Amino Acids

296 Pages·1986·21.25 MB·English

by  John A. Sturman

NEUROMETHODS 0 3
Amino Acids
NEUROMETHODS
Prouram Editors: Alan A. Boufton and Glen 8. Baker
Series 1: Neurochemistry
1. General Techniques
Edlted by Alan A. Boulton and Glen B. Baker, 1985
2. Amines and Their Metabolites
Edited by Alan A. Boulton, Glen B. Baker, and Judith M, Baker,
1985
3. Amino Acids
Edlted by Alan A, Boulton, Glen B. Baker, and James D. Wood,
1985
4. Receptor Binding
Edited by Alan A. Bouiton, Glen B. Baker, and Pave1 D. Hrdina,
1986
5. Enzymes
Edited by Alan A. Boulton, Glen B. Baker, and Peter H. Yu, 1986
NEUROMETHODS
Series I: Neurochemistry
Proaram Editors: Alan A. Boulton and Glen B. Baker
NEUROMETHODS 0 3
Amino Acids
Edited by
Alan A. Boulton, Glen B. Baker,
and James D. Wood
Humana Press Clifton, New Jersey
l
Library of Congress Cataloging in Publication Data
Mam entry under title
Ammo acids
(Neuromethods, 3 Series I, Neurochemistry)
Includes bibliographies and index.
1 Ammo acids-Analysis 2 Neurochemlstry-
Technique. I. Boulton, A. A (Alan A.) II Baker,
Glen B., 1947- III Wood, J. D. IV Series
Neuromethods, 3 V Series Neuromethods Series I,
Neurochemistry. [DNLM 1, Ammo acids-analysis
2. Neurochemistry-methods Wl NE337G v 3 / QU 60 A5161
QP 561.A468 1985 591 19’245 85-24862
ISBN O-39603-077-6
0 1985 The Humana Press Inc
Crescent Manor
PO Box 2148
Clifton, NJ 07015
All rights reserved
No part of this book may be reproduced, stored m a retrieval system,
or transmitted m any form or by any means, electronic, mechanical,
photocopymg, microhlmmg, recording, or otherwise without writ-
ten permission from the Publisher
Printed in the Umted States of America
Foreword
Techniques in the neurosciences are evolving rapidly. There are
currently very few volumes dedicated to the methodology em-
ployed by neurosclentlsts, and those that are available often seem
either out of date or limited m scope. This series is about the
methods most widely used by modern-day neurosclentists and 1s
wrltten by their colleagues who are practicing experts.
Volume 1 will be useful to all neurosclentists since It concerns
those procedures used routinely across the widest range of
subdisciplines. Collecting these general techniques together m a
single volume strikes us not only as a service, but will no doubt
prove of exceptIona utilitarian value as well. Volumes 2 and 3 de-
scribe all current procedures for the analyses of amines and their
metabolltes and of ammo aads, respectively. These collections
will clearly be of value to all neuroscientlsts working m or
contemplating research m these fields. Similar reasons exist for
Volume 4 on receptor binding techniques since experimental de-
tails are provided for all types of llgand-receptor bmdmg,
including chapters on general prmciples, drug discovery and de-
velopment, and a most useful appendix on computer programs
for Scatchard, nonlinear, and competitive displacement analyses.
Volume 5 provides procedures for the assessment of enzymes in-
volved m biogenic amme synthesis and catabolism.
Volumes in the NELJROMETHODS series will be useful to
neurochemists, -pharmacologists, -physiologists, -anatomlsts,
psychopharmacologists, psychlatnsts, neurologists, and chemists
(organic, analytical, pharmaceutical, medicinal), in fact, everyone
involved m the neurosclences, both basic and clinical.
V
Preface
Although it was m 1913 that Aberholden and Weil described
the individual amino acid content of hydrolyzates of gray matter,
white matter, spinal cord, and peripheral nerve, many years were
to pass before a defmitive analysis of the levels of the various free
amino acids in the central nervous system was reported. The de-
velopment of microbiological assays for various amino acids led in
1950 to the studies of Schurr and coworkers that provided data on
the levels of 12 different ammo acids m brain tissue However,
the procedures used m these mvestigations did not allow the de-
termination of tissue levels of glutamate, glutamine, aspartate,
glycme, alanme, or serme, among whrch group of amino acids are
some of particular interest to neuroscientists because of their role
or potential role as ammo acid transmitter substances.
The microbial assays were, however, rapidly superseded by
the advent of paper and ion-exchange chromatographic tech-
niques that were based on the initial studies m the 1940s by
Consden, Gordon, and Martin. Ion-exchange chromatography m
particular was sublect to vigorous technical improvements re-
sulting m the use of both cationic and anionic resins for the esti-
mation of the ammo acids. By the early 1950s Partridge and
coworkers m Britain, and Moore and Stem in the Umted States,
had developed these ion-exchange chromatographic procedures
to the point that the methods were adopted by a multitude of lab-
oratones. Thus came about the “mformation explosion” on the
ammo acid levels m innumerable species and tissues, as well as
on the effects of drugs and experimental conditions on these lev-
els. The 1950s also saw the development of gas chromatography
by James and Martin and of improvements in thin layer chroma-
tography by Stahl, both methodologies eventually being incorpo-
rated into the battery of chromatographic techniques used in the
determination of ammo acids.
This volume deals with the maIor procedures currently being
applied to ammo acid analysis, some of them relying essentially
VII
Preface
on long-established methodologres (e.g., automated ammo acid
analysis; gas chromatography), others depending on compara-
tively recent developments based on earlier-established tech-
niques (e.g , HPLC; gas chromatography-mass spectrometry;
double-isotope dansylation), and yet others based on entirely
new approaches (e g , radioreceptor assays). In addition, this
volume contains chapters on related methodologies that are vital
tools for researchers working m the field of amino acids and the
central nervous system Examples of these are the techniques
used to study the uptake and release of ammo acids from nervous
tissue, the location of the y-aminobutyric acid (GABA) system m
the central nervous system by autoradlography or by immunocy-
tochemistry, as well as the turnover of ammo acid transmitters
Some duplication may be evident between chapters However,
each author vrews the situation from a different perspective, and
the presentation of the material from both viewpomts can be re-
freshing and informative.
The chapter on automated amino acid analysis describes
long-established procedures, but also provides the reader with an
insight mto the various problems and limitations encountered
with the procedures Two chapters are devoted to the analysis of
amino acids by gas chromatography procedures, one describing
the use of gas chromatography per se, and the other describmg
this technique in combination with mass spectrometry. The latter
combined procedure couples the excellent separative power of
gas chromatography with the direct identification and quantita-
tion capabilities of mass spectrometry. Unfortunately, the high
cost of the equipment does not make it readily available to many
researchers
Another methodology described in this volume IS the use of
I-dimethylammonaphthalene-5-sulfonyl chloride (dansyl chlo-
ride) in the estimation of ammo acid levels. A double-isotope
dansyl microassay for cerebral amino acids IS described in detail,
including the use of thin-layer chromatographic techniques for
the separation of the dansyl derivatives The usefulness of the
double-isotope labeling in overcommg problems inherent m the
dansyl procedure IS discussed by the author. Apart from a liquid
scintillatron counter, which IS available m most laboratones, no
sophisticated or expensive equipment is required for the
microassay On the other hand, extensive and time-consuming
manual manipulations are required for the procedure.
Preface lx
The application of high performance liquid chromatography
(HPLC) to the analysis of amino acids forms the sublect matter of
another chapter HPLC is one of the more recent developments in
ammo acid determmations, and the use of precolumn fluorogenic
derivatization m combination with reversed-phase liquid chroma-
tography has simplified and improved the separation of ammo
acids. This system combmes simplicity of sample preparation
with high sensitivity and speed of analysis. Not surprismgly,
HPLC is the system of choice for amino acid analysis by an ever-
increasing number of laboratories.
Estimation of ammo acid levels by radioreceptor binding
techniques is a relatively new approach. The chapter on this topic
reviews the underlying prmciples of ligand binding assays and
details the practical considerations fundamental to the successful
utilization of these techniques. Although this type of assay is ex-
tremely sensitive, it is beset with problems of specificity, and only
one ammo acid (GABA) can currently be determined accurately
by the method Future developments should, however, extend its
applicability to some of the other ammo acids, but it is likely to
remam a method for determining a particular ammo acid, rather
than one to be utilized for the measurement of a spectrum of
ammo acids.
The application of immunocytochemistry to ammo acid
transmitter systems m the central nervous system has provided
valuable mformation on the cellular and subcellular locations of
these transmitter systems It is particularly suited to the detection
of the GABA system, and this topic forms the focal point of the
chapter on immunocytochemical techniques. Although the im-
munocytochemical approach is extremely valuable m specific situ-
ations, the method is of limited applicability m the amino acid
field, only the GABA and taurme systems having been mapped
by these procedures.
A related topic, the m vitro autoradiographic localization of
amino acid transmitter systems, is dealt with m a subsequent
chapter. This technique mvolves the detection of ammo acid re-
ceptors and high-affinity uptake sites using radioactively labeled
ligands and light microscopy. This approach IS, of course, limited
to ammo acids with transmitter function, and the GABA,
glutamate, and glycme systems are dealt with m the chapter
The turnover of ammo acids acting as neurotransmitters has
been the focus of much attention and the source of much contro-
X Preface
versy. The chapter on this sublect explains how the complex
compartmentation of amino acid metabolism in brain tissue gives
rise to problems in interpreting “turnover” data The results of
previous studies are discussed within the framework of the
known and postulated compartmentation of the ammo acids
The final chapter of this volume deals with the various tech-
niques used to measure amino acid uptake and release both m
vitro (slices and synaptosome preparations) and m viva (cortical
cups and push-pull cannulae).
In conclusion, this volume provides a detailed account of the
various techniques used to determine ammo acid levels, together
with the attendant problems, pitfalls, advantages, and disadvan-
tages of the methods. It also provides the reader with background
mformation and methodological approaches used to locate ammo
acid neurotransmitter systems and to study the dynamics of the
systems. It is intended that this volume serve as a reference
source for the established mvestigator in the amino acid field and
also as a guide to the new investigator with respect to the selec-
tion of the appropriate techniques.
Alan A. Boulton
Glen B. Baker
James D. Wood
Contributors
DEREK A. APPLEGARTH Bzochermcal Dtseases Laboratory, De-
l
partment of Pedtatncs, Brz tush Columbra Chddren’s Hospz tal,
Vancouver, Brtttsh Columbza, Canada
ROGERF.BUTTERWORTH Laboratory of Neurochemlcal Clnz~al
l
Research Center, Hiipz tal Samt-Luc, Unmersl ty of Mon trial,
Montreal, Quebec, Canada
D. L. CHENEY Neurosczence Research, Research Department
l
CUBA-GEIGY Corporatzon, Pharmaceuttcals Dtviszon, Summtt,
New Jersey
RONALDT COUTTS Neurochemxal Research Unzt, Department
l
of Psychtatry and Faculty of Pharmacy and Pharmaceutzcal Set-
ences, Unzverstty of Alberta, Edmonton, Alberta, Canada
JOHN WILLIAM FERKANY 9 Nova Pharmaceu ttcal Corporation,
Balttmore, Maryland
FRODEFONNUM Norwegian Defence Research Establishment, Dr-
l
vtszon for Environmental Tox~ology, Kleller, Norway
ANDERS HAMBERGER lnstttute of Neurobrology, Unfversrty of
l
Goteborg, Goteborg, Sweden
CHIN-TARNGLIN Department of Phystology, College of Medlcme,
l
The Pennsylvanza State Umverslty, Hershey, Pennsylvania
PETERLINDROTH Department of Analytical and Marme Chemzs-
l
try, Unzverstty of Goteborg, Goteborg, Sweden
MATS SANDBERG Instztute of Neurobzology, Unzverstty of
l
Goteborg, Goteborg, Sweden
JOHN A STURMAN Department of Pathological Btochemlstry,
l
New York State lnstz tu te for Basic Research tn Developmental Dtsa-
bthttes, Staten Island, New York
WOLFGANG WALZ Department of Pharmacology, Unrverszty of
l
Saskatchewan, Saskatoon, Saskatchewan, Canada
PAUL L. WOOD Neuroscience Research, Research Department
l
CIBA-GEIGY Corporatton, Pharmaceuttcals Dtvzszon, Summit,
New ]ersey
xi