Table Of ContentNEUROMETHODS I-J 2
Amines and Their Metabolites
NECIROMETHODS
Program Editors: Aian A. Boulton and Glen 8. Baker
Series 1: Neurochemistry
1. General Techniques
Edited by Alan A. Boulton and Glen 5. Baker, 1985
2. Amines and Their Metabolites
Edited by Alan A. Boo/ton, Glen 5. Baker, and Judith M. Baker,
1985
3. Amino Acids
Edited by Alan A. Boulton, Glen 5. Baker, and J. D. Wood, 1985
4. Receptor Binding
Edlted by Alan A. Boulton, Glen 5. Baker, and P. Hrdina, 1986
5. Enzymes
Edited by Alan A. Boulton, Glen 5. Baker, and P. H. Yu, 1986
NEUROMETHODS
Series I: Neurochemistry
Program Editors: Alan A. Boulton and Glen B. Baker
Amines and Their Metabolites
Alan A. Boulton, Glen B. Baker, and Judith IV. Baker
Humana Press Clifton, New Jersey
l
Lrbrary of Congress Cataloging-in-Publication Data
Main entry under trtle
(Neuromethods ,2 Series I, Neurochemrstry)
includes blbllographies and index
1 Biogenic amines--Analysis 2 Neurotransmitters
--Analysis 3 Neurochemistry--Technique I Boulton,
A A (Alan A ) II Baker, Glen B , 1947-
Ill Baker, Judith M IV Series Neuromethods , 2
V Series Neuromethods Series I, Neurochemistry
[DNLM 1 Biogenic Amines--analysis 2 Neurochemistry
--methods WI NE337G v 2 / QU 60 A5141
QP801 B66A45 1985 599’0188 8524868
ISBN o-89603-076-8
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 in any form or by any means, electromc, mechamcal,
photocopying, microfilmmg, recordmg, or otherwise without wrrt-
ten permission from the Publisher
Printed in the United States of America
Foreword
Techniques u-r the neurosciences are evolving rapidly. There are
currently very few volumes dedicated to the methodology em-
ployed by neuroscrentists, and those that are available often seem
either out of date or limited in scope This series is about the
methods most widely used by modern-day neuroscrentrsts and 1s
written by their colleagues who are practicing experts
Volume 1 will be useful to all neuroscientists since it concerns
those procedures used routinely across the widest range of
subdiscrplines Collectmg these general techmques together in a
single volume strikes us not only as a service, but will no doubt
prove of exceptronal utilrtarian value as well Volumes 2 and 3 de-
scribe all current procedures for the analyses of ammes and their
metabolrtes and of ammo acids, respectively. These collectrons
will clearly be of value to all neuroscrentrsts working m or
contemplating research in those fields. Similar reasons exist for
Volume 4 on receptor bmdmg techniques since experimental de-
tails are provided for all types of lrgand-receptor binding,
including chapters on general principles, drug discovery and de-
velopment, and a most useful appendix on computer programs
for Scatchard, nonlinear, and competitrve displacement analyses.
Volume 5 provides procedures for the assessment of enzymes m-
volved m brogemc amme synthesis and catabolrsm.
Volumes in the NEUROMETHODS series will be useful to
neurochemists, -pharmacologists, -physrologrsts, -anatomrsts,
psychopharmacologrsts, psychratrrsts, neurologrsts, and chemists
(organic, analytrcal, pharmaceutrcal, medicinal), m fact, everyone
involved m the neurosciences, both basic and clinical.
V
Preface
Orgamc ammes have been considered for many years to be rm-
portant to the functlonmg nervous system. The observations of
Gaddum and Schrld in 1934 led to the fluorescence measurement
of adrenalin m body fluids and these early studies were subse-
quently expanded by other mvestlgators to include a host of
catecholammes, mdolalkylammes, phenolrc amines, polyammes,
and their acidic, basic, and neutral metabohtes. A volummous ht-
erature has been published on the levels of ammes and metabo-
lites m body fluids and organs in health and disease “Amine the-
ories” have assumed especial importance m speculation
concernmg the causes of various psychiatric disorders; for exam-
ple mania, depression, and schlzophrenla.
Early investigators were able to use methods such as
chromatographlc separation, followed by colorimetrlc or
fluorimetrrc detection, to confirm the presence or absence of a
particular compound. Next, these methods were refined to allow
quantltatlon of the substance of interest. Assays were then devel-
oped that could be used to measure more than one amine and/or
their metabolites at a time. Finally, newer techniques were elabo-
rated that were capable of simultaneous analysis of large numbers
of compounds at much improved sensitlvmes and with a great
deal more specificity than the earlier methods could attam.
In preparing their chapters for this volume of Neuromefhods,
the authors have attempted to include a comprehensive literature
review pertinent to each topic and to make practical suggestions
that may help others to avoid technical difficulties. The methodo-
logical examples may be especially useful for investigators at-
tempting a particular technique for the first time, whereas the lit-
erature reviews should prove useful to the experienced and
novice alike. Rather than simply concentrating on the classrcal
biogemc ammes, such as noradrenalme, dopamme, and
5-hydroxytryptamme (and their metabolrtes), the contributors
have been encouraged to include, If appropriate, accounts of the
applicability of their methodologies to the study of other classes
vtti Preface
of amines, e.g , the “trace” ammes (P-phenylethylamme, the
tyrammes, the octopammes, the synephrmes, phenylethanola-
mine, and tryptamme), hrstamme and Me-methylhistamme, the
polyammes (cadaverme, putrescme, spermme, and spermrdme),
and choline and acetylcholme
This volume provides a useful descrrptron of the “state-of-
the-art” with regard to analysis of brogenlc ammes and their me-
tabolites Hrstochemrcal fluorescence and receptor bmdmg proce-
dures (other than autoradrographrc techniques) have been
omitted; because of the vast amount of work that has been done
n-r these two areas, rt was felt that each of these topics warranted
separate volumes m the Neuvo~ethods series
Smce fluorescence procedures for analyzing levels of amines
and their metabolrtes have been used extensively m tissues and
body fluids for a number of years, the chapter dealing with these
techniques gives a hrstorrcal perspective and attempts to con-
dense the extensive literature on the sublect With the help of this
review the reader should be able to identify the methods most
suited to his or her particular requirements Gas chromatography
has proven to be a very versatile tool for analyzing a large number
of compounds of interest. The chapter dealing with this technique
points out that gas chromatography may be used to quantrtate
numerous ammes and their metabolrtes in small amounts of trs-
sue using relatively inexpensive equipment.
The radioenzymatrc methods reviewed m Chapter 3 pro-
vided another element of specrfrcrty and sensmvity on their intro-
duction in the mid-1960s and early 1970s. Since minor variations
in techniques can greatly affect the results obtained using these
methods, the drscussion u-r this chapter should provide an mvalu-
able aid to those embarking on a radroenzymatrc assay for the first
time The author provides important information about the prep-
aration of enzymes required for the assays, the separation tech-
niques used to isolate the radrolabeled ammes, and the specl-
frcitres of the varrous methods Also included IS a brief section m
which references are given for the applrcatron of the techniques to
assays of enzymes involved m the synthesis and degradation of
brogenrc ammes
One of the areas of analysis that IS developmg most rapidly
and finding widest applrcatron at this time is that of high-pressure
(high-performance) liquid chromatography There IS a great need
for comprehensive literature reviews of applications such as that
provided m this chapter to be used both as teaching tools for re-
search students and as updates for more experienced personnel
In addition, the commentaries on the dlffrcultres and possibrllties
Preface
IX
of the techniques given m this chapter should spark further ad-
vances in the field
The sophisticated methods of m vivo voltammetry, immu-
nohistochemistry and radioimmunoassay, gas chromatogra-
phy-mass spectrometry, high-resolution and metastable mass
spectrometry, and autoradiography are discussed m Chapters 5
to 9, respectively. There has been a plethora of papers in recent
years on the sublect of m vivo voltammetry for the study of
ammes and their metabohtes in nervous tissue. These techniques
have the distinct advantage of providing mformation from freely
moving animals Immunohistochemistry and radioimmunoas-
says provide extremely sensitive means of mvestigatmg ammes in
tissues and/or body fluids. Radioimmunoassays have been widely
applied for analyses of a wide variety of drugs and naturally
occurring substances, such as peptides and steroids, and, al-
though the low molecular weight ammes present special prob-
lems, the results to date are very encouraging indeed. In both m
vivo voltammetry and immunological assays, there are potential
specificity problems, and the authors of these chapters have pro-
vided mterestmg discussions of how these problems have been
dealt with m practical situations.
Mass spectrometric techniques, although of limited availabil-
ity because of the high costs involved for purchase of equipment,
nevertheless have the important advantage of high specificity,
and it has almost become universally accepted that any new tech-
nique developed for quantitative analysis of biogeruc ammes
and/or their metabolites must give values m good agreement with
those obtained using mass spectrometric procedures. Such tech-
niques, combined with either gas chromatography or thin-layer
chromatography, have provided mvaluable mformation not only
about the catecholamines and 5-hydroxytryptamine, but also
about other ammes that are present in much lower absolute con-
centrations m nervous tissue.
The chapter on autoradiography deals with the application of
in vitro autoradiographic techniques to the localization of amine
receptor sites m neural tissue. The authors have provided exten-
sive methodological details and have also described the similari-
ties and differences between homogenate receptor binding tech-
niques and the autoradiographic methods. They have also
indicated that the two techniques complement one another, with
autoradiography providmg a means of determining distribution
of receptors anatomically, i.e., m histological sections
Chapters 10 and 11 deal with assessments of turnover rates
of cerebral ammes and neuronal transport of ammes m vitro. An
X Preface
understanding of both of these areas is necessary m order to eval-
uate the importance of functional deficits m various disease states
and the effects of pharmacologic treatments
In conclusion, we believe that the contents of this volume
will be a valuable addition to any library. For the experienced SCI-
entrst rt will provide an up-to-date evaluation of the literature
relatmg to the analysis of a wide variety of ammes and their
acidic, basic, and neutral metabolites. In addition, for those who
are beginning their study of these areas or who are broadening
their interests, the methodologrcal commentaries and the practi-
cal suggestions contained m the volume should provide mvalua-
ble assistance m the laboratory itself.
Judith M. Baker
Glen B. Baker
Alan A. Boulton
