NEUROMETHODS 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few volumes dedicated to the methodology em- ployed by neuroscrentists, and those that are available often seem either out of date or limited in scope This series is about the methods most widely used by modern-day neuroscrentrsts and 1s written by their colleagues who are practicing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 Contributors GEORGE M. ANDERSON Department of Laboratory Medicine, l Child Study Center, Yale Unrverslty, New Haven, Connectaxt GLEN B. BAKER Department of Psychiatry, Unzverstty of Alberta, l Edmonton, Alberta, Canada JUDITH M. BAKER Alberta Pharmacy, Edmonton, Alberta, l Canada ALAN A. BOULTON Psychtatrtc Research Dwzon, Untverslty of l Saskatchewan, Saskatchewan, Canada GREGORYM.BROWN Department of Neurosczences and Psychza- l try and Brain Behavior Program, McMaster Untverstty, Hamilton, On tano, Canada ROGER F. BLJTTERWORTH Laboratory of Neurochemrstry, Hopnat l Sarnt-Luc, Unwersrty of Montve’al, Montre’aI, Que’bec, Canada RONALD T. COUTTS Faculty of Pharmacy and Pharmaceu ttcal l Sctences, Unzverszty of Alberta, Edmonton, Alberta, Canada WILLIAMG.DEWHURST Department of Psychza try, Untverst ty of l Alberta, Edmonton, Alberta, Canada DAVID A. DURDEN Department of Psychiatry, Untverszty of l Saskatchewan, Saskatchewan, Canada LILLIAN E DYCK Psychiatrrc Research Dzvtston, Unzverstty of l Saskatchewan, Saskatchewan, Canada LEE J. GROTA Department of Psychiatry, Unwerszty of Rochester, l Rochester, New York JOSEPH B. JUSTICE, JR Department of Chemtsty, Emory Umver- l szt y, Atlanta, Georgia FAROUK KAROUM National lnstttute of Mental Health, St l Elizabeth’s Hospital, Washnzgton, DC JACOB KORF Department of Bzologtcal Psychiatry, Groningen, l The Netherlands XI