln (via
glutamate) is one of the three precursor amino acids of GSH (L-g-glutamyl-L-cysteinyl-
glycine). A significant correlation between reduced Gln supply and diminished intra-
cellular GSH was observed in cultured peripheral blood mononuclear cells,30 activated
T-cells,31 Peyer’s patches,32 and critically ill patients.33 These data clearly show that leu-
kocytes are unable to maintain their GSH levels when Gln is depleted.
GSH is one of the three redox couples that determines the antioxidative capacity of a
cell: nicotinamide adenine dinucleotide phosphate (NADPH/NADP+), thioredoxin
(TRXred/TRXox), and glutathione (GSH/GSSG). Although glutathione is (due to its high
concentration) the most important determinant of the cellular redox state, the GSH/GSSG
ratio is clearly affected by the redox ratios of the other couples. Under physiological
conditions, GSH levels are 10- to 100-fold higher than GSSG levels. Gln starvation-induced
reduction of GSH content is likely to show a strong effect on the cellular redox state.
Shifting the redox state toward oxidizing conditions affects the activation of several sig-
naling molecules, including protein kinase B, protein phosphatases 1 and 2A, calcineurin,
nuclear factor (NF)-kB, c-Jun N-terminal kinase (JNK), apoptosis signal-regulated kinase
1 (ASK1), and p38 mitogen-activated protein kinase (MAPK).34,35 These mediators are
named redox-sensitive kinases. Oxidizing conditions activate pathways that reduce pro-
liferation and cytokine production and increase apoptosis.36–38

11.2.4 Apoptosis
Apoptosis can be induced by a range of environmental, physical, or chemical stresses.
Recent studies have established that the survival-promoting effects of Hsp70 can be partly
attributed to the suppression of apoptosis.39 Thus, the reduced Hsp70 expression in Gln-
starving cells, together with their impaired antioxidative capacity, is likely to make them
more sensitive to induction of apoptosis. We showed in premonocytic U937 cells that the
number of apoptotic cells increased only marginally after Gln starvation for 4 h. However,
Gln starvation renders these cells more susceptible to specific apoptosis triggers.40 The
number of apoptotic cells was significantly higher (50 to 60%) in Gln-starving cells when
exposed to Fas-ligand or heat shock. No effect of Gln starvation was found on UV irra-
diation-induced apoptosis. Hence, Gln starvation sensitizes cells to apoptosis induction,
probably by an impaired stress response. However, the increased susceptibility depends
on the apoptosis trigger.
Apoptosis is initiated by two principal pathways. The intrinsic pathway (stimulated,
for example, by tumor necrosis factor-a (TNF-a)) involves mitochondria, whereas the
extrinsic pathway is directly activated by the ligation of death receptors such as Fas. Binding
of Fas-ligand or agonistic antibodies to Fas receptor initiates the activation of the caspase
cascade and promotes JNK activation through interaction with ASK1, a member of the
mitogen-activated protein kinase kinase kinase (MAPKKK) family.41 These activation steps
finally lead to apoptosis. Studies in myelocytic HL-60 cells showed an increase in the Fas-
mediated apoptosis pathway by Gln starvation.42 Gln starvation for 12 h induced a reduc-
tion of cell volume by about 40%. This shrinkage promoted a ligand-independent activation
of the Fas-mediated apoptosis pathway. Accordingly, cell shrinkage did not induce apop-
tosis in CD95 receptor-negative lymphoma L1210 cells. Replacement of glutamine with
surrogate compatible osmolytes counteracted cell volume decrement and protected the
CD95-expressing cells from apoptosis. A similar increase in Fas-mediated apoptosis was
observed in Gln-starving HeLa cells.43 Fas-ligation activated ASK1 and JNK in Gln-starving
cells but not in normal cells. The authors found a Gln-dependent association of ASK1 with
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Chapter eleven: Glutamine metabolism 179

glutaminyl-tRNA synthetase (Gln-RS). The ASK1 activity was inhibited by the interaction
with Gln-RS. This antiapoptotic function of Gln-RS was weakened during Gln starvation.
Thus, Gln directly interferes with the signal transduction pathway of Fas-mediated apop-
tosis. Isolated human neutrophils react differently to Gln starvation.7 When exposed to Fas-
ligand, neutrophil apoptosis is increased by a reduction in the extracellular concentration
of glucose but is unaffected by Gln concentration.
A crucial step in the apoptotic mechanism of TNF-a is the perturbation of mitochon-
drial functions leading to the formation of ROS. Thus, the cytotoxicity of TNF-a depends
on the antioxidative capacity of the cell, as well as on the mitochondrial activity. In mouse
fibrosarcoma L929cells, Gln is utilized as a major energy source and drives mitochondrial
ATP formation, while glucose is mainly converted to lactate through glycolysis. Gln
starvation desensitizes these cells to TNF-a cytotoxicity, while lack of glucose in the
medium does not alter the TNF-a response.44 Thus, Gln starvation protects these cells
from TNF-a cytotoxicity by reducing mitochondrial activity and thereby preventing ROS
formation. A similar relation of Gln metabolism and TNF-a-induced cytotoxicity was
described in Ehrlich ascites tumor-bearing mice.45 Feeding mice with a Gln-enriched diet
led to a high rate of Gln oxidation, which promoted a selective depletion of mitochondrial
GSH by about 58%. The increase in ROS production by TNF-a further depleted mitochon-
drial GSH and finally induced apoptosis. Taken together, these data clearly show that Gln
starvation affects cellular susceptibility to apoptosis in dependence of the cell type and of
the apoptosis trigger.

11.2.5 Osmosignaling
In recent years, it has become increasingly evident that cell volume represents a major
determinant of cellular function in a variety of cell types. In mammalian cells, hydration
may change dynamically in response to hormones, ethanol, aniso-osmotic environments,
and oxidative stress, or by cumulative substrate uptake. Gln has been shown to increase
hepatocyte hydration due to the cumulative uptake of Gln into the cells, which activates
osmosignaling pathways involving MAPK.46 Gln-induced cell swelling also activates
extracellular signal-regulated kinases (ERK) and p38 (MAPK). A similar relation of Gln
to osmosignaling pathways is described also in other cell types, including myelocytic
HL-60 cells42 and adipocytes.47 Therefore, it seems that the osmoregulatory effect of Gln
is not restricted to Gln-consuming cells (more details on osmosignaling are given in
Chapters 16 through 18).

11.2.6 Immune response


In addition to their impaired proliferative responsiveness to mitogens, Gln-starving T-lym-
phocytes show remarkable changes in their function. It was found that the expression of
the cell surface activation markers CD25, CD45RO, and CD71 and the production of
interferon-g and TNF-a are dependent on the concentrations of Gln. Gln starvation arrests
the cells in the G0/G1 phase and reduces lymphokine-activated killer cell activity. The
growth arrest can be reversed by addition of Gln by a cAMP-inhibitable and Raf-inde-
pendent activation of the JNK MAPK.48
In contrast to the rapidly proliferating lymphocytes, macrophages are terminally
differentiated cells that have lost their ability to divide. Gln starvation reduces the expres-
sion of human leukocyte antigen HLA-DR on monocyte-derived macrophages, and
decreases tetanus toxoid-induced antigen presentation. In addition, low Gln levels down-
regulate the expression of intercellular adhesion molecule-1 (ICAM-1/CD54), Fc receptor
for immunoglobulin (Ig) G (Fc gamma RI/CD64), and complement receptors type 3 (CR3;
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180 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

CD11b/CD18) and type 4 (CR4; CD11c/CD18). These reductions are associated with a
decreased phagocytosis of IgG-sensitized ox erythrocytes or opsonized Escherichia coli.
Monocyte expression of CD14, CD71, and Fc gamma RIII/CD16 and the capacity to
phagocytose latex beads are not affected by Gln starvation.49 Gln has an important effect
on the maturation of myelomonocytic U937 cells along the monocytic pathway. Gln star-
vation reduced DNA synthesis and enhanced the development of vacuoles.
Direct evidence for the influence of Gln starvation on gut immunology was shown in
a mouse model. Parenteral feeding of mice with Gln-free solutions increased translocation
of bacteria and toxins from the gut into the circulation, caused intestinal immunoglobulin
(Ig) A deficiency, and reduced lymphocyte yield in the gut-associated lymphoid tissue.
The addition of Gln to the feeding solution improved the outcome of endotoxin-challenged
animals and normalized intestinal IgA levels and lymphocyte numbers in the PP, the
lamina propria, and the intraepithelial layer (see Chapter 42 for more details). We could
show in recent in vivo studies that restoring plasma Gln levels by Gln supplementation
in patients undergoing elective surgery is associated with a diminished decrease in HLA-
DR50 and with an increased TNF-a production in response to ex vivo lipopolysaccharide
(LPS) stimulation.40 Thus, maintaining plasma Gln levels seems not only to be essential
for the leukocyte energy status but also for their function and is therefore important for
the inflammatory response.

Acknowledgments
We thank Christine Brostjan, Maja Munk Eliasen, Andreas Spittler, Maria Zellner, and
Susanne Oehler for helpful discussions and careful reading of the manuscript.

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section B

Control of and by amino acids


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chapter twelve

Insulin and the regulation


of amino acid catabolism
and protein turnover
Jean-Pascal De Bandt
Université Paris 5

Contents
Introduction..................................................................................................................................185
12.1 The physiological role of insulin ....................................................................................186
12.1.1 Mechanisms of insulin action .............................................................................186
12.1.2 Insulin and the regulation of metabolism ........................................................187
12.1.3 Effect on gene transcription ................................................................................188
12.2 Insulin and amino acid catabolism ................................................................................188
12.3 Insulin and protein turnover ...........................................................................................190
12.3.1 Control of protein metabolism at the cellular level........................................190
12.3.1.1 Regulation of protein catabolism ........................................................190
12.3.1.2 Regulation of protein synthesis ...........................................................191
12.3.2 Insulin and the control of protein metabolism in vivo in humans...............193
12.3.2.1 Regulation of protein catabolism ........................................................193
12.3.2.2 Regulation of protein synthesis ...........................................................194
12.4 Conclusion ..........................................................................................................................195
References .....................................................................................................................................195

Introduction
Insulin is a key hormone, the various effects of which have long been known. The main
metabolic effects of insulin include the regulation of glycemia, energy metabolism, protein
accretion, and growth. We all know from our student textbooks that insulin promotes
glucose uptake and storage by various cell populations, and amino acid transport and
utilization for protein synthesis. But even though we have made tremendous strides in
understanding the mechanisms of insulin action, some areas remain uncharted, including
certain aspects of the regulation of amino acid catabolism and protein turnover.

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186 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

This knowledge gap has many causes: studies of insulin action in situations of acute
insulin withdrawal or of insulin resistance, while yielding interesting data, may have
underestimated compensatory mechanisms; extrapolation of data from experimental stud-
ies to human beings is sometimes difficult; organ specificity of insulin action, for example,
according to the composition of muscle fibers, is only now being explored. The issue is
further complicated by the fact that both insulin and amino acids exert regulatory action
on glucose and amino acid metabolism and on protein turnover via interfering signaling
pathways (see Chapters 16 and 18 for details).
From a teleological point of view, the importance of these interactions that hamper
our understanding was only to be expected. Insulin is one of the main hormones of energy
storage and homeostasis and of protein accretion. Storage and utilization of amino acids,
and even more so of essential amino acid, as an energy source, is not cost-effective. For
example, we are all well aware of the importance of peripheral amino acid mobilization
during the metabolic response to stress; however, when sustained during prolonged stress
situations, this has adverse consequences in terms of malnutrition and increased morbidity.
The study of the kinetics and dose–response relationship of amino acid effect on protein
metabolism has shown that “the mechanisms of control of muscle protein synthesis contain
a physiological device that limits the amount of protein that can be stored in muscle.”1
Thus, when the protein pool is at its highest, amino acid excess must be dealt with.
According to amino acid supply levels, amino acid availability therefore ought to act to
control the anabolic response to insulin. For example, at high amino acid supply levels,
peak amino acid utilization for protein synthesis can be reached and excess amino acid
then has to be degraded; this requires a relative decrease in the insulin effect on the former
process and a relative insulin resistance in order to promote amino acid oxidation at the
expense of glucose utilization. Conversely, if amino acid supply levels are low, the pro-
moting effect of insulin on protein synthesis has to be blunted.

12.1 The physiological role of insulin


12.1.1 Mechanisms of insulin action
After binding of insulin, insulin receptors become tyrosine-phosphorylated through an
autophosphorylation reaction. The receptor then phosphorylates a number of intracellular
substrates such as the insulin receptor substrate (IRS)-1 and IRS-2 and Shc, initiating
various signaling pathways (Figure 12.1). However, many insulin responses are mediated
through IRS-1 and IRS-2. These proteins are characterized by the presence of an NH2-
terminal pleckstrin homology (PH) domain, which can mediate interaction with phospho-
lipids, and a phosphotyrosine-binding domain, which allows binding to the insulin recep-
tor.2,3 IRS proteins couple insulin receptors to the phosphatidylinositol 3-kinase (PI3-
kinase) and extracellular signal-regulated kinase (ERK) cascades:

• PI3-kinase belongs to a family of enzymes that phosphorylate phosphoinositides.


Among PI3-kinases, class IA enzymes consist of a p110 catalytic subunit and a p85
regulatory subunit. P85 presents two SH2 domains (SH2 for Src-homology-2) en-
abling binding to phosphorylated tyrosine residues. Insulin specifically activates
PI3-kinase type IA, the products of which include phosphatidylinositol-3,4-bisphos-
phate (PtdIns(3,4)P2) and phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3).
These phospholipids activate various signaling kinases, including the mammalian
target of rapamycin (mTOR), alternate protein kinase C isoforms, and phosphoi-
nositide-dependent kinase (PDK). PDK phosphorylates and activates protein ki-
nase B (PKB)/akt, which in turn can phosphorylate several substrates, including
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Chapter twelve: Insulin and the regulation of amino acid catabolism and protein turnover 187

insulin

Insulin receptor

GLUT4 translocation Cbl

CAP
Shc IRS1
Grb2 PI3K
ras--GDP
SOS
p85 p110
PtdIns(4)P,
ras--GTP PtdIns(4,5)P2
raf

PtdIns(3,4)P2,
MEKK
PtdIns(3,4,5)P3
MAPKinase
cascade MEK PDK1
aPKC
PKB/Akt

ERK
mTOR Forkhead
GSK3
transcription factors

p70s6k

Gene expression, Glucose metabolism,


Cell growth, Glycogen/lipid/protein synthesis
Differentiation Specific gene expression

Figure 12.1 Insulin signaling cascade. The insulin receptor undergoes autophosphorylation and
catalyzes the phosphorylation of cellular proteins such as IRS, Shc, or Cbl. These proteins then
interact with SH2 domain-containing molecules. Activation of phosphatidylinositol-3-OH kinase
(PI3K) results in the generation of phosphatidylinositol-3,4-diphosphate (PtdIns(3,4)P2) and phos-
phatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3). PtdIns(3,4)P2 and PtdIns(3,4,5)P3 activate a va-
riety of downstream signaling kinases, including phosphoinositide-dependent kinase 1 (PDK1),
protein kinase B (PKB/Akt), alternate protein kinases C (aPKC: PKC z and l), and the mammalian
target of rapamycin (mTOR). Tyrosine phosphorylation of Shc proteins, which in turn interact with
the adapter protein Grb2, recruits the Son-of-sevenless (SOS) exchange protein for activation of Ras.
Ras stimulates a serine kinase cascade (MAPkinase) through stepwise activation of Raf, MEKK (MEK
kinases), MEK (MAP/ERK kinases), and ERK (extracellular signal-regulated kinases), leading to the
phosphorylation of transcription factors. (Adapted from Saltiel, A.R. and Kahn, C.R., Nature, 414,
799, 2001; White, M.F., Am. J. Physiol., 283, E413, 2002.)

glycogen synthase kinase 3 (GSK-3) and mTOR. This pathway seems to be involved
in the control of various biological processes such as glucose transport and me-
tabolism, protein synthesis, and cell survival.2,3
• ERK cascade activation involves the GRB-2/SOS complex, which can bind activat-
ed Shc and IRS proteins, and in turn interacts with Ras, thus yielding the active
form of Ras (Ras-GTP). Ras association with Raf-1 enables the activation of the
latter and initiation of the MEK/ERK signaling cascade. These mitogen-activated
protein kinases (MAPKs) have a wide range of potential substrates, including
transcription factors and other protein kinases.2,3

12.1.2 Insulin and the regulation of metabolism


Insulin is a multifunctional anabolic hormone. While on a short-term basis it regulates
protein and energy balance, it also stimulates cell growth and differentiation.2
Insulin plays a prominent role in the moment-to-moment adaptation of protein and
energy metabolism to nutrient supply. It increases glucose uptake in muscle and adipose
tissue and inhibits hepatic glucose production. It also promotes the storage of nutrient in
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188 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

adipose tissue, liver, and muscle by stimulating lipogenesis, glycogenesis, and, at least in
vitro, protein synthesis and by inhibiting lipolysis, glycogenolysis, and protein breakdown.
The role of insulin in promoting growth is an evolving research field. The role of
distinct forms of PKB/akt has been emphasized: mice lacking akt2 are insulin resistant
and undergo a diabetes-like syndrome, and the absence of akt1 is associated with growth
stunting.4 The role of insulin is complicated by the fact that insulin, insulin-like growth
factor-1 (IGF-I), growth hormone, and thyroid hormones are interdependent, with complex
interrelationships in growth and aging.5 Moreover, while the physiological roles of insulin
and insulin-like growth factors are distinct — these hormones acting, respectively, on fuel
uptake and metabolism and on cell growth and differentiation — ligand interaction with
the insulin receptor and IGF-I receptor activates very similar signaling pathways, so that
it may also result in very similar biological effects.3 It has even been suggested that in vivo
specificity of insulin and IGF is in part related to the level of receptor expression in target
tissues with high levels of insulin receptors and low levels of IGF-I receptors in hepato-
cytes, adipocytes, and skeletal muscle and the opposite in fibroblasts and various undif-
ferentiated progenitor cells.3

12.1.3 Effect on gene transcription


Insulin can affect the transcription of different genes via two different mechanisms. First,
via ERK and MAPK activation, insulin can activate genes involved in cell growth.6 Second,
insulin acts via PKB-mediated forkhead transcription factors. Forkhead transcription fac-
tors are normally located in the nucleus where they activate transcription of genes, includ-
ing phosphoenol pyruvate carboxykinase and tyrosine aminotransferase. PKB activation
by insulin leads to the phosphorylation of these transcription factors and to the inactivation
of these genes.6 Cis-acting elements or insulin response elements (IREs) that mediate the
action of insulin on gene transcription mediate the insulin-dependent transcriptional
inhibition of several hepatic genes such as those encoding phosphoenolpyruvate carboxy-
kinase (PEPCK), insulin-like growth factor-binding protein 1 (IGFBP-1), tyrosine amino-
transferase, apolipoprotein CIII, and glucose-6-phosphatase.6 The other consensus IREs
are the Ets motif and the serum response element, which mediate stimulatory effects of
insulin on several genes.6 For example, insulin has been demonstrated to increase IGFBP-3
gene transcription7 or cytoskeletal gamma-actin gene.8

12.2 Insulin and amino acid catabolism


Overall, insulin favors amino acid uptake by various organs. It modulates the activity of
several amino acid transport systems such as systems A, ASC, CAT, N, and Nm.9–12 System
A is a key target of hormonal regulation. It has been shown that insulin up-regulates
system A by various mechanisms according to the tissue: it activates system A in a gene
transcription-dependent manner in hepatocytes, and through a rapid mechanism in mus-
cle.9 This effect is independent of protein synthesis and electrochemical gradient of sodium.
Surprisingly, system A activity is also up-regulated in situations of insulin deficiency or
insulin resistance. This is eventually related to another mechanism of system A regulation
called adaptive regulation, i.e., the derepression of system A in the absence of amino acids.9
In skeletal muscle cells, insulin induces plasma membrane abundance of SAT2, one
of the cloned members of the system A transporter family. This occurs in a PI3-kinase-
dependent manner,13 as demonstrated by its inhibition by the PI3-kinase inhibitor, wort-
mannin, but does not involve mTOR or MAPK pathways.14 Hyde et al.13 demonstrated
that this increase results from increased recruitment of the transporter from an endosomal
compartment to the cell membrane. Notably, these authors also observed that SAT2 was
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Chapter twelve: Insulin and the regulation of amino acid catabolism and protein turnover 189

present in a specific population of endosomal vesicles, from which the glucose transporter
GLUT4 was excluded.13
In adipocytes, insulin stimulated glutamine transport via the ASCT2 members of the
ASC transporter. This effect may involve both the ERK/MAPK pathway and the insulin-
induced modifications of ion transport through the cell membrane.15
Concerning amino acid oxidation, it has been largely demonstrated in the liver that
the regulation of the urea cycle depends on substrate availability and hormones.12 Beliveau
Carey et al.16 demonstrated that substrate availability exerts moment-to-moment control
over the velocity of urea synthesis. However, while insulin increases amino acid transport
to the liver as stated above, it preserves amino acids from catabolism, as suggested, for
example, by the absence of any increase in 15N-urea from 15N-glutamine in isolated rat
hepatocytes compared with the stimulating effect of glucagon.17
Among amino acid-degrading enzymes only phenylalanine hydroxylase and the
branched-chain alpha-keto acid dehydrogenase (BCKAD) complex are regulated by phos-
phorylation/dephosphorylation mechanisms and are thus potential targets of insulin
action. Most other amino acid-degrading enzymes show adaptive induction in conditions
of increased gluconeogenesis.18
Phenylalanine hydroxylase, the major regulatory step of the phenylalanine degrada-
tion pathway, catalyzes the conversion of phenylalanine to tyrosine. Insulin has been
shown to antagonize the glucagon-stimulated phosphorylation of phenylalanine hydrox-
ylase in hepatocytes.19 This may be relevant to the potential use of phenylalanine as a
gluconeogenic precursor. Induction of diabetes in rats is associated with a significant
increase in the abundance of both phenylalanine hydroxylase protein and phenylalanine
hydroxylase-specific mRNA.20
Given the importance of branched-chain amino acids (BCAAs) and particularly of
leucine in the control of protein turnover, their metabolism and regulation by insulin have
been the focus of much research. BCAAs form the bulk of the amino acids released by the
splanchnic bed after a meal. The first two enzymes of the catabolic pathway of BCAAs are
BCAA-aminotransferase (BCAT) and BCKAD. In humans, transamination and oxidative
capacities are highest (66% of total capacities) in skeletal muscle.21 This is notably different
from rats in which, besides a considerably (nearly 10-fold) higher total oxidative capacity,
oxidation is mainly hepatic (60%). Hormonal regulation of BCAAT expression has not been
demonstrated except in the mammary gland.22 However, in type I diabetic patients, Nair
et al.23 have observed that insulin treatment decreases leucine transamination. BCKAD
complex represents the key step of BCAA catabolism and is subject to tight regulation.
BCKAD is subject to end-product inhibition, but the most important mechanism is its
regulation by phosphorylation/dephosphorylation. Inactivating phosphorylation is per-
formed by a BCKAD kinase and dephosphorylation by a BCKAD phosphatase, which act
on the E1 component of the enzyme complex.18 Little is known about the regulation of the
phosphatase. Evidence of BCKAD regulation by insulin is essentially indirect, deduced
either from in vivo experiments or from studies in the diabetic situation. In diabetic animals,
Lombardo et al.24 observed an increase in the hepatic BCKAD activity related to an increase
in its protein mass and decreased mass of its associated kinase. These alterations appear
to occur posttranscriptionally, since diabetes had no effect on the gene expressions of
BCKAD subunits or BCKAD kinase. On the other hand, the protein expression of BCKAD
kinase has been shown to be downregulated posttranscriptionally in the skeletal muscle
but upregulated pretranscriptionally in the cardiac muscle.25 In type I diabetic patients,
insulin withdrawal increases and insulin administration decreases leucine oxidation,23,26–29
while in streptozotocin diabetic rats, an increase in nitrogen exchange between leucine and
alanine has been demonstrated together with an increase in total alanine release by mus-
cle.30 In perfused rat hindquarter, insulin decreased leucine oxidation.31 Insulin and leucine
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190 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

both stimulate BCKAD activity, promoting dephosphorylation in the adipose tissue


enzyme.32 However, in normal fasted adults 13C-leucine oxidation measured after a 12-h
fast33 was insensitive to increased insulin with leucine clamped at a low concentration, and
oxidation increased with the physiological increases in leucine concentration after a 4-day
fast, while insulin decreased.34 This suggests that in the whole body the overriding regu-
latory influence on BCKAD is substrate availability. At increasing protein supply but
constant energy level eliciting insulin responses,35,36 leucine oxidation ranged from inhibi-
tion at the lowest to marked stimulation at the highest protein intakes, consistent with the
changes in plasma leucine. It would thus appear that there is an equilibrium between the
effect of insulin on BCKAD and protein feeding-related induction of the enzyme when the
BCAA supply is from the diet and disposal is a priority.

12.3 Insulin and protein turnover


12.3.1 Control of protein metabolism at the cellular level
12.3.1.1 Regulation of protein catabolism
Pioneering studies, in particular by Mortimore’s group,37 demonstrated that autophagic
protein degradation, which is largely responsible for liver protein degradation during
fasting, is inhibited by amino acid and insulin (see also Chapter 16). Interestingly, these
authors indicate that insulin failed to inhibit protein breakdown in the absence of amino
acids or in the presence of maximally suppressive amino acids, while it virtually sup-
pressed the zonal loss of effectiveness of regulatory amino acids at amino acid levels
matching those in the portal vein in fed rats (Figure 12.2).37

400
Valine release (nmol/min,100g rat)

350

300

250

200

150

100

50

0
0 2 4 6 8 10
Multiples of plasma amino acid concentration

Figure 12.2 Insulin and amino acid control of hepatic proteolysis. Isolated rat livers were perfused
with either regulatory amino acids (leucine, tyrosine, glutamine, proline, methionine, histidine,
tryptophan) with () or without (●) insulin or a complete plasma amino acid mixture () at multiples
of their concentration in normal plasma. Protein degradation was evaluated by the measurement
of valine release. (Adapted from Mortimore, G.E. et al., Diabetes Metab. Rev., 5, 49, 1989.)
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Chapter twelve: Insulin and the regulation of amino acid catabolism and protein turnover 191

Blommaart et al.38 showed, in isolated hepatocytes, that insulin and hypo-osmotic


conditions similarly enhance the sensitivity of autophagic proteolysis to inhibition by
amino acids. While hepatic cell swelling has been demonstrated to be an important
regulator of liver metabolism and protein turnover, insulin stimulates Na+-H+ exchange,
Na+-K+-2 Cl– cotransport, and the Na+-K+-ATPase in the liver, leading to intracellular K+,
Na+, and Cl– accumulation and thus cell swelling39 (see Chapter 16 for more details). In
parallel, insulin through its known stimulatory effect on amino transport via the Na+-
dependent system A may further enhance amino-induced cell swelling. As both cell
swelling and insulin activate PI3-kinase, a possible action via PDK1 and the mTOR path-
way may provide a common ground for these effects. Notably, distinct classes of PI3-kinase
are involved in the signaling pathways that control macroautophagy. Class IA enzymes,
which are stimulated by insulin, inhibit macroautophagy, whereas class III enzymes trigger
autophagic sequestration.40 It should also be mentioned that Petiot et al.40 did not observe
any involvement of PKB/akt in the control of these processes. On the other hand, phos-
phorylation of ribosomal protein S6 has been shown to be inhibitory for autophagy in rat
hepatocytes; both hypo-osmolarity and insulin increased phosphorylation of S6 at low
amino acid concentrations.38
An inhibitory effect of insulin on ATP and ubiquitin-dependent protein degradation
has also been demonstrated. In vitro, in rat L6 myotubes, insulin lowered levels of 14-kDa
ubiquitin-conjugating enzyme E2(14k) mRNA.41 Bennett et al.42 showed that insulin inhib-
ited protein (lysozyme) degradation by the proteasome in reticulocyte extracts by more
than 90%. This effect was also observed in HepG2 cells.42 Mitch et al.43 observed in acutely
diabetic rats that insulin reversed muscle proteolysis and that this was associated with a
normalization of the mRNAs encoding the ubiquitine–proteasome system component.
However, the mechanisms of the inhibition of proteolysis by insulin may vary according
to the fiber composition of the muscle studied.44 In a study of the influence of a euglycemic
hyperinsulinemic clamp on proteolysis measured in vitro, Larbaud et al.44 observed that
the antiproteolytic effect of insulin was suppressed by inhibitors of the lysosomal path-
ways in the slow-twitch red-fiber soleus, but by a proteasome inhibitor in the fast-twitch
mixed-fiber extensor digitorum longus.

12.3.1.2 Regulation of protein synthesis


Insulin regulates protein synthesis in mammalian cells. Besides its effect on gene expres-
sion, insulin acts at the level of mRNA translation. In the adrenalectomized streptozotocin
diabetic rat, global translation rate is inhibited by 40% even with in vivo infusions of
glucose and amino acids, and this inhibition is entirely restored by insulin infusion at
physiological levels.45 Furthermore, the increased insulin secretion within the first 20 min
of refeeding in a 4-day fasted rat is causally associated with increased skeletal muscle
protein synthesis.46 However, in vivo studies indicate that insulin effects on protein syn-
thesis may be tissue specific. Boirie et al.47 have studied, in miniature swine, fractional
synthesis rates (FSR) of mitochondrial and cytoplasmic proteins in liver, heart, and skeletal
muscle, and myosin heavy chain in muscle. Insulin infusion increased the FSR of muscle
mitochondrial proteins but had no effect on FSR of myosin. In the heart, insulin only
increased FSR of cytoplasmic protein. In liver, insulin stimulated neither mitochondrial
nor cytoplasmic protein FSR.
Only the steps of the peptide chain initiation process (Figure 12.3) affected by insulin
will be addressed here (see Chapters 16 and 18 for more details on this process). Insulin
induces a global increase in the rate of translation but also a marked increase in the
translation of specific mRNAs such as ribosomal proteins and elongation factor.48 It acts
on several steps in the translation process:
192

insulin PKB/Akt - GSK3

eIF2B eIF2B--P

GDP GTP

eIF2 eIF2 eIF2


GTP GDP GDP
Met-tRNAi
eIF4E eIF4A
eIF4G
60S
eIF3 eIF2 eIF3
eIF2 GTP 40S
1382_C12.fm Page 192 Tuesday, October 7, 2003 6:31 PM

40S eIF3
GTP Met-tRNAi
40S m7GTP-mRNA
Met-tRNAi
40S
43S preinitiation complex
80S eIF3 80S initiation complex
m7GTP-mRNA
60S
eIF4E eIF4A
eIF4G ELONGATION
60S eIF4F

eIF4E eIF4A
eIF4E
eIF4G
eIF4E-BP1
+
insulin PKB/Akt mTOR eIF4E-BP1 -P

Figure 12.3 Initiation of protein synthesis in mammalian cells. Regulation by insulin. Dissociation of the 80S ribosome into its components, 40S and 60S,
requires the eukaryotic initiation factor (eIF) 3. The binding of the initiator Met-tRNA to the 40S subunit is mediated by eIF2 (only when it is complexed
with GTP). At the following stage, eIF4F (complex of eIF4E, eIF4G, and eIF4A) binds to mRNA (m7GTP-mRNA), and the eIF4F-mRNA complex then
binds to the 43S preinitiation complex. The last stage is the addition of the 60S subunit to form the 80S initiation complex, which can enter elongation.
This stage involves the hydrolysis of the GTP bound to eIF2 and the release of eIF2-GDP along with the other eIFs. Insulin acts via PI3-kinase/PKB and
phosphorylation/inactivation of GSK-3, and enables eIF2B to promote GTP exchange on, and thus activation of, eIF2. On the other hand, insulin favors
the dissociation of eIF4E from its binding protein, eIF4E-BP1, enabling its association with the other proteins of the eIF4F complex and the subsequent
interaction of this complex with the 43S preinitiation complex. (Adapted from Proud, C.G. and Denton, R.M., Biochem. J., 328, 329, 1997; Kimball, S.R.
et al., J. Appl. Physiol., 93, 1168, 2002.)
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Chapter twelve: Insulin and the regulation of amino acid catabolism and protein turnover 193

• Regulation of eukaryotic initiation factor (eIF) 2 activity: eIF2 exists as an inactive


eIF2-GDP and an active eIF2-GTP complex, eIF2B promoting the exchange of GDP
by GTP on eIF2. eIF2B is inhibited by its phosphorylation by GSK-3. Insulin acts
via PI3-kinase and PKB/akt, which phosphorylates and inactivates GSK-3, thus
alleviating its inhibitory effect on eIF2B and leading to enhanced nucleotide ex-
change on eIF2. Increased availability of active eILF2-GTP thus promotes transla-
tion initiation at the stage of the 43S preinitiation complex.48
• Regulation of the eIF4F complex: Insulin favors the dissociation of eIF4E from
E-BP1, enabling its association with the other proteins of the eIF4F complex and
the subsequent interaction of this complex with the 43S preinitiation complex.48
This may occur via PI3-kinase and PKB/akt in the insulin signaling cascade and
modulation of the phosphorylation state of eIF4E-binding protein 1 (eIF4E-BP1)
and ribosomal protein S6 kinase (p70S6K). However, amino acids are required for
the maintenance of the insulin effect on eIF4E-BP1 and p70S6K 49 (see Chapters 16
and 18 for more details).
• Regulation of eEF2: Translation elongation requires eukaryotic elongation factors
(eEFs). eEF2, which mediates ribosomal translocation, is phosphorylated/inacti-
vated by eEF2-kinase, which is regulated by both MAPK and the mTOR signaling
pathway.50 Insulin decreases the activity of eEF2-kinase.48 This effect is influenced
by amino acids.51
• Activation of p70S6K by insulin leading to the phosphorylation of the ribosomal
protein S6 has been suggested to play an important role in the regulation of specific
mRNAs encoding for ribosomal and elongation factors, the so-called 5'-terminal
oligopyrimidine (TOP) mRNAs.52

12.3.2 Insulin and the control of protein metabolism in vivo in humans


Thus from in vitro or cells-in-culture studies it seems reasonable to assume that insulin
exerts a marked inhibitory effect on proteolysis and a stimulatory one on synthesis. While
the in vivo antiproteolytic effect of insulin has been repeatedly demonstrated, the anabolic
effect of insulin on protein synthesis is largely debated.
The main difficulty in interpreting whole-body studies arises from the combination
of direct and indirect actions of insulin and of the associated changes in amino acid
concentrations. The direct effect of amino acids on protein synthesis and catabolism and
the hypoaminoacidemia induced by insulin administration can largely account for the
discrepancies in insulin effects.53 Given that the role of insulin is to preserve amino acids
for protein synthesis, insulin-induced or insulin-independent changes in amino acid avail-
ability may affect the response of protein metabolism because of its sensitivity to amino
acid concentration.

12.3.2.1 Regulation of protein catabolism


In humans, the rate of whole-body protein degradation falls in response to physiological
increases in plasma insulin.54 Conversely, whole-body protein degradation is increased in
insulin-withdrawn type 1 diabetic patients.23,28,29 When the fall in plasma amino acid is
prevented either by amino acid perfusion or by an oral protein supply, an inhibition of
proteolysis by insulin is observed in type I diabetes.27,55 Of note, Biolo et al.53 observed
that whole-body proteolysis was suppressed during meal ingestion in type I diabetic
patients despite insulin withdrawal; this finding suggests that the influences of amino
acids and insulin on proteolysis are separate and additive. In normal subjects, insulin-
induced inhibition of proteolysis is enhanced by higher levels of amino acids.34
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194 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

The tissue location of insulin action on proteolysis is a subject of debate. While several
investigations indicate that insulin is most effective on skeletal muscles,56–58 this preference
has not been consistently observed.59,60 In a study in type I diabetic patients, Nair et al.23
observed that insulin also causes an inhibition of protein breakdown in splanchnic tissues.

12.3.2.2 Regulation of protein synthesis


On a whole-body basis, the anabolic effect of insulin on protein synthesis has not been
unequivocally demonstrated.28,61,62 On the contrary, some studies have even suggested an
insulin-induced reduction in protein synthesis.23,26 In diabetic patients, some studies indi-
cate that both protein breakdown and synthesis increase during insulin deprivation; net
protein loss may thus be due to a greater increase in catabolism.63
While limitations of method may be responsible, as suggested by Biolo et al.,64 who
observed a stimulating effect of insulin using arteriovenous catheterization and muscle
biopsy approaches, one explanation could be the sensitivity of protein synthesis to amino
acid availability:

• A study using varying intakes of protein eliciting similar insulin levels indicates
that the response of protein synthesis depends on plasma leucine levels.35 Volpi et
al.,65 measuring whole-body protein kinetics in healthy volunteers during intra-
gastric infusion of water, glucose–lipid–amino acid meal, or isocaloric glucose–lip-
id meal, showed that dietary amino acids account for approximately 90% of post-
prandial protein anabolism and insulin for only 10%. When amino acid levels are
clamped, increasing insulin up to levels inducing maximal suppression of proteol-
ysis did not influence whole-body protein synthesis.34,57 However, during an insu-
lin–glucose clamp with amino acid infusion, Bennet et al.61 observed in type I
diabetic patients that protein synthesis was unaltered by insulin, but that there
was a concomitant decrease in the intramuscular concentrations of a number of
amino acids, including leucine. This suggests that the decrease in amino acid
availability has hampered the effect of insulin on protein synthesis. In another
study in healthy subjects, the same authors62 reported that insulin, given with a
sufficient amount of amino acids, may stimulate leg and whole-body protein
synthesis.
• It has been suggested that the relative sensitivity of protein synthesis and proteol-
ysis to insulin and amino acids may differ between rats and humans; this might
thus also account for the lack of stimulating effect of insulin in humans.66 In
humans, the inhibition of proteolysis could be the major response to insulin; a
reduction in intracellular amino acid concentrations and specifically of leucine
would thus inhibit protein synthesis.
• This question is further complicated by possible pathology-related or amino acid-
induced alterations in the sensitivity to insulin action. Deficient protein turnover
sensitivity to insulin action has been described both in type I and type II diabe-
tes.27,62,67,68 On the other hand, while the stimulatory effect of specific amino acids
such as leucine and arginine on insulin secretion has been largely demonstrated
(see Chapter 20 for details), several in vivo studies have pointed to a deterioration
of tissue response to insulin action in various situations, including high-protein
diet. Infusion of amino acids during a euglycemic hyperinsulinemic clamp in
normal fasted volunteers decreases whole-body glucose oxidation, nonoxidative
glucose disposal, forearm glucose disposal, and responsiveness of hepatic glucose
production to insulin.69 Patti et al.70 have shown that this occurs via decreased
insulin-stimulated tyrosine phosphorylation of IRS-1 and a marked inhibition of
insulin-stimulated PI3-kinase. They suggested that amino acids act as specific
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Chapter twelve: Insulin and the regulation of amino acid catabolism and protein turnover 195

positive signals for maintenance of protein stores, while inhibiting other actions
of insulin on glucose metabolism. Tremblay and Marette71 further demonstrated
in L6 myotubes that amino acids speed up the time-dependent deactivation of IRS-
1-associated PI3-kinase via a rapamycin-sensitive increase in serine/threonine
phosphorylation of IRS-1 and decreased binding of PI3-kinase to IRS-1.

Thus, while protein synthesis is clearly regulated by amino acid levels, the exact
contribution of insulin remains to be clearly delineated.

12.4 Conclusion
While we gradually map the pathways of insulin action on amino acid metabolism, new
knowledge concerning the interference of nutrients comes and blurs the picture. Although
insulin is demonstrably a major contributor to the regulation of amino acid and protein
metabolism, the precise delineation of the pathways involved, along with the possible
species-to-species and tissue-to-tissue particularities, remains to be established. Building
on initial studies, which indicated that insulin acts via PI3-kinase while numerous medi-
ators also act on this pathway, we now know that a specific class of PI3-kinase is induced
by insulin. The possibility of subtypes of other intermediates of insulin signaling cannot
be ruled out, while the unraveling of the complex world of transcription factors will
probably yield interesting findings. For example, peroxisome-proliferator-activated recep-
tor (PPAR) a activators promote BCAA oxidation by increasing expression of components
of the BCKAD complex and decreasing expression of the BCKAD kinase,72 while PPARg
agonist potentiates insulin-stimulated Akt phosphorylation in adipose, muscle, and liver
tissues.73 The effects of exercise on muscle insulin signaling and action are also of great
interest in the context of the present “diabesity” pandemic. Muscle insulin action on
glucose transport, glycogen synthesis, and amino acid transport has been demonstrated
even after a single bout of exercise.74 Moreover, some data suggest that insulin in combi-
nation with prior contractions induces a stimulation of protein synthesis at physiological
concentrations of amino acids.75

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30. Meynial-Denis, D., Chavaroux, A., Foucat, L., Mignon, M., Prugnaud, J., Bayle, G., Renou,
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infusion of somatostatin and insulin, Eur. J. Clin. Nutr., 45, 515, 1991.
37. Mortimore, G.E., Pösö, A.R., and Lardeux, B.R., Mechanism and regulation of protein deg-
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De Kreutzenberg, S.V., and Tiengo, A., Effects of insulin on whole body and forearm leucine
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chapter thirteen

Control of amino acid metabolism


by counterregulatory hormones
Jan Wernerman
Karolinska Institutet

Contents
Introduction..................................................................................................................................201
13.1 Plasma concentration of amino acids.............................................................................202
13.2 Muscle free amino acids...................................................................................................204
13.3 Protein metabolism............................................................................................................204
13.4 Conclusion and perspective.............................................................................................206
References .....................................................................................................................................207

Introduction
The metabolic alterations seen following trauma were early recognized to be attributable
to increased levels of stress hormones, predominantly adrenaline, cortisol, and glucagon.1,2
In animal experiments a hypoglycemia model was used that, to some extent, produced
alterations, preferable in carbohydrate metabolism, which had similarities to what was
seen following trauma.3 The hypoglycemic event also created elevations of adrenaline,
cortisol, and glucagon, and thereby these hormones were given the name counterregula-
tory hormones. In particular, glucagon has also been used in clinical contexts to counteract
hypoglycemia. In carbohydrate metabolism the counterregulatory hormones induce a state
of hyperglycemia, enhanced endogenous glucose production as well as enhanced gluco-
neogenesis. The obvious links between carbohydrate and amino acid metabolism soon
led to a series of experiments where the hypoglycemic event was exchanged by an infusion
of the three most important hormones: adrenaline, cortisol, and glucagon.4 The triple
hormone cocktail soon became a standardized experimental setup that could be used in
experimental animals or in healthy volunteers to study metabolic events associated with
trauma and sepsis in a standardized manner.
In carbohydrate metabolism it was quite obvious that the three counterregulatory
hormones had a synergistic effect upon, for example, plasma glucose levels, compared to
when the singular hormones were given individually.5 For both cortisol and glucagon,

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202 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

metabolic effects were seen at a much lower plasma level than when these hormones were
given isolated. The mechanisms for these actions are perhaps still not fully understood,
but cortisol may act on the adrenergic receptors to down-regulate them and also to sustain
the effects for longer periods of time. In general, cortisol induces net protein breakdown
and hyperaminoacidemia.6 The regulation of glucagon effects is less well understood, but
glucagon facilitates the uptake of substrates into the liver, preferably by system A.7 In high
concentrations, glucagon causes hypoaminoacidemia. In general terms, stress hormones
increase the whole-body metabolic rate.8,9 When it comes to the effects of the counterreg-
ulatory hormones upon amino acid metabolism and protein metabolism, interest was
initially focused upon alanine as one of the important precursors for gluconeogenesis. In
parallel to the effects upon plasma glucose, the effects on plasma alanine concentration
are mainly confined to the time interval when the plasma concentrations of the counter-
regulatory hormones are elevated.10 This singular finding clearly indicates that the hor-
mone cocktail infused only partially mimics the actual events in conjunction with trauma
and sepsis. Also, concerning glutamine kinetics and the exchange of alanine across the
leg, there are distinct differences between a triple hormone infusion and catabolic
patients.10–12

13.1 Plasma concentration of amino acids


When looking upon the effects of hormones on the plasma amino acid concentrations,
there are two major things to consider: the direction of changes and the time pattern.
Sometimes the direction of changes is not consistent over time, which makes this compli-
cated but also fascinating when trying to understand the underlying mechanisms. The
major part of the effects elicited by a triple hormone combination is produced by adren-
aline alone,10,13 and is therefore abolished by a simultaneous beta-blockade.13 During the
infusion of counterregulatory hormones for up to 6 h, there is a general pattern in which
the concentrations of most of the essential amino acids decrease.10,14,15 The only exception
is alanine, which starts to increase after a short period of stress hormone infusion.10 When
the infusion of counterregulatory hormone stops, this is the end of the story, and the
plasma amino acid concentrations go back to normal.10 However, there are a number of
exceptions. Glutamine, for example, decreases under the influence of counterregulatory
hormones, but afterwards, glutamate exhibits an overshot above the basal value before
coming back to normal (Figure 13.1). Another pattern is shown by the branched-chain
amino acids (leucine, valine, and isoleucine), which initially go down but later increase
to be significantly elevated in plasma 24 h after the end of the infusion. The increased
plasma concentrations of branched-chain amino acids are also seen on the third day after
surgical trauma.16 However, also following elective surgery the biphasic pattern is seen
with a decrease of the plasma concentrations of branched-chained amino acids at 12 h
postoperatively, coming back to normal 24 h following the operation.17 Among the other
essential amino acids, the aromatic amino acids show high plasma levels at 24 h; this is
marginally significant, but on the 3rd and even 10th days postoperatively, values are
high.16,17 In general, plasma amino acid patterns following elective surgical trauma in
otherwise healthy individuals show increased amino acid concentrations in plasma during
a 2-week period following surgery. It is therefore obvious that the counterregulatory
hormones as well as the surgical trauma initiate changes over time that are much longer
than those during which the plasma levels of counterregulatory hormones are elevated.
During prolonged periods of stress, as for intensive care patients, plasma amino acid levels
are normal or close to normal for most amino acids.18,19 The exception is glutamine, which
holds a low plasma concentration, also related to outcome.20 On the other hand, the levels
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Chapter thirteen: Control of amino acid metabolism by counterregulatory hormones 203

700
*
600
plasma conc . (µ mo l/L)
500

400
** *
300
**

200
**
100
** glutamine
BCAA
st ress hormon e infusi on

0
0 6 12 18 24

time (h)

Figure 13.1 Concentrations of glutamine and the sum of branched-chain amino acids (BCAA) in
plasma of healthy volunteers during and after a combined stress hormone infusion of adrenaline,
cortisol, and glucagon. * and ** denote significantly different from basal values, p < 0.05 and p < 0.01,
respectively. (Data from Wernerman, J. et al, Clin. Nutr., 4, 207–216, 1985; Shamoon, H. et al., Diabetes,
29, 875–881, 1980; Wernerman, J. et al., Clin. Physiol., 13, 309–319, 1993.)

of aromatic amino acids, in particular phenylalanine, are usually higher than normal.19 In
experiments with a prolonged infusion of counterregulatory hormones over several days,
slight increases in the plasma levels of alanine and phenylalanine were the only changes
seen.21 As for the exchange of amino acids across muscle tissue, a prolonged stress hormone
infusion does not elicit any changes, compared to the basal situation.21 In burn patients
there is a considerable efflux of alanine,12 while the efflux of glutamine is sometimes
reported elevated but in other studies not found different from what is seen in healthy
individuals.12,22,23
For glutamine a decrease in plasma concentration is seen in response to adrenaline
alone. This slight decrease is accompanied by increases in the rate of appearance as well
as the rate of disappearance.24,25 The rate of appearance corresponds to an increased release
from skeletal muscle, while the rate of disappearance corresponds to an increase in renal
gluconeogenesis.24 In response to physiological concentrations of cortisol or glucagon, the
plasma concentration of glutamine, as well as the rates of appearance and disappearance,
is not altered.26 For alanine the plasma concentration increases slowly in conjunction with
a triple hormone infusion. In parallel, the efflux from the leg is only marginally increased
after 60 min of stress hormone infusion but later on is unaltered, compared to basal.10,12
This is in contrast to the marked increase in alanine concentration in skeletal muscle
described below, which is seen accompanying adrenaline alone as well as a triple hormone
combination.10 The increase of essential amino acids, in particular the branched-chain
amino acids and the aromatic amino acids seen in response to counterregulating hormones
as well as following trauma and sepsis, is still obscure. However, a relation to an increase
in protein degradation has been suggested.27
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204 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

13.2 Muscle free amino acids


In general, tissue free amino acids are believed to stay in balance with plasma concentra-
tion. However, this is an oversimplification, and many times plasma is even a poor
reflection of what is happening inside the cells. As the tissue free amino acid pool is the
precursor pool for protein synthesis as well as the substrate pool for oxidative processes,
changes in the tissue free amino acid pool are perhaps a better reflection of metabolic
events. It must be emphasized that the intracellular free amino acid concentrations vary
considerably between different tissues. Following an infusion of the counterregulatory
hormones, the changes come much slower, compared to plasma.10,14
In muscle, glutamine and alanine are the two amino acids responding already within
an hour, glutamine going down, while alanine increases. Supposedly this reflects an
enhanced export of gluconeogenetic precursors from muscle. The branched-chain amino
acids respond with a biphasic pattern similar to that seen in plasma (Figure 13.2).
Glutamine concentration, on the other hand, shows a steady decrease in muscle during
the 24 h after a 6-h triple hormone infusion without any signs of restitution.15 Following
elective surgery, a similar pattern is seen over the first 3 or 4 days.16,17 After that, a gradual
restitution back to normal is seen up to the 7th or 10th day (Figure 13.3). Following severe
septic events, glutamine is shown to be the last amino acid to be restored back to normal
concentration in muscle.16,28 This slow restoration stands in contrast to the rapid restoration
of intracellular free glutamine depletion that occurs during refeeding after short-term
starvation.29 The regulating mechanisms for glutamine depletion as well as repletion are
not well understood. Muscle glutamine concentration drops rapidly in septic patients in
the ICU, most often down to 25% of normal levels.18,19 In general, nonessential amino acids
stay rather stable in plasma concentration during counterregulatory hormone infusion,
but in muscle, several nonessential amino acids such as glutamine, alanine, and glutamate
exhibit distinct changes of concentrations. Glutamate, which holds a comparatively high
concentration in muscle, compared to the low concentration in plasma, shows a rapid
decrease during hormone infusion and a gradual normalization over the next 18 h. Muscle
alanine concentration increases during the counterregulatory hormone infusion, but is
rapidly normalized after that. The majority of these experiments are performed in healthy
volunteers in the basal state. Simultaneous food intake given as intravenous nutrition
abolishes these changes.30 The metabolic handling of the nutrients, on the other hand, is
only marginally affected by elevated concentrations of stress hormones, at least in terms
of amino acids.31,32 During a prolonged stress hormone infusion, however, intracellular
glutamine concentration in muscle decreases despite food intake.21

13.3 Protein metabolism


As amino acids are the brick stones and currency of protein metabolism, amino acid
metabolism and protein metabolism are closely related. It was early shown that an infusion
of counterregulatory hormones induces a negative whole-body nitrogen balance in healthy
volunteers.33,34 This is, of course, a reflection of what also happens after a surgical trauma.
On the tissue level, muscle protein synthesis as reflected by the concentration and size
distribution of ribosomes decreases following a triple hormone infusion.35 The same hap-
pens during infusions of adrenaline or cortisol only, but the combination gives a more
profound decrease. Also, an in vitro assay of incorporation of a labeled amino acid into
polypeptide chains in a cellular fraction of isolated ribosomes shows a decrease in response
to stress hormones.36 The effects on skeletal muscle concern both synthesis and degrada-
tion of muscle proteins.37 In quantitative terms, the muscle protein synthesis rate is only
marginally decreased, by 15%, at the end of a 6-h stress hormone infusion.38 However,
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Chapter thirteen: Control of amino acid metabolism by counterregulatory hormones 205

15 .75

muscle conc. (mmol/k g)


**
10 0.50 *

***

*
5 0.25

**
glutamine
BCAA
st ress hor mone i nfusi on

0
0 6 12 18 24
time (h)
Figure 13.2 Concentrations of glutamine and the sum of branched-chain amino acids (BCAA) in
muscle of healthy volunteers during and after a combined stress hormone infusion of adrenaline,
cortisol, and glucagon. The left scale on the y-axis applies for glutamine, while the right scale applies
for the sum of branched-chain amino acids. *, **, and *** denote significantly different from basal
values, p < 0.05, p < 0.01, and p < 0.001, respectively. (Data from Wernerman, J. et al, Clin. Nutr., 4,
207–216, 1985; Wernerman, J. et al., Clin. Physiol., 13, 309–319, 1993.)

after normalization of plasma hormonal levels 18 h later and then continuing decrease of
the muscle protein synthesis rate, a drop of 30% is seen, compared to the preinfusion
levels. This has also been investigated in circulating lymphocytes, which also reveal a 40
to 60% decrease of in vivo protein synthesis following a 6-h triple hormone infusion.38,39
This goes together with an increase in the count of circulating leukocytes but a decrease
in the circulating numbers of lymphocytes. All subfractions of lymphocytes show a similar
decrease as the total numbers, except for the natural killer cells, which respond with an
increased fraction. Cortisol alone elicits none of these changes.40 For the circulating lym-
phocytes, the time pattern is different from that of the skeletal muscle. The decrease in
protein synthesis rate seen at the end of the 6-h stress hormone infusion is totally abolished
18 h later, when the decrease seen in the muscle protein synthesis rate is even more
pronounced.38 This also has some resemblance to the response seen following elective
surgery, when a depression of the muscle protein synthesis rate occurs in the early post-
operative period and is not reversed on the third postoperative day regardless of nutrition
support.41 For protein synthesis in lymphocytes, the postoperative time course has not
been fully characterized, but 24 h postoperatively, an enhanced protein synthesis rate in
circulating lymphocytes is seen regardless of postoperative nutrition or a preoperative
glucose load.42 Elevated stress hormone concentrations also influence the synthesis rate
of albumin, which is unaffected at the end of a 6-h infusion.38 Subsequently, at 18 h
postinfusion a 15% increase in albumin synthesis rate is seen. This is in accord with the
increased albumin synthesis rate seen 24 h postoperatively and in ICU patients.43–45 How-
ever, the possibility of an early decrease in albumin and total liver protein synthesis rate
1382_C13.fm Page 206 Tuesday, October 7, 2003 6:33 PM

206 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

16 1.00

**
muscle conc. (mmol/k g) ** *
12 0.75 *

**
* ***
8 0.50

4 0.25

glutamine
elective BCAA
su rgery

0
0 24 48 72 96 120 144 168 192 216 240

time (h)
Figure 13.3 Concentrations of glutamine and the sum of branched-chain amino acids (BCAA) in
muscle of metabolically healthy patients undergoing elective abdominal surgery of medium size.
The left scale on the y-axis applies for glutamine, while the right scale applies for the sum of
branched-chain amino acids. *, **, and *** denote significantly different from basal values, p < 0.05,
p < 0.01, p < 0.001, respectively. (Data from Petersson, B. et al., Br. J. Surg., 79, 212–216, 1992; Essén,
P. et al., Clin. Physiol., 12, 163–177, 1992.)

in response to stress hormones is suggested by the findings of a sharp decrease in total


liver protein synthesis rate as well as albumin synthesis rate already during a laparoscopic
surgical procedure.46 Presently, however, there are no data concerning albumin synthesis
during the early phase of a stress hormone infusion.

13.4 Conclusion and perspective


Amino acid metabolism is regulated to a great extent by hormones, which also means that
protein metabolism is influenced.47 In conjunction with trauma and sepsis, the stress
hormones are of particular importance, and it is their combined effect that works on amino
acid and protein metabolism. In addition to the mainly catabolic hormones discussed here,
hormones with anabolic or partly anabolic actions, such as insulin, growth hormone,
insulin-like growth factor I, and sex steroids, are also active and make the final picture
even more complicated. As emphasized above, it is the direction of changes and the time
pattern of changes that are important. Furthermore, as indicated above, individual tissues
may respond quite differently to the same hormonal challenge. Although a lot of work
was done on basic physiology 30 to 40 years ago, there are still many questions that need
to be answered, especially when several hormones appear simultaneously. New tech-
niques, in particular the use of stable isotopes, also provide the possibility of reassessing
old dogmas, which sometimes appear to be oversimplifications of the underlying mech-
anisms.
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Chapter thirteen: Control of amino acid metabolism by counterregulatory hormones 207

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14. Wernerman, J., Hammarqvist, F., Botta, D., and Vinnars, E., Stress hormones alter the pattern
of free amino acids in human skeletal muscle, Clin. Physiol., 13, 309–319, 1993.
15. Hammarqvist, F., Ejesson, B., and Wernerman, J., Stress hormones initiate prolonged changes
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16. Petersson, B., Vinnars, E., Waller, S.-O., and Wernerman, J., Long-term changes in muscle
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17. Essén, P., Wernerman, J., Sonnenfeld, T., Thunell, S., and Vinnars, E., Free amino acids in
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163–177, 1992.
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23. Vesali, R.F., Klaude, M., Rooyackers, O.E., Tjäder, I.I., Barle, H., and Wernerman, J., Longi-
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24. Gerich, J.E., Meyer, C., and Stumvoll, M.W., Hormonal control of renal and systemic
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Nutr., 23, 279–287, 1999.
26. Battezzati, A., Simonson, D.C., Luzi, L., and Matthews, D.E., Glucagon increases glutamine
uptake without affecting glutamine release in humans, Metabolism, 47, 713–723, 1998.
27. Fürst, P., Regulation of intracellular metabolism of amino acids, in Nutrition in Cancer and
Trauma Sepsis, Bozetti, F. and Dionigi, R., Eds., Karger, Basel, Switzerland, 1985, pp. 21–53.
28. Askanazi, J., Carpentier, Y.A., Michelsen, C.B., Elwyn, D.H., Furst, P., Kantrowitz, L.R.,
Gump, F.E., and Kinney, J.M., Muscle and plasma amino acids following injury: influence
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acid metabolism in skeletal muscle, Metabolism, 43, 1158–1163, 1994.
31. Fong, Y.M., Albert, J.D., Tracey, K., Hesse, D.G., Calvano, S., Matthews, D.E., and Lowry,
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36. Wernerman, J., von der Decken, A., Hammarqvist, F., Botta, D., and Vinnars, E., Incorporation
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37. Gore, D.C., Jahoor, F., Wolfe, R.R., and Herndon, D.N., Acute response of human muscle
protein to catabolic hormones, Ann. Surg., 218, 679–684, 1993.
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39. Januszkiewicz, A., Essén, P., McNurlan, M., Ringdén, O., Wernerman, J., and Garlick, P.,
Protein synthesis of circulating human T lymphocytes does not respond to a cortisol chal-
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combined stress hormone infusion decreases in vivo protein synthesis in human T lym-
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43. van Acker, B., Hulsewé, K.W.E., Wagenmakers, A.J.M., Deutz, N.E.P., von Meyenfeldt, M.F.,
and Soeters, P.B., Effect of surgery on albumin synthesis rate in humans, Clin. Nutr., 17
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Albumin and fibrinogen syntheses increase while muscle protein synthesis decreases in head-
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45. Essén, P., McNurlan, M.A., Gamrin, L., Garlick, P.J., and Wernerman, J., Tissue protein
synthesis rate in critically ill patients, Crit. Care Med., 26, 92–100, 1998.
46. Barle, H., Nyberg, B., Ramel, S., Essén, P., McNurlan, M., Wernerman, J., and Garlick, P.,
Inhibition of liver protein synthesis during laparoscopic surgery, Am. J. Physiol., 277,
E591–E596, 1999.
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chapter fourteen

Nitric oxide
Eric J. Mahoney
Rhode Island Hospital
Jorge E. Albina
Rhode Island Hospital

Contents
14.1 The biology of nitric oxide...............................................................................................211
14.1.1 The nitric oxide synthases...................................................................................212
14.1.1.1 nNOS (NOS I).........................................................................................214
14.1.1.2 iNOS (NOS II).........................................................................................214
14.1.1.3 eNOS (NOS III) ......................................................................................215
14.1.2 Nitric oxide: mechanisms of action ...................................................................215
14.2 Nitric oxide: physiology and pathophysiology in mammalian cells .......................217
14.2.1 Nitric oxide and the vasculature........................................................................217
14.2.2 Nitric oxide, wound repair, infection, and septic shock ................................220
14.2.3 Nitric oxide and apoptosis ..................................................................................223
14.2.4 Nitric oxide and cancer........................................................................................224
14.2.5 Nitric oxide and cellular energy metabolism...................................................226
14.2.6 Nitric oxide and the nervous system ................................................................226
14.3 Conclusion ..........................................................................................................................228
Acknowledgments ......................................................................................................................228
References .....................................................................................................................................228

14.1 The biology of nitric oxide


Nitric oxide (NO) and its biochemistry have had an almost unprecedented effect on the
way in which cellular interactions are understood. In historical perspective, the chemical
properties of nitric oxide were described by Joseph Priestley1 in 1772 after exposing various
metals to nitric acid. More recently, nitric oxide was allocated to the fields of food pres-
ervation in the curing of meat as well as an environmental pollutant from combustion
engines. However, upon its discovery as an effector molecule in mammalian physiology,2–6
nitric oxide was catapulted to a position of extreme importance. Its functions have been
shown to include signal transduction, cytotoxicity, vascular tone, and inflammation, to

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212 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

name only a few. This recognition was demonstrated by it being named “molecule of the
year 1992” in Science7 and culminated in Drs. Furchgott, Murad, and Ignarro being
awarded the Nobel Prize for Medicine in 1998 for the discovery and subsequent physio-
logic characterization of nitric oxide.
The reader is advised that the information presented herein has been obtained from
various animal and cell models. Like many other physiologically relevant molecules, there
can be dramatic differences in function across species and even across organ systems
within the same species. Further complicating this picture, the actions of nitric oxide can
change according to additional factors such as NO concentration, redox status of the target
cell, or even which reactive nitrogen species is active given the specific milieu. Although
the term NO is used for simplicity, what NO synthase (NOS)-containing cells contribute
to their environment is proposed to be a mixture of NO, NO2, N2O3, NO2–, NO3–, nitro-
samines, nonprotein nitrosothiols, and S-nitrosylated proteins.8 This chapter will attempt
to provide a cogent view of this highly protean molecule and its role in a biological system.

14.1.1 The nitric oxide synthases


The nitric oxide synthases are a family of three isoforms9 that catalyze the conversion of
L-arginine to nitric oxide and citrulline.

L - arginine æ ææÆ nitric oxide + citrulline


NOS

Based on initial description, these three isoforms were divided into two groups:
constitutive, containing nNOS (NOS I) and eNOS (NOS III), and inducible (iNOS, NOS II).
However, as will be discussed, it is now realized that the expression of nNOS and eNOS
can be induced under certain physiological conditions. Similarly, iNOS may function
constitutively under other conditions.
The three isoforms do share the same domain structure and catalytic mechanism.
Specifically, the overall amino acid sequence homology for the three human NOS isoforms
is approximately 55%, with particularly strong sequence conservation noted in regions
involved in catalysis.10 Across species, the homology between equivalent isoforms aver-
ages 90 ± 6%.9
Each of the NOS isomers is a homodimer made up of monomers containing a reductase
domain and an oxygenase domain, with the active site being an iron protoporphyrin IX
(heme) within the oxygenase domain. Each domain has absolute cofactor requirements:
the C-terminal reductase domain requires NADPH, FAD, and FMN; the N-terminal oxy-
genase domain requires heme, tetrahydrobiopterin, and L-arginine. Connecting these two
domains is a calmodulin consensus-binding domain.11 Along with these requirements,
each NOS is functionally active only in dimeric form because the active site requires two
hemes.12 In order to dimerize, each monomer must incorporate the heme moiety, and
dimerization is promoted and stabilized by the binding of tetrahydrobiopterin and L-argi-
nine.13,14 Further stabilizing dimerization, each isoform has been shown to contain zinc,15
whose binding site is at the dimeric interface.16 The binding of zinc has been shown to
function structurally rather than catalytically and is important for optimum enzymatic
activity.15
The flow of electrons within NOS has been established, from NADPH to FAD to