Amines & Their Metabolites (NEUROMETHODS) (Neuromethods 2 Series I Neurochemistry)

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 Main entry under trtle

Data

(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 , 1947Ill 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 Crescent Manor PO Box 2148 Clifton, NJ 07015

Press Inc.

All rights reserved No part of this or transmitted photocopying, ten permission Printed in the

book may be reproduced, stored m a retrieval system, in any form or by any means, electromc, mechamcal, microfilmmg, recordmg, or otherwise without wrrtfrom the Publisher United States of America

Foreword Techniques u-r the neurosciences are evolving rapidly. There are currently very few volumes dedicated to the methodology employed by neuroscrentists, and those that are available often seem either out of date or limited in scope This series is about the methods most widely used by modern-day neuroscrentrsts and 1s written by their colleagues who are practicing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 describe 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 details are provided for all types of lrgand-receptor binding, including chapters on general principles, drug discovery and development, and a most useful appendix on computer programs for Scatchard, nonlinear, and competitrve displacement analyses. Volume 5 provides procedures for the assessment of enzymes mvolved 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 rmportant 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 subsequently expanded by other mvestlgators to include a host of catecholammes, mdolalkylammes, phenolrc amines, polyammes, and their acidic, basic, and neutral metabohtes. A volummous hterature has been published on the levels of ammes and metabolites m body fluids and organs in health and disease “Amine theories” have assumed especial importance m speculation concernmg the causes of various psychiatric disorders; for example 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 developed that could be used to measure more than one amine and/or their metabolites at a time. Finally, newer techniques were elaborated 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 methodological examples may be especially useful for investigators attempting a particular technique for the first time, whereas the literature 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

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Preface

of amines, e.g , the “trace” ammes (P-phenylethylamme, the the synephrmes, phenylethanolatyrammes, the octopammes, 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-ofthe-art” with regard to analysis of brogenlc ammes and their metabolites Hrstochemrcal fluorescence and receptor bmdmg procedures (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 condense 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 trssue using relatively inexpensive equipment. The radioenzymatrc methods reviewed m Chapter 3 provided another element of specrfrcrty and sensmvity on their introduction 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 mvaluable aid to those embarking on a radroenzymatrc assay for the first time The author provides important information about the preparation of enzymes required for the assays, the separation techniques used to isolate the radrolabeled ammes, and the speclfrcitres 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 research 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 advances in the field The sophisticated methods of m vivo voltammetry, immunohistochemistry and radioimmunoassay, gas chromatography-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 radioimmunoassays 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, although the low molecular weight ammes present special problems, 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 provided mterestmg discussions of how these problems have been dealt with m practical situations. Mass spectrometric techniques, although of limited availability 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 technique 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 techniques, 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 concentrations 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 extensive methodological details and have also described the similarities and differences between homogenate receptor binding techniques 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 evaluate 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 SCIentrst 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 practical suggestions contained m the volume should provide mvaluable assistance m the laboratory itself. Judith M. Baker Glen B. Baker Alan A. Boulton

Contributors GEORGE M. ANDERSON

Department of Laboratory Medicine, Child Study Center, Yale Unrverslty, New Haven, Connectaxt Department of Psychiatry, Unzverstty of Alberta, GLEN B. BAKER Edmonton, Alberta, Canada JUDITH M. BAKER Alberta Pharmacy, Edmonton, Alberta, Canada Psychtatrtc Research Dwzon, Untverslty of ALAN A. BOULTON Saskatchewan, Saskatchewan, Canada GREGORYM.BROWN Department of Neurosczences and Psychzatry and Brain Behavior Program, McMaster Untverstty, Hamilton, On tano, Canada Laboratory of Neurochemrstry, Hopnat ROGER F. BLJTTERWORTH Sarnt-Luc, Unwersrty of Montve’al, Montre’aI, Que’bec, Canada RONALD T. COUTTS Faculty of Pharmacy and Pharmaceu ttcal Sctences, Unzverszty of Alberta, Edmonton, Alberta, Canada WILLIAMG.DEWHURST Department of Psychza try, Untverst ty of Alberta, Edmonton, Alberta, Canada DAVID A. DURDEN Department of Psychiatry, Untverszty of Saskatchewan, Saskatchewan, Canada Psychiatrrc Research Dzvtston, Unzverstty of LILLIAN E DYCK Saskatchewan, Saskatchewan, Canada l

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LEE J. GROTA

Department of Psychiatry, Unwerszty of Rochester,

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Rochester, New York

JOSEPH B. JUSTICE, JR szty, Atlanta,

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Department

of Chemtsty,

Emory Umver-

Georgia

FAROUK KAROUM

National lnstttute of Mental Hospital, Washnzgton, DC Department of Bzologtcal Psychiatry, JACOB KORF The Netherlands l

Health,

St

Elizabeth’s

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Groningen,

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COI-ltFibUtOFS

RONALD A LESLIE

Department of Anatomy, Dalhousre UnzverNova Scotza, Canada ADRIAN C MICHAEL Department of Chenustry, Emory Unzverszty, Atlanta, Geovgza KATHLEEN M. MURPHY Department of Pharmacology, Dalhousre Unzverslty, Hallfax, Nova Scotia, Canada ADIL J NAZARALI Department of Surgery, Unwerslty of Alberta, Edmonton, Alberta, Canada DARRYL B NEILL Department of Psychology, Emory Unzversrty, Atlanta, Georgza ROBERTSON Department of Pharmacology, HAROLD A Dalhousze Unzverslty, Haltfax, Nova Scotza, Canada JUAN M. SAAVEDRA Natzonal lnstztute of Mental Health, Bethesda, Maryland CHRIS SHAW Department of Psychology, Dalhouste Unzversl ty, Halifax, Nova Scotia, Canada l

szty, Hallfax,

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Contents Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..v Preface

. . . . . . . . . . . ...*.

. . . . . . . . ..*......*.....*

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CHAPTER 1 FLUORESCENCE ANALYSIS OF AMINES AND THEIR METABOLITES Judith M. Baker, Roger F. Butterworth, and Willlam G. Dewhurst . ... . ... 1 1. Introduction . . .... ... .. ... .. . . . . . . . ... 1 1.1. Theory and Instrumentation .... 1.2. Some Methodological Problems Encountered m Fluorescence Techniques . . . . . . . . . . . . . . . . . . . 3 2. Fluorescence Techniques for Detection and Qualitative Analysis of Ammes and Their Metabolites. . . . . . . . . . . . . . 5 3. Fluorescence Techniques for Quantitation of Ammes and .. ..... .. ... 6 Their Metabolites .. .... .... . ..... 3 1. Derwatization. . . . . . . . . . . . . . . . . . . . . , . . . . . . . . , 6 3 2. Native Fluorescence . . . . . . . . . . . . . . . . . , . . . . . . 18 3 3. Fluorescence Detection Combined With Another . . . . . . . . . . . . . . . . . . . 18 Quantitation Method . . 3 4 Micromethods . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 4. Advantages and Disadvantages of Fluorescence 21 Techniques . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . 22 5. Examples of Fluorimetric Methods. , . . . . . 5.1. Determination of HVA and DOPAC . . . : : : : . . . . . . 22 5.2. Simultaneous Determination of DA, NA, and 5-HT. 25

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xiv 6 Summary. . References

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30 31

CHAPTER 2 GAS CHROMATOGRAPHY OF AMINES AND THEIR METABOLITES IN TISSUES AND BODY FLUIDS Ronald T. Coutts, Glen B. Baker, and Adll J. Nazarah 1. Introduction ... .... . . .. ... . . . . . . . 45 1.1. GC Columns. . ...... . . . , . . . . . . . . . , . 46 1.2. Support Materials . . . . . .. . . . . . . . . . . . 46 47 1.3. Stationary Phases .. . 1 4 Capillary Columns 48 .... .. ... .. 48 1 5. Detectors .... . . . . . . , . . , . , . 51 1.6. Inlection Systems. . . . 2. Isolation of Ammes and Their Metabohtes From Tissues and Body Fluids and Derivatization for Gas Chromatography ..... ..... .... . . . . . . . . 52 3. Analysis of Specific Types of Ammes and Their . 53 Metabohtes Using Gas Chromatography . 3.1. Catecholammes and Their Metabolites. . . . . . . . . 53 3.2. 5-Hydroxytryptamme (5-HT; Serotonm) . . . . . , . . 58 3 3. Trace Amines and Their Acid Metabolites . . . . . 59 3.4. Histamine, tele-Methylhistamme, Spermidme, .. . 62 Spermine, Putrescme, and Cadaverme . . . 64 3 5. Acetylcholme (ACh) and Choline (Ch) . . 4. Examples of Some Protocols Used for Analysis of ... 64 Ammes and Their Metabolites. .. ... .. 4.1. Simultaneous Analysis of PEA, m-TA, p-TA, NMN, and 3-MTA . . . . . . . . ... .. ... . . 64 4.2. Simultaneous Analysis of Tryptamme (T) and 5-Hydroxytryptamme (5-HT) . . . . . . . . . . . . . 66 4.3. Simultaneous Measurement of Noradrenaline (NA), Dopamine (DA), and 5-HT in Brain Tissue . . . , , .67 4.4. Determination of m- and p-Hydroxyphenylacetic Acids m Mouse Brain. . . . . . . . . . . . . . . 69 5. Advantages and Disadvantages of Gas Chromatography Relative to Other Methods Available for Analysis of ,.. . . . 70 Biogemc Ammes and Their Metabohtes ... References ..... .. . . .. . 72

Contents

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CHAPTER 3 RADIOENZYMATIC MICROMETHODS OF BIOGENIC AMlNES IN BRAIN Juan M. Saavedra

1. 2 3. 4. 5.

6

7. 8.

9. 10

FOR THE QUANTITATION

87 Introduction ..................... ..... 89 General Procedure for Radioenzymatic Methods 91 The “Punch” Dissecting Technique ................... 94 General Procedures for Enzyme Purification ..... .......... 96 Indoleammes .............. 96 5 1. General Procedure for the Assay of Indoleammes ........... 98 5.2. Assay of N-Acetylserotonin 101 5 3 Assay of Serotonin ............ .................. 103 Catecholammes and Derivatives 6.1 General Procedure for the Assay of Catecholamines ............................. 103 6.2 Assay of Dopamme, Noradrenalme, and ...................... 104 Adrenaline. 6.3. Radioenzymatic Assays for Catecholamme ........... Derivatives ............ 106 ......................... Histamine 109 7.1 General Procedure for the Histamine Assay. ...... 109 110 7.2. Assay of Histamme. .............. Phenylethanolamine, B-Phenylethylamme, Octopamine, 112 ...................... and Tyramme. 112 8.1. General Procedure for the Assay. ........ .... ........... 113 8.2. Assay of Phenylethanolamme . . 115 8.3. Assay of B-Phenylethylamme. .......... 8.4. Assay of Octopamine ........................... 117 .............. ............ 118 8.5. Assay of Tyramine Assays for Enzymes of Synthesis and Degradation of 120 Biogenic Ammes .................................. 121 Conclusions , . .......................... References ....................................... ..12 2

CHAPTER

4

LlQUID CHROMATOGRAPHIC ANALYSlS OF MONOAMINES THEIR METABOLITES George M. Anderson . ..* . . . . . 1. Introduction . . . . . . . . . . . . . . . . . 2. Brain. . .

AND

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129 130

Contents

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4.

5

6.

7

2 1 Catecholammes m Bram ........ . 2.2. Catecholamme Metabolites and Precursors Brain ................................ 2 3 Indoles m Brain .......................... ........................ 2.4. Pineal Indoles. Blood ...................................... 3.1. Catecholammes m Blood 3.2. Catecholamme Metabolites m Blood. ............. 3.3 Indoles m Blood. Cerebrospmal Fluid (CSF) ........................ 4.1. Catecholamme Metabolites in CSF ............... 4.2. Indoles m CSF ........... .................. Trace Amines and Metabolites 5.1. Tryptamine and Metabolites. ................... ....... 5 2 Phenolic Trace Ammes .............. Urme 6.1. Urine Catecholammes 6.2. Catecholamme Metabohtes m Urme, ........ ............. 6.3. Indoles in Urme ..... Conclusions . . . ...,,,..... .. ... References . . . . . , . . .

CHAPTER 5 IN VW0 VOLTAMMETRY Joseph B. Justlce, Jr., Adrian

C. Michael,

and Darryl

. m

130

..13 6 144 147 149 . 149 154 158 162 162 164 164 164 168 169 ....... 169 174 180 . 182 I83

B. Nell1

197 Introduction ... 198 Introduction to Voltammetry ....... . . 202 Electrochemistry of Catecholammes . .. Voltammetrrc Techniques ,... .......... .. 204 . . 205 4.1. Chronoamperometry ....... ...... 208 4.2. Normal Pulse Voltammetry 209 4.3. Differential Pulse Voltammetry ...... .. .. 4 4 Differential Double Pulse Voltammetry . , . . . . 211 4.5. Linear Sweep and Cyclic Voltammetry (LSV and . . . . . . 1.. 211 CV) . . . . . . . * . . . 4 6. Linear Sweep Voltammetry With . . . , , . , . . . 213 Semidifferentration .. . . . * . * 214 5 Instrumentation. . . ... . . . . 214 5.1. Electronics

1 2 3 4

XVII

Contents ................................... 5.2. Electrodes ............... 5 3 Electrode Modification 5.4. Model of Electrode Response In Vivo ........... 5 5. Calibration of Electrodes ................... ........................... 6. Interpretation ........................... 6.1. Introduction .... 6.2. Dopamine, DOPAC, and Ascorbic Acid .................. 6.3. Serotonm and 5-HIAA . ............. 6.4. Neurotransmrtter Detection ...................... 7. Apphcations ............................. 7.1. Introduction 7.2. Clearance of Released Dopamine From Extracellular Fluid ................. .... 7 3 Dopamme Release m the Median Emmence ......... 7.4. Catecholammes in the Locus Ceruleus 7.5. Catecholammes and Ascorbic Acid. ............. 7.6. Serotonmerglc Pathways ............... ........ 7 7. Voltammetry and Iontophoresis ............ 7.8. Catecholammes and Behavior ............................ References. CHAPTER 6 IMMUNOHISTOCHEMISTRY AND RADIOIMMUNOASSAY BRAIN AMINES Gregory M. Brown and Lee J. Grota

216 221 225 227 230 230 232 238 239 241 241 241 242 243 243 245 248 248 257

OF

. ...,... ., .... 267 1 Introduction . .,.. 267 2. Antigens................... . . . . . . . . . . ..,....... 267 2 1 Couplmg Reactions. ... ....... ........ . , . 268 2.2. Indolealkylammes and Their Derivatrves. . , , 273 2.3 Catecholammes and Their Derrvatlves. ., . .. .......... 275 2 4 Other Antigens ..... 2 5. Productron and Characterization of Antisera . , . , . 276 .... 277 3 Radlormmunoassay . . . ...... , . , . . . . . . 277 3 1. Basic Considerations ........ 3.2. Indolealkylammes and Related Substances . . . . . 277 . . . . 283 3.3. Catecholamines and Related Substances . ....... . 286 4 Immunohrstochemlstry ..,....... 4.1. Basic Considerations . . . . . . . . . . . . . . . . . 286 . . . 287 4.2. Indolealkylamme Immunohlstochemlstry .

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4.3. Catecholamme Immunohistochemistry , . , . . , . . , 292 4.4. Immunohistochemistry of Other Amine .. .. .*. . ..* 297 Neurotransmitters . . . 295 5. Conclusions . . . . . . . . . . . .... .. . References. , . . . . . . . . . . . . . . . . . . . . . . . . . . 296 CHAPTER 7 COMBINED GAS CHROMATOGRAPHY-MASS SPECTROMETRY IN THE ANALYSIS OF BIOGENIC AMINES IN HUMANS Farouk

Karoum

... . 305 . . ... ... .. .. . . ... .. 1. Introduction ... . ... 306 2 Material and Methods 2.1. Mass Fragmentography . . . . . .. . .... . . . . . 306 307 2.2. Derivatization . . . . 307 2.3. Pentafluoropropionate Derivatives (FFP). . . * * : . 2.4 Ethylester/Pentafluoropropionate Derivatrve . . . . 307 (EEIPFP) . . . .. ... .... . . . . . 309 2 5. Pentafluoropropionate Ester .... .... . . . , . . 309 2.6. Quantification. , . , . . . . . . . . . ... .. 311 2.7 Extraction. .. . . *.. . 2.8. Assay of Catecholammes and Metabolites m Human Brain Tissue . , . , . . . . . . . . . . . . . . . . . . . , . . . . 311 2.9. Assay of Catecholamine Metabolites m Plasma and . . . . 312 Cerebrospmal Fluid (CSF) . . . ... .... 2 10 Assay of Catecholammes and Their Metabolites in Urine. . . . . . . . .. . .. . . . . . . . . . . . 312 2 11 Assay of Indole Ammes and Metabohtes m Urine . 313 313 2 12 Assay of Phenylethylamme (PEA) m Urine . . . 313 2 13 Assay of Phenylalanine and Tyrosme in Urine 2.14 Assay of Phenylacetic Acid (PAA) u-t Urine . . , . , 314 . * . .314 2.15. Assay of PAA m Plasma and CSF. . . . . . . ... . . *. * 315 3 Conclusions . .... ... . 321 References . . . , , . . . . . . . . . . CHAPTER 8 HIGH RESOLUTION OF TRACE BIOGENIC Dawd A. Durden

AND METASTABLE MASS SPECTROMETRY AMINES AND METABOLITES

1. Introduction . . . . 2. Mass Spectrome try

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. . . . . . . . . * . . . 325 . . . . . . . . . . . . 327

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2.1. Low Resolution Mass Spectrometers . . . . . . . . . . . . 328 2.2. High Resolution Mass Spectrometers .. . 329 2 3. Other Mass Spectrometrrc Techniques. . . . . . . . . . 332 . 2.4. Sample Introduction and Associated .... , 333 2 5. Ion Formation. ........ .. . .. 335 2.6. Quantitative Mass Spectrometry ..., .,. 338 3. Derivatives of Ammes and Metabohtes. . . . . . . . . , . . , . 340 3 1 Derivatives of Ammes .... . *. .. 341 3 2. Derivatives of Acids . . . . . . . . . . . . . . . . . . . 344 3.3. Derivatrves of Alcohols .. . . . . . . . . . . . . . . . 346 4. Protocols . . . . . 347 4.1. AmmesbyTLG-&M&&I .: .:..::::::::.347 4.2. Acids and Alcohols by CC-MS . . . . . . . . . . . . . . . . . 356 5. The Case for HRMS and HRGC . . . . . . . . . . . . . . . . . . . . 359 . ............. .. .... References . . . 360 CHAPTER 9 AUTORADIOGRAPHIC METHODS FOR THE LOCALIZATION AMINE RECEPTOR SITES IN NEURAL TISSUE R A Leslie, C. Shaw, H A Robertson, and K. M. Murphy

OF

1 Introduction .a.......* .. *... . . . . . . . . 373 1.1. Why the Autographrc Method Is Used . . . . . . . . . . 374 2 Procedures ......... . .. ............ . 377 2.1. The Choice of Autoradlographlc Techniques . . . . 377 2.2 I’reparation of Tissues for Autoradiography. . . . . . 379 2.3. Determmation of Appropriate Llgands and Binding Parameters. . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . , . 383 2.4 Incubation Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . 387 2 5. Exposure of the Labeled Sections to Nuclear Emulsion ... . ... .. .. . . . . . . 390 3. Assessment of Autoradiograms. . . . . . . . . . . . . . . . . . 394 3.1. Quahtatlve Assessment . . . . . . . . . . . . . . . . . . . . . . . . 394 3.2. Quantification of Autoradiographic Results . . . . 397 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402 References. . . . . . . . . . . . . . . . . . . . . . . . . . . 403 CHAPTER 10 TURNOVER RATE ASSESSMENTS OF CEREBRAL NEUROTRANSMII-I’ER AMINES AND ACETYLCHOLINE J. Korf 1. Introduction

....

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Contents

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2. Scope of the Review ............................. 3. Turnover Methods for 5-Hydroxytryptamme ..... 3.1. Some Biochemical Features. .................. ................... 3.2. Nonsteady-State Methods ..................... 3.3. Steady-State Methods. ......................... 3.4. Conclusions ................. 4. Turnover Methods for Dopamme 4.1. Some Biochemical Features. ................ .......... 4.2. Nonsteady-State Methods ............ .................... 4.3. Steady-State Methods. ................................ 4.4. Conclusions ............ 5. Turnover Methods for Noradrenaime 5.1. Some Biochemical Features. ............. 5.2. Nonsteady-State Methods ................ 5.3. Steady-State Methods. ..... ..... ............... ., 5.4. Conclusions ............ 6. Turnover Methods for Adrenaline ........ 7. Turnover Rate Methods for Acetylcholme 7.1. Some Biochemical Features. ................ .................. 7.2. Nonsteady-State Methods. ................... 7.3. Steady-State Methods. ............................... 7 4. Conclusions ............. 8. Turnover Methods for Other Ammes ............................. 8.1. Tryptamine ............................. 8.2. Histamine 8.3. Other Ammes. ................... ..... References ............ ..... ........ CHAPTER 11 NEURONAL TRANSPORT Glen B. Baker and Lhan

OF AMINES E. Dyck

410 410 410 411 413 418 419 419 420 422 422 423 423 426 427 . 429 429 433 433 435 436 440 441 441 442 443 444

IN VITRO

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457 1.1. Uptake and Release of Neurotransmitters. . . . . . 457 1.2. Tissue Preparations Used to Study Uptake and Release Processes . . . . . . . . . . . , . . . . . . . . . . . . . . 458 1.3 Experimental Conditions for Studying Uptake and Release........................... . . . . . . . ...459 472 2 Uptake and Release of Specific Amines .... .... 2.1. Catecholammes ... ............ .... . . . 472 2 2. 5-Hydroxytryptamme (5-HT; Serotonm) . ... 473 474 2 3 Choline and Acetylcholine .. . ..

3. 4. 5. 6.

Index

476 2 4. Histamine and Polyammes , . . . . . . . . . . . . . . 2.5. Trace Ammes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477 Extraneuronal Transport of Biogenic Amines . . . . . . 478 Presynaptic Receptors and Interactions Among Putative . . . . . . . . . . . . 489 Neurotransmitters. . . ... .... . Binding of Antidepressants and Uptake of Biogemc .... . .... ... . . . . . . . . . . . . . . . . 492 Ammes . Typical Protocols Employed in Neuronal Transport . 498 Studies InVitro . . . . . .. . . . . . . . . . . . . . . . . . . . . . 6 1. Effects of Drugs on the Uptake of Radiolabeled DA, NA, or 5-HT Into Prisms Prepared From . . . . . . . . . . . . 498 Rat Brain . . . . . . . . . . . . . . . . . . . 6 2. Superfusion Apparatus to Study Effects of a Drug on the Release of Radiolabeled DA, NA, or 5-HT in Prisms Prepared From Rat Brain Areas . . 498 6.3. Transfer Procedure to Investigate the Release of Radiolabeled p-TA and DA From Rat Striatal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499 Slices. .. .... .. . . . . . . 502 References . . . . ... . ......

....

.........

.

. . . . . . . . . . . 535

Chapter 1

Fluorescence Analysis of Amines and Their Metabolites JUDITHM. BAKER,ROGERF. BUIIERWORTH,AND WILLIAMGDEWHURST 1. Introduction 1.1. Theow and Instrumentation The term fluorescence commonly refers to the phenomenon m which light from the ultraviolet (UV) spectrum is absorbed by a substance and emitted in the visible spectrum. Certain organic molecules absorb light energy, resulting in various interatomic bonds being raised to higher energy levels. This energy may be dissipated in several ways, one of which is the emission of light. Under normal circumstances, the emitted radiation is of a longer wavelength (lower energy) than that absorbed. The number of photons of light emitted is proportional to the number of molecules involved; that is, the concentration of fluorescent substance(s) present m the sample under consideration. The fluorescence intensity of a particular substance is determined by the difference in energy between the excited and ground states and the relative importance of other types of energy dissipation, such as collisional deactivation. It is important to note that the chemical and physical properties (e.g., pK,, dipole moment, mteratomic distances) of the excited state may be much different from those of the ground state. These changes may result in special difficulties m the analysis of certain compounds (see section 1.2). In a typical instrument for measurement of fluorescence, UV radiation is produced by a light source and passes through a pri1

2

Baker, Butterworth,

and Dewhurst

mary filter system so that a particular wavelength 1s directed onto the cuvet contammg the sample. Light emitted from the sample passes through a secondary filter system, which results m light of only a specified wavelength reaching the detector. In general, detection systems are composed of a photomultiplier tube, an amplifier, and an indicator or recorder that provides a reading in arbitrary fluorescence units Whereas older instruments had filters that had to be mserted and changed manually, many newer models have gratings that can be adjusted by an external dial, allowing for greater freedom m wavelength selection. Some mstruments will scan either the excltatlon or emission wavelength, thus providing two spectra of responses for the substance of interest. The light source and detector are usually positioned at right angles to each other, resulting m decreased interference from the source at the detector. Such an arrangement provides an increase m sensitlvlty of approximately three orders of magnitude over that achieved by measurement of absorbed light (absorption spectrophotometry). Procedures utlhzmg measurement of fluorescence may provide speclflclty because: (a) many compounds do not absorb UV light, or if they do, do not fluoresce after absorption of light; (b) even when other fluorescent substances are present, wavelengths for maximal excitation (A,,) for the particular compound of mterest may not correspond to those of other substances present; in the event that A,, is the same for two substances, A,, may be sufficiently different so that there is no Interference problem; and (c) many procedures utihze prepurlflcation steps The measurement of fluorescence may be used as a tool for quantltatlon or detection m three situations. Compounds that are naturally fluorescent may be measured directly m solution Since these compounds are usually not strongly fluorescent, the sensitivity of such methods is hmited. More frequently, compounds of interest are converted to fluorescent denvatlves. Such reactions, m addition to producing strongly fluorescent products, often increase the specificity of the assay method since some potentially interfering compounds may not form fluorescent derivatives under the conditions used. Finally, certain types of compounds may be detected or quantltated because of the fact that they quench the fluorescence of other strongly fluorescent compounds. For example, with fluorescent detectors used m thin-layer chromatography, compounds may be vlsuahzed under UV hght as dark spots on a fluorescent background. The usefulness of fluorescence measurement as a method for quantitatlon may be limited by certain characteristics of the

Fluoresence Analysis ofAmines and TheirMetabolites

3

sample itself. The light used for excitation may cause or accelerate chemical changes in the compound of interest. Photochemical decomposition is usually more significant at higher excitation energies (shorter A,,), so for situations m which such reactions occur rt IS important to choose the longest wavelength possible for excitation. Variations m sample viscosity may also cause changes in measured fluorescence; usually increased viscosity leads to an mcrease u-t fluorescence. Finally, quenching of fluorescence may occur as a result of such factors as temperature, drssolved oxygen, and impurities (see section 1.2). Strict control over sample processing, purity of chemicals used, and cleanliness of glassware may be required to limit quenching.

1.2. Some Methodological Problems Encountered in Fluorescence Techniques Some common methodological problems will be covered m this section. For more detailed discussrons, the reader should refer to general reference texts, such as the one by Guilbault (1973). Since fluorescence is usually measured m solutron, solvent effects may assume significant importance in certain assay methods. Polar molecules show an increase in dipole moment of the excited state that ~111 be stabilized by the use of a polar solvent, resulting m a decrease m fluorescence intensity. Therefore when polar substances are being analyzed, fluorescence mtensrty ~111 be inversely proportional to solvent polarity. Polar compounds dissolved m polar solvents also display a shift in X,, and h,, to longer wavelengths (lower energy). As the dielectric constant of the solvent increases, this bathochromic shift becomes more pronounced since the magnitude of the shift is dependent upon the strength of solute-solvent interactions. As a general rule, A,, shifts more than A,,, leading to a larger separation between the excitation and emrssron spectra. This alteration of A,, by changing solvent polarity may be used to advantage m separating the emission spectra of substances of differing polarities. Solvent effects are usually of little significance if either the solute or the solvent is nonpolar. Solvents containing a heavy atom often result m enhancement of phosphorescence at the expense of fluorescence The sensitivity of an analytical procedure utrlrzmg fluorescence may be increased by avoiding, or removmg any excess of, solvents containing a heavy atom (e.g., ethyl iodide). Fluorescence measurements may also be affected by the pH of the solutron under consrderatron. The drfference between A,,

4

Baker, Butterworth,

and Dewhurst

illustrates and X,, (St o k e’s shift) for solutions of 5-hydroxyindole this point: at pH7, h,, is at 330 nm, and in strong acid, A,, is at 550 nm but there is no change m A,,. Such effects occur since the fluorescence mtensrty and A,, of the ionized and unionized species differ, and because of this a pH should be selected such that virtually 100% of the substance to be measured exists as a smgle species. Because the pK, of the excited state is different from that of the ground state, the appropriate pH range may be different from that which would be predicted on the basis of the properties of the ground state. Adjustment of pH may be employed to mcrease the sensitivity and specificity of an assay by decreasing the formation of the fluorescent derivatives of interfering substances (see section 3.1.1). Intermolecular hydrogen bonding may lead to a decrease m fluorescence intensity, similar to the situatron resulting from polar solute-solvent mteractions. Therefore, the choice of a solvent that does not hydrogen bond with the substance to be analyzed may result m increased sensitivity. Such a generalization may not be applicable to aromatic carboxyl compounds and nitrogen heterocycllcs because with such substances hydrogen bonding tends to decrease WIT* energy transitions and increase T-IT* transitions that are more apt to result m fluorescence emission. It is usually necessary to employ some prepurification steps before analysis of complex samples since the presence of other solutes may result in quenching or interference. Oxygen is the most common fluorescence quencher, but interference from this source may be overcome by degassing the sample or the addition of antioxidants (e.g., ascorbic acid). Complexmg agents [e.g., ethylenedlamme tetraacetic acid (EDTA)] may be Included to prevent fluorescence quenching by metal ions. Some metals are more problematic than others-Fe2+ is an efficient quencher, whereas Mg2+, Zn2+, and Cd2+ are poor Linearity should be given important consideration m fluorescence assays. It is often found that the fluorescenceconcentration curve levels off or decreases at high concentrations (concentration quenching). Therefore it is important to ensure that the concentration of the substance of interest in the sample falls within the linear portion of the fluorescence-concentration curve that IS prepared for quantitation purposes using known amounts of standard. Elevated temperatures may result m mcreased quenching and chemical instability. Gerst et al. (1966) reported changes in the fluorescence of the trihydroxymdole derivatives of adrenaline (A) and noradrenaline (NA) amounting to 14%/“C as a result of

Fluoresence Analysis ofAmlnes and TheirMetabokes

5

the chemical instability of the fluorescent molecules at increased temperatures. Such effects may be particularly important when an instrument 1sbeing used m which the light source can cause an increase m sample temperature while measurements are being conducted. If temperature lab&y does prove to be a problem, samples should be left m the instrument for the minimum time required to obtain a fluorescence reading

2. Fluorescence Techniques for Detection and Qualitative Analysis of Amines and Their Metabolites Amines and their metabolites may be detected and identified m tissue extracts and biological fluids by means of fluorescence methods. Usually these procedures involve the formation of a fluorescent derivative; however, detection of native fluorescence may be feasible for certain compounds Fluorescence detection is often used in combmatlon with paper or thin-layer chromatography (TLC). Histochemlcal procedures have been widely used to investigate ammergic pathways m nervous tissue, but these techniques ~~11be covered m another volume m this series. Qualitative fluorescence procedures for several biogemc ammes and their metabohtes have been available for some time Gaddum and Schild (1934) detected adrenaline (A) m biological fluids after addition of sodium hydroxide and observation under UV light. Shore and Olm (1958) identified noradrenalme (NA) in various tissue extracts using separation by paper chromatography followed by spraying with ferricyamde and au oxidation. The dopamme (DA) metabolrte homovanillrc acid (HVA) was identified on paper chromatograms of brain tissue extracts by the formation of a fluorescent dihydroxyindole derrvative after treatment with ferric chloride and sodium hydroxide (Sharman, 1963). In 1957 Duggan et al. reported that it might be possible to detect a number of compounds of biological interest utillzmg their native fluorescence at various pH values Some of the substances they investigated included DA, 3,4-dlhydroxyphenylacetic acid (DOPAC), p-hydroxymandelic acid, p-hydroxyphenylacetic acid, 5-hydroxymdole-3-acetic acid (5-HIAA), NA, 5-hydroxytryptamme (5-HT, serotonm), tryptamme (T), and p-tyramme (p-TA). Bell and Somerville (1966) converted a number of ammes of biological interest to fluorescent derivatives on paper chromatograms by reaction with formaldehyde. Noradrenaline, A, DA, 5-HT, T, 3-hydroxy-4-methoxyphenylethylamme, 3-

6

Baker,

Buttenvorth,

and Dewhurst

methoxytyramme (3-MTA), 3,4-dimethoxyphenylethylamine (DMPEA), octopamme (OA), and bufotenm were among the compounds detected. Another method that permits identification of a large number of compounds IS that described by Dolezalova et al. (1973). Using l-dlmethylammonaphthalene-5-sulfonyl (dansyl) denvatrves, the authors detected putrescme (PUT), 5-HT, T, TA, A, NA, DA, 6-hydroxytryptamme, 5,6-drhydroxytryptamme, spermme (SPM), and spermrdme (SPD) on twodlmenslonal TLC Crevelmg et al (1968) also used dansyl denvatrves combmed with TLC to identify the following amines m mammalian heart plpertdine, dlmethylamine, phenylethylamine (PE), methylamme, phenylethanolamme (PEOH), PUT, SPD, SPM, and TA. Jepson and Stevens (1953) prepared fluorescent denvatlves of 5-HT and other tryptammes by spraying paper chromatograms with nmhydrm. Urinary tryptamme has been identified by means of its native fluorescence spectra at pH 10 (Sjoerdsma et al., 1959). o-Phthalaldehyde (OPT) has been a popular spray reagent for detecting mdoleamines such as bufotenin and 5-methoxy-N,Ndimethyltryptamine on TLC (Narasimhacharr and Plaut, 1971) Axelsson and Nordgren (1974) reported the detection of a variety of mdoles m blood plasma by utilrzmg either a formaldehyde spray or OPT followmg separatron on TLC. Dewhurst and McKrm (1979) employed OPT combined with two-dimensional TLC to develop a sensitive procedure to investigate the presence of 5-methoxytryptamme in rat brain. Formation of fluorescent derivatives has been utlllzed m the rdentrflcation of other amines. Histamine (HA) has been detected after TLC or electrophoresis by forming the OPT denvatrve (Shelley and Juhlm, 1966). Srmilarly, the OPT derivative of SPD has been identified as a substance interfering with the assay of brain HA (Kremzner and Pferffer, 1966). Seiler and Demrsch (1978) have reviewed m detail the properties of a number of fluorescent derrvatlves suitable for detectron of ammes.

3. Fluorescence Techniques for Quantitation and Their Metabolites

of Amines

3.1, Derivatization 3.1.1

Catecholamines

and Alkalrne

Metabohtes

The observation by Gaddum and Schrld (1934) that A exposed to oxygen m the presence of strong alkali formed a fluorescent deriv-

Fluoresence Analysis ofAmlnes and ThevMetabolltes

7

atrve led to the subsequent development of numerous fluorescence methods for the determination of catecholamines in body fluids and tissues. Trihydroxyindole derivatives of A and NA are formed as shown m Fig. 1. The formatron of an adrenochrome by oxidation 1s followed by rearrangement to an adrenolutine. Ascorbic acid is commonly used as a reducing agent to prevent breakdown of the lutines m the presence of oxygen and thus stabilize the fluorescence (Ehrlen, 1948, Lund, 1949, von Euler and Flodmg, 1955; Cohen and Goldenberg, 1957; Anton and Sayre, 1962). Other compounds that have been utilized for this purpose include throglycohc acid (Merrrlls, 1963), P-thiopropromc acid (Palmer, 1963), drmercaptoethanol in sodmm sulfite (Haggendal, HO

CHOH-CH*-NH-R

HO la

I

-2H

lb -2H

OH

lc

R -OH t

1 Formation of trlhydroxymdole derwatlves (Id) from % catecholammes (la) noradrenalme (R = H) and adrenaline (R = CH3), (lb) noradrenochrome (R = H) and adrenochrome (R = CH3), (lc) noradrenolutme (R = H) and adrenolutme (R = CH3)

8

Baker,

Buttenvorth,

and Dewhurst

1963), and B-mercaptoethanol (Well-Malherbe and Bigelow, 1968). When ascorbic acid IS used, diaminoethane (von Euler and Lishajko, 1961) or sodium borohydride (Gerst et al., 1966) are added to overcome the problem of conversion of the acid to fluorescent products m alkaline solutron. The oxidizing agent manganese dioxide has also been employed to produce the oxidation reactions shown m Fig. 1 (Lund, 1949; Cohen and Goldenberg, 1957) Von Euler and Flodmg (1955) used potassium ferricyamde and iodine, but concluded that iodine was not suitable because of the extended time required to prepare a faded blank. Iodine results m iodmation of the derivatives in the 2 position of the mdole ring. These iodinated derivatives have also been reported to be more stable and to permit more complete separation of A and NA than do the hydroxymdole derivatives resulting from the other methods (Crout, 1961). Metal ions such as Zn2+ (von Euler and Floding, 1955) and Cu2+ (Haggendal, 1963; Weil-Malherbe and Bigelow, 1968) may be used as catalysts to facilitate the oxidation reaction. In order to prevent the formation of a calcium-magnesium phosphate gel that may quench fluorescence, Crout (1961) has proposed that EDTA be added to urine before analysis. Gerst et al. (1966) have mdicated that the temperature at which the fluorescence of trihydroxymdole derivatives is measured can assume great importance. In a series of detailed papers, Anton and Sayre (1962, 1964, 1966) have discussed a variety of factors that can affect fluorescent measurements with such derivatives Hahn (1980) has suggested that the use of reduced volumes and microcuvettes can result in a tenfold increase in sensitivity of catecholamine assays. In order to provide differential estimation of A and NA, oxidation may be performed at two different pH values, measurement of fluorescence may be made at two different wavelengths, or both of these approaches may be combmed (Lund, 1950; von Euler and Flodmg, 1955; Price and Price, 1957; Cohen and Goldenberg, 1957, Bertler et al , 1958; von Euler and Llshalko, 1959; Haggendal, 1963, Vendsalu, 1960; Anton and Sayre, 1962; 1968). Merrrlls (1963) used the Weil-Malherbe and Brgelow, reducing agent thloglycolrc acid to protect only noradrenolutine, whereas ascorbic acid was utilized to protect both adrenolutme and noradrenolutme. Solvent extraction (Shore and Olm, 1958, Anton and Sayre, 1968), adsorption onto alumina (Lund, 1949; Cohen and Goldenberg, 1957; von Euler and Lishajko, 1959, Anton and Sayre, 1962; Merrills, 1963), or extraction with cation-exchange

Fluoresence

Analysis ofAmlnes

and ThelrMetabolites

9

resins (Bertler et al,, 1958; Vendsalu, 1960, Haggendal, 1963) has been employed for prepurifrcation to increase sensitivity and specificity of the subsequent chemical techniques. Dolphin et al. (1975) have shown that the drug a-methyl-p-tyrosine (a-MPT) will Interfere with the assay for NA if the solvent extraction procedure of Maickel et al. (1968) is employed, but not if prepurification is carried out using Dowex 50 or alumina. Dopamme measurements were unaffected by the drug. An automated method for differential analysrs of NA and A was reported by Merrills (1963) In an analogous situation to the trihydroxymdole derivatives formed from NA and A, DA has been analyzed by the production of a dihydroxyindole derivative The final derivative formed from DA is shown in Fig. 2 Carlsson and Waldeck (1958) employed this method by using rodme as an oxidizing agent and catalyzmg formatron of the final derivative by exposure to UV light. Under these conditions 3,4-dihydroxyphenylalanme (DOPA) formed the same derivative as DA and thus had to be removed when present m appreciable amounts. A number of modifications to the procedure were made m an effort to improve sensitivity and/or reproducibility and to eliminate mterfermg substances (Uuspaa, 1963; Anton and Sayre, 1964; Greenland and Michaelson, 1974; Atack, 1973). These changes are discussed m somewhat more detail m a recent review from our laboratories (Baker and Dewhurst, 1982). Formation of hydroxyindole derivatives has also been utilized for analysis of the methoxy derivatives of the catecholamines (normetanephrme (NMN), metanephrme (MN), and 3-MTA). The reaction products formed from these substances possess spectral characteristics identical with those of then corresponding catecholamines, and this has necessitated the development of procedures for removal of interference from NA, A, or DA as appropriate. Modlficatrons employed have included differential 0x1dations using a variety of pH conditions and/or selective oxidizing agents and separation using ion-exchange or alumina columns (Bertler et al., 1959; Smith and Well-Malherbe, 1961; Haggendahl, 1962; Carlsson and Lindqvlst, 1962; BrunIes et al., 1964, Taniguchr et al., 1964; Anton and Sayre, 1966; Carlsson and Waldeck, 1964,

t-i Fig.

2

Dlhydroxymdole

derivative

formed

from

dopamme

10

Baker, Butterworth,

and Dewhurst

Guldberg et al., 1971, Kehr, 1974, Reviews. Well-Malherbe and Smith, 1966, Baker and Dewhurst, 1982). Calverley et al (1981) have pointed out that certain MAO inhibitors can interfere with fluorescence assays for NMN and 3-MTA, and this should be kept m mind when dealing with samples taken from sublects treated with such drugs The hydroxyindole derivatives have now been widely utrllzed for simultaneous analysis of a variety of catecholammes and/or related metabohtes (Drulan et al., 1959; Sourkes and Murphy, 1961; McGeer and McGeer, 1962; Chang, 1964, Laverty and Taylor, 1968; Westermk and Korf, 1977; Hamall and Sekl, 1979). Furthermore, various procedures have been described for slmultaneous measurement of catecholammes and their metabohtes by formatron of hydroxymdole derivatives and various indole amines by further derlvatlve formation (Malckel et al., 1968; Ansell and Beeson, 1968; Miller et al., 1970; Shellenberger and Gordon, 1971, Haubrlch and Denzer, 1973; Cox and Perhach, 1973; Butterworth et al , 1975, Karasawa et al , 1975; Jacobowltz and Richardson, 1978; Szabo et al., 1983) or by native fluorescence (Karrya and Aprrson, 1969, Metcalf, 1974; Holman et al., 1976) The second prmcrpal method for formatron of fluorescent derivatives of catecholammes and metabolites IS oxidation followed by condensation with ethylenediamine (Fig. 3). Following the report of Natelson et al. (1940) that A m ammonia solution will condense with ethylenedlamine, butylamine, aniline, or o-phenylenediamme to produce fluorescent derlvatrves that can be extracted mto butyl or amyl alcohol, Well-Malherbe and Bone (1952) suggested that this reaction could be used to measure A and NA in blood after adsorptron onto alumina. These authors reacted the catecholamines with ethylenediamine and extracted them mto lsobutanol for fluorescence readings. They reported that such derlvatrves are more stable than those produced by the trihydroxymdole method. The procedure was later adapted for analysis of catecholammes in tissues (Montagu, 1956, 1957) Weil-Malherbe (1961) proposed modifications to his origlnal method to increase its sensmvity and specificity and to allow quantltation of DA m addition to NA and A. Laverty and Sharman (1965) and Sharman (1971) reported the use of acetylatlon, paper chromatography, elutlon, and ethylenediamme condensation for analysis of DA Crawford and Yates (1979), using a similar procedure, reported the effects of prior acetylatlon on sensitlvlties for DA and 3-MTA (increased), for DOPA, NA, and NMN (decreased), and for A and MN (no change)

Fluoresence

Analysis

ofAmlnes

and Their Metabobtes

11

R = H, noradrenallne R = CH,,adrenallne

I

-4H

OH

Fig. 3 Ethylenedlamme fluorescence measurements.

condensatron

of catecholammes

for

The ethylenedramme condensatron method has been crrtrcized by various workers for factors such as presence of interfering substances m urine (von Euler et al., 1955), overestrmatron of NA concentratrons (Valk and Price, 1956), and mstabihty of NA derrvatrves (Mangan and Mason, 1957) Nadeau and Joly (1958) stated that the method resulted m multiple derrvatrves of NA and A. However, Well-Malherbe (1960) indicated that under the condrtrons mrtrally proposed (Well-Malherbe and Bone, 1952), only one product was formed from A and two were formed from NA. A small number of derrvatrzatron methods other than the two principal ones mentioned above have been reported for the fluorescence analysis of catecholammes and their metabohtes. Goldenberg and White (1962) suggested the measurement of MN and NMN by oxrdatron to vanillm followed by condensatron with throsemrcarbazrde at pH 12 to form a fluorescent derrvatrve Bell and Somervrlle (1966) reacted formaldehyde and catecholammes

12

Baker,

Butterworth,

and Dewhurst

on paper chromatograms and eluted the resultant derivatives for fluorescence measurement. Sekl and Hamall (1979) have measured DA after oxldatlon by hexacyanoferrate in the presence of p-aminobenzolc acid. A dansyl derivative was employed by Oberman et al. (1970) to quantltate DA m urine, but the method was only sufficiently sensitive to be used m cases of high DA excretlon (e.g., in subjects receiving L-DOPA). A more sensitive procedure might be developed from the technique of Davis (1978) in which some ammes and amino acids can be dansylated in aprotic solvents in which “naked” fluoride anion, solubllized by means of 18-crown-6, activates ammo and hydroxyl hydrogen atoms to displacement by the dansyl group. This method could be used for TA, 3-MTA, HA, PE, DA, OA, PEOH, NMN, T, and y-ammobutync acid (GABA). 3.1.2. Other

Metabolltes

of Catecholamines

Various other catecholamme metabolites have been assayed as fluorescent denvatlves. Homovanllllc acid was first measured in brain tissue in 1963 by Anden et al. and Sharman. Both methods rely on formation of a fluorescent derivative after oxidation by potassium ferricyamde (Anden et al , 1963) or ferric chloride (Sharman, 1963). Ferric chloride oxidation has been reported to have a wider application (Sharman, 1971), whereas ferricyamde results in the development of a more intense fluorescence. Due to its greater simplicity, the method of Anden et al. (1963) has been used more often. A modified ferricyamde oxldatlon technique has been published by Juorio et al. (1966), but Sharman (1971) reported problems in reproducing this method. Glessing et al. (1967) have reported that other compounds such as vanyllactlc acid and vanylacetlc acid would interfere with the assay of Anden et al for HVA, and have suggested that a separation by twodlmenslonal paper chromatography could yield an assay procedure for these compounds. Gerbode and Bowers (1968) modified the procedure of Anden et al. by using an ethyl acetate extraction and a more dilute ferncyanide reagent. Prasad and Fahn (1974) have reported an automated method for 3-methoxy-4-hydroxyphenylalanine, 3-MTA, and HVA by a modiflcatlon of the method of Anden et al. A more complex automated analysis reported by Westerink and Korf (1977) allows measurement of NA, DA, 3-MTA, HVA, and dlhydroxyphenylacetic acid (DOPAC) using ferricyamde oxidation. Dlhydroxyphenylacetlc acid has been measured by Rosengren (1960) followmg condensation with ethylenediamine m 4M ammonium chloride Sharman et al. (1967) used an ion-

Fluoresence

Analysjs

ofAmines

and Tbelr Metabolites

13

exchange resin to separate HVA (for analysis according to Anden et al.) from DOPAC that was acetylated, separated from catecholammes and other neutral compounds, and reacted with 1,2-diaminoethane to form a yellow fluorescence. After being made acidic and then neutralized, a unique blue fluorescence develops that was used to measure DOPAC. A slightly modified version of this method was reported by Murphy et al (1969) (see section 5 1), and was further combined with the procedure of Con tractor (1966) to assay 5-hydroxymdoleacetic acid (5-HIAA) by Ahtee et al. (1970) 3.1.3. lndoleamines

and Their Metabolites

Three principal fluorescence procedures have been utilized for analysis of indoleamines. In early procedures, the mdoleammes were condensed with formaldehyde and oxidized to form the fluorescent norharman derivative (structure shown m Fig. 4). Tryptamme (T) (Hess and Udenfriend, 1959; Martin et al., 1972) and tryptophan (TP) (Denckla and Dewey, 1967) have been measured using such a reaction. Maickel and Miller (1966) analyzed 5-HT and 5-methoxytryptamme followmg condensation with o-phthalaldehyde (OPT). Curzon and Green (1970) found that the sensitivity of this method for 5-HT and 5-HIAA was increased by the addition of cysteme. Korf and Valkenburgh-Sikkema (1969) used the OPT reaction to measure 5-HIAA in urine after first destroymg 5-HT with periodate. Welch et al. (1972) automated the analysis of 5-HT based on the OPT condensation reaction. In 1973, Atack and Lmdqvist measured 5-HT, 5-HIAA, and 5-hydroxytryptophan (5-HTP) in brain using a complicated dual assay. Samples were first assayed by measurement of native fluorescence and then condensed with OPT and the fluorescence determined again. The authors claim that the dual assay allows the differentiation of 5-HIAA from other substances eluted with it. Good agreement was reported between the values of both assays for 5-HT and 5-HIAA. 5-Hydroxytryptophan could not be detected by either means The OPT reaction has also been used

Fig 4 Norharman formed from tryptamme after condensation with formaldehyde and oxidation.

14

Baker, Butterworth, and Dewhurst

for quantitation of bufotenin, 5-methoxy-N,N-dimethyltryptamme (Narasimhachari and Plaut, 1971) and 5-MT (Prozialeck et al., 1978). A further refinement of the OPT method has been proposed by Dombro and Hutson (1980) for analysis of 5-HIAA in urine and cerebrospmal fluid (CSF) They have reported poor reproducibility and recovery using the methods of Korf and Valkenburgh-Sikkema (1969), Ashcroft and Sharman (1962), and Atack and Lmdqvist (1973) due to substances m CSF and urine that quench the fluorescence of the OPT-5-HIAA condensate. They therefore propose an mltial separation on an anlonexchange column before reactmg with OPT. The third derivative commonly employed m the analysis of mdoleamines is that formed by reaction with nmhydrm Vanable (1963) modified the procedure of Jepson and Stevens (1953) to provide for determination of 5-HT m solution Snyder et al. (1965) measured 5-HT concentrations m tissues usmg the ninhydrm reaction and have found that this method provides an eightfold mcrease m sensitivity over usmg native fluorescence m strong acid. Quay (1968) reported that nmhydrm formed fluorescent derivatives with bufotenm, 5-HIAA, N-acetyl-5-hydroxytryptamme, and 5-HTP, and mvestigated the temperature and time requu-ements for this formation for each of these substances. 3.1.4. Histamine and Its Metabolites The orrgmal fluorescence assay for HA was that proposed by Shore et al. (1959) mvolvmg condensation with OPT. This procedure was scaled down by Noah and Brand (1961) for analysis of plasma and an initial separation on a decalso column was mtroduced to decrease interference from another substance suggested to be histidme. In 1962 the original procedure of Shore et al. (1959) was modified to include an extraction with butanol/chloroform mstead of butanol to decrease histidine interference (Burkhalter, 1962) Beall (1963) employed the scaled-down procedure of Noah and Brand (1961) without the decalso column for analysis of plasma and obtained a value of 5.6 kg/L Noah and Brand the same year (1963) published a simplified method using a lower concentration of OPT reagent for plasma analysis and obtained a value of 2.09 pg/L. Thompson and Walton (1964) introduced some modifications of the original procedure of Shore et al. to increase fluorescence intensity by 50%. In 1964 as well, Green and Erickson proposed the use of a Dowex 50 column to separate interfering substances prior to an OPT condensation reaction. Von Redlich and Glick (1965) have published a review of previous methods

and propose

a modified

method

for small

amounts

of

Fluoresence

Analysrs ofAmrnes

and Their Metabolltes

15

trssue or body flurds that mcorporates the findings of several authors. In accord with Shore et al. (1959), these authors report negligible interference by 5-HT. Kremzner and Pfeiffer (1966) first reported the identification of SPD as a substance interfering with the fluorescence assay of HA following OPT condensation Graham et al. (1968) have reported a method for measurement of HA in plasma using separation on a decalso column followed by a butanol extraction and OPT condensation This method produces plasma levels of HA of 0.62 pg/L as compared with 2.09 pg/L from Noah and Brand (1963), 5.6 pg/L from Beall (1963), and 7.7 Fg/L by Thompson and Walton (1964). The authors also state that some interference by SPD could be possible m tissues or fluids in which this polyamine is present. In 1969, Anton and Sayre proposed another modification of the original assay of Shore et al. (1959) to overcome interference from SPD and histidine and decrease variability introduced by the pH sensitivity of the OPT-HA fluorophore. The method mcludes a series of solvent extraction steps prior to the OPT condensation step. Harvey (1973) has mvestigated this method of Anton and Sayre (1969) and concluded that the overall recovery as determined by use of radiolabeled HA is 25%, not 70-75% as reported by Anton and Sayre. Furthermore, Harvey (1973) has suggested that whereas the rsopentanol extraction with dibasic potassium phosphate does favour HA over SPD, three times more SPD is carried over than reported by Anton and Sayre, and this discrepancy could contribute to the variability noted using their method. Von Redlich and Ghck (1969) have provided a further review and micromethod for HA and 5-HT and have shown evidence that HA may bmd to glassware during sample processing. Hakanson et al. (1972) have provided a careful analysis of the reaction conditions for formation of the OPT-HA fluorophor that will result in mmimal SPD interference. These authors claim that their procedure is three times more sensitive than that originally suggested by Shore et al. (1959). Rohde et al. (1980) used a Dowex 50 column prior to OPT condensation and reported that most of the variability in analysis of biopsies of human gastric mucosa is a result of the imprecision of sample-taking and not that of the biochemical assay. Two procedures for assay of tissue HA have been reported by Lewis et al. (1980) and Lewis and Fennessy (1981). The first, for brain tissue, employs a Bio Rex 70 column extracted with NaCl followed by OPT-condensation, and the second employs an HCl column extraction. This latter method is faster than the former and has a higher recovery, but is suitable only for peripheral tissues since it also extracts SPD.

16

Baker, Buttenvortb, and Dewhurst

Myers et al. (1981) attempted to measure urinary HA by cation exchange, organic solvent extractron, and OPT condensation, but found that the values obtamed were higher than those reported for other techniques. They therefore found it necessary to digest part of the sample with diamine oxidase and take the difference as being the amount of HA origmally present. Using this technique they obtained normal value of 13 ng/mL. Endo (1981) has proposed a method for srmultaneous analysis of HA, PUT, SPD, and SPM using a cellulose phosphate column and elutron by borate buffers of different iomc strengths. Parkm et al. (1982) have used a Dowex 50 ion-exchange resin plus butanol extraction to remove nonHA fluorescence m the analysis of human gastric asprrate. In addition to these manual methods, the OPT condensation reaction has been utilized m several automated analyses for HA (Evans et al., 1973, Martin and Harrison, 1973; Siraganian, 1975, Remders et al , 1980, Wilhelms, 1980; Assem and Chong, 1982). In addition to the OPT condensation product, various other fluorescent derivatives could be used for analysis of HA. Ghosh and Whitehouse (1968) have reported a new reagent that could be used for histamine analysis: 7-chloro-4-mtrobenzo-2-oxa-1,3diazole Seller and Wiechmann (1970) have reviewed the use of dansylation, a reaction that can also be carried out m aprotic solvents (Davis, 1978). The dansyl derivative was also used by Yamatodam et al. (1977) for analysis of HA and methylhistamme (MHA). Alkon et al. (1971) have employed a reaction with N-bromosuccimmide and condensation with o-phenylenediamme to form fluorescent derivatives of MHA, HA, 3-methylhistidme, hrstidine, imidazoleacetic acid (IMAA), and methylimidazoleacetic acid Finally, Seller et al. (1973) have suggested that bansyl (5-di-n-butylammonaphthalene-l-sulfonyl chloride) derivatives may have some advantages over dansyl derivatives for analysis of some ammes, mcludmg HA 3.15 Putresclne, Spermine, and Spermidine Putrescme and the polyamines SPM and SPD have been successfully identified and analysed using a number of fluorescent derivatives. Smce this group of compounds does not exhibit any unique structural features, derivatives must be formed before detection or measurement is possible Furthermore, all methods must include a suitable procedure for separation of the derivatives formed. Specificity of the procedure will depend on the quality of separation achieved and sensitivity of the assay on the derivatives formed.

Fluoresence

Analysis of Amines and Their Metabolites

17

Fluorescamme has been used to form intensely fluorescent conjugates of PUT and the polyammes (Abe and Samejima, 1975; Endo, 1981). OPT derivatives have been successfully used for fluorimetric determinations (Kremzner, 1966; Elliott and Mrchaelson, 1967; Kremzner et al., 1970, Mach et al,, 1981). Finally, a number of workers have used dansyl derivatives (Creveling et al., 1968; Seiler and Wrechmann, 1970, Seiler and Demrsch, 1978) or the closely related dansyl conjugates (Seiler et al., 1973). Seiler (1977) has provided a comprehensive revrew of the methodology avarlable for the analysis of di- and polyamines. 3.1.6. Other Biogenic

Amines

and Metabolites

A variety of other biogenic amines have been analyzed following formation of fluorescent derivatives. Seiler and Weichmann (1970) have reported on the use of dansyl derivatives for quantitanon of primary and secondary ammes, imrdazoles, and phenols by fluorescence methods. An example of a dansyl derivative is given in Fig. 5. Direct fluorescence scanning of these derivatives on thin-layer chromatograms may be used, and a linear quantrtatron range of lo-’ to lo-r2 mol has been reported for this procedure. Reactions with mtrosonaphthol (Oates, 1961; Spector et al., 1963) or dansyl chloride (Kostyukovsku and Melamed, 1981) have been employed for analysis of urinary and tissue tyramine. For measurement of B-phenylethylamme, fluorescent products have been formed by reaction with alloxan (Boulton and Mrlward, 1971), p-dimethylammocinnamaldehyde (Spatz and Spatz, 1972), and ninhydrm m the presence of L-leucyl- L-alanine (Suzuki and

CH,CH,N-SO,

I

Fig 5. The dr-dansyl denvatrve formed by reactlon of dansyl chlorrde wrth the phenolic amine p-tyramme.

18

Baker, Butterworth,

andflewhurst

Yagi, 1976). Methods utilizing fluorescence also have been reported for the quantitation of 3,4-dimethoxyphenylethylamme (DMPEA) (Narasimhachari et al , 1972), and for mescaline (3,4,5-trimethoxyphenylethylamme) (Cohen and Vogel, 1970).

3.2. Native Fluorescence As mentioned previously, certain substances are naturally fluorescent and thus require no derivative formation before quantitatron. As a general rule, the use of native fluorescence provides lower sensitivity and reduced specificity compared to the use of fluorescent derivatives. The presence of interfering substances in the sample and a low fluorescence intensity are often problems with assays employing native fluorescence Many indoles exhibit native fluorescence m strong acid, and this property has been used for analysis of T (Sloerdsma et al., 1959; Oates, 1961), 5-HT (Bogdanski et al., 1956; Oates, 1961, Ashcroft and Sharman, 1962, von Redlich and Glick, 1969), 5-HIAA (Ashcroft and Sharman, 1962; Contractor, 1966, Chilcote, 1972; Haubrich and Denzer, 1973), and mdoleacetic acid (IAA) (Weissbach et al., 1959; Chilchote, 1972). Differential solvent extraction and measurement of fluorescence in HCl were utilized by Quay (1963) for the quantitation of 5-hydroxymdole, 5-methoxymdole, 5-HIAA, 5-HT, 5-HTP, bufotenm, melatonm, 5-MT, N-acetylserotonin, and 5-methoxymdole-3-acetic acid. Similar methods have been used by Naraslmhachari et al. (1971) and by Cohen and Vogel (1972) to mvestrgate N-methylated tryptammes.

3.3, Fluorescence Detection Combined With Another Quantitation Method 3.3.1. High Pressure f iquld Chromatography (HPLC) Analytical procedures may combme fluorescence detection with another maIor instrumental technique. In recent years HPLC methods have become increasingly popular, and many of these procedures employ fluorescence detection and quantitatlon [see the chapter by Anderson m this volume and reviews by Anderson and Young (1981) and Hartwick and Brown (1980)]. Davis et al. (1978) reported the use of precolumn derivatization with OPT followed by HPLC with fluorescence detection to measure NA, DA, 5-HT, NMN, OA, and TA, and in 1979 Davis et al added HA to this impressive list of compounds to be analyzed in a single sample. OPT derivatives have also been used m HPLC techniques to measure ammo acids, including GABA (Lindroth and Mopper, 1979), OA (Mel1 and Carpenter, 1980), mdoles (Anderson et al.,

Fluoresence

Analysis ofAmlnes

and The/r Metabohtes

19

1981), and HA (Skofitsch et al., 1981; Lebel, 1983). An OPT-ethanethiol derivative and a laser excitation source have been used by Todoriki et al. (1983) for analysis of NA and DA. The proposed structure for this unique derivative IS given m Fig. 6. HPLC followed by detection of native fluorescence has been used for quantitation of a number of mdoleammes and their metabolites including 5-HT, 5-HIAA, IAA, melatonm, N-acetyl5hydroxytryptamme, and a number of others (Young et al., 1980; McKim and Dewhurst, 1980; Flatmark et al., 1980; Cross and Joseph, 1981; Jackman et al., 1980; HOJO et al., 1982; Young and Anderson, 1982, Yamada et al., 1983; Wolf and Kuhn, 1983, Peat and Gibb, 1983). Precolumn derivatization with dansyl-Cl (Yamada and Aizawa, 1983) and postcolumn reaction with 2-cyanoacetamide (Honda et al., 1983) have been used for measurement of NA, A, and DA by HPLC with fluorescence detectron. 3.32

Other Methodology

A number of other methods have been combined with fluorescence detection for quantitatlon of biogenic ammes and their metabohtes Radlolabeled dansyl-Cl has been employed to measure catecholammes and 5-HT (Recasens et al., 1977) and 0-methylcatecholamme metabolites (Saller and Kopin, 1980). Dansyl and closely related derivatives have also been used for quantitation using combined thin-layer chromatography-hlghresolution mass spectrometry (seeChapter 8), a method applicable to many noncatecholic biogenic amines (Durden et al., 1974; Davis, 1979). Finally, Andermann and Andermann (1979) have reviewed methods combining fluorescence and densitometry for vanillylmandelic acid (VMA), HVA, 5-HIAA, A, NA, DA, and 5-HT.

3.4. Micromethods Micromethods represent specialized applications of fluorescence procedures for the assay of compounds in single cells (Osborne,

Fig. 6. Proposed structure for the OPT-ethanethiol derivative of dopamine (Todonki et al., 1983)

20

Baker, Butterworth, and Dewhurst

1974). A variety of substances have been measured, mcludmg NA, DA, 5-HT, T, A, OA, and ammo acids. McCaman et al. (1973) extracted 5-HT and DA from single cells of H~udo medrcina2is using a specific liquid cation exchanger and then assayed these amines using micromodifications of the assay methods of Maickel and Miller (1963) and Shellenberger and Gordon (1971). Sensitivity limits of 2 and 4 pmol were reported for 5-HT and DA, respectively. Osborne (1974) modified the method of Bell and Somerville (1966) for the semiquantitative estimation of DA, 5-HT, and NA m single cells Sensitivities of 6, 5, and 7 ng were reported for DA, 5-HT, and NA, respectively, but it was found that protein mterferes with amme analysis to a greater extent than it does m methods utilizing dansylation Dolezalova et al. (1973) have reported a method for detection of PUT, SPM, SPD, 5-HT, T, TA, A, NA, and DA as their dansyl derivatives. As little as 10-‘2-10-‘4 mol could be detected, but the method could not be used for quantitation. In his detailed 1974 review, Osborne (1974) carefully outlined microprocedures involving reaction with dansyl-Cl to measure the amines 5-HT, T, NA, A, OA, GABA, and other amino acids. Such derivatives are highly fluorescent and allow detection of as little as 5 pm01 on thin-layer chromatograms by direct fluorimetry. If radiolabeled dansyl-Cl is used and measured by autoradiography, as little as 1 pmol may be detected. Although such methods are very sensitive, they are not without problems. Quantitation is often difficult because of the occurrence of undesirable side reactions, variability m the degree to which individual compounds react, and variability caused by changes m the ratio of reagent to substrates. Osborne has stated that the identities and structures of all substances to be dansylated must be known before quantitatlon can be carried out. Because of these limitations, Internal standards can be used only when the ammo acid and amine contents of all samples are not significantly different. Quantitation can be improved by selectively isolating the compounds of Interest before the dansylation reaction; however, a decrease in sensitivity of up to a factor of 1000 may result. These micromethods have been applied to the measurement of single cell content, in vivo synthesis, and turnover of 5-HT m the giant serotonin cells of the cerebral ganglion of Helzx pomatiu, detection and measurement of T m nervous tissue of rat, mouse, snail, and crab, and measurement of NA, A, and OA in minute tissue samples.

Fluoresence

Analysis

of Amlnes

and Their Metabolites

4. Advantages and Disadvantages Techniques

21

of Fluorescence

Advantages of quantitative methods using fluorescence measurements include versatility and relatively low costs for instrumentation and reagents compared to those required for techmques such as mass spectrometry. Often large numbers of samples can be processed, and sophisticated training of personnel is not required. Fluorescence techniques also have important disadvantages. A great deal of care must be taken in order to ensure adequate sensitivity and specificity. Anton and Sayre (1962, 1964, 1966) have discussed m great detail the factors that can affect the outcome of fluorescence assays. A frequent criticism of fluorescence assays is lack of specificity, and there are a number of examples m the literature in which amine concentrations in tissues and body fluids measured by fluorescence methods are higher than those found with more specific techniques, such as mass spectrometry. Direct comparisons with other methods have now been reported in the literature. Three authors have compared traditional fluorescence measurements with methods using HPLC with electrochemical detection (ED). Marsden (1981) found that the OPT condensation procedure of Curzon and Green (1970) for 5-HT consistently gave values 30% higher than those obtained using HPLC wrth ED, although there was good correlation between the two methods. The authors suggest that the higher fluorescence values may be caused by the lack of a true tissue blank in the OPT assay. Curzon et al. (1981) have found that a slight modification of the OPT assay gave better agreement with the HPLC-ED method. Westermk (1982) has compared values for DA obtained by measurement of fluorescence after ethylene diamme condensation, DOPAC and HVA measured by the fluorescence procedure of Westerink and Korf (1977), and 5-HIAA assayed as its OPT condensate with values for the same four compounds obtained using HPLC-ED. He found a good correlation between the two methods for DOPAC and HVA, a poorer correlation for DA, and an unacceptable agreement for 5-HIAA, with the fluorescence methods being suspect m the latter two cases. Seller and Wiechmann (1979) found that the use of dansyl descanning riva tives combined with direct of thin-layer chromatograms resulted m a sensitivity comparable to that obtaining using 14C or 3H tracers (0.005 nmol). Carlmi and Green (1963) compared the guinea pig ileum bioassay for HA with the

22

Baker, Butterworth, and Dewhurst

OPT-fluorescence method (Shore et al., 1959) m rat bram and concluded that the bioassay was more specrfrc because the fluorescence assay appeared to be measuring other substances m addition to HA. Hakanson et al. (1972) reported that therr rmproved OPT method for analysrs of HA had a sensrtrvrty equal to that of the radloenzymatrc procedure used by Snyder et al. (1966), a claim shared by Siragaman (1975) and Beaven et al. (1982) Warren et al (1983) suggest that urmary HA may be more accurately measured by the fluorescence method than by the radioenzymatrc assay smce the latter procedure may be affected by urinary salt content Giacobmr (1975) compared the sensitrvitres of varrous analytical methods and reported a sensmvrty range of lo-“-lo-‘* mol for standard fluorimetric techniques This was compared wrth the followmg sensrtrvrtres for other techniques: colorimetrlc (lO-‘-lO-‘“), gas chromatographlc (lo-‘“lo-‘*), radlometrlc (10-‘2-10-13), mrcroTLC-dansyl (lo-‘*-lo-‘*), gas chromatographrc-mass sfectrometrrc (10-‘2-10-‘4), and fluorimetry-cyclmg (10-14-10-’ ).

5. Examples of Fluorimetric 5.1. Determination

Methods

of HI44 and DOPAC

The following procedure represents an amalgamatron of the methods of Sharman et al. (1967) for DOPAC and Anden et al. (1963) for HVA as reviewed by Murphy et al. (1969) and Sharman (1971) 5.1.1. Materials All reagents and chemrcals used are of analytical reagent quality unless otherwise stated. Glass-distrlled water L-Cysteine hydrochlorrde (recrystallized from ethanol) 1,2-Draminoethane (drstilled three times and stored at 4°C) n-Butyl acetate (distilled once and washed once with water) Microanalytical reagent-grade hydrochloric acrd Concentrated perchlorrc acid (sp. gr 1 72) Trrs solution (6 g/L m dlstllled water) 1,2-Draminoethane reagent (consistmg of 35 mL drstrlled water, 1 mL 2N HCl, and 1.5 mL 1,2-drammoethane)

CHEMICALS AND SOLVENTS

Fluoresence

Analysis ofAmines

and Their Metabobtes

23

Cysteine solution (freshly prepared at a concentration of 1 mg/mL) Potassium ferncyanide m 5N ammonmm hydroxide (20 m@) Hydrochloric acrd solution (1:l v/v dilution of cont. HCl 136% v/v] m distilled water) 1,2-Diammoethane solution (1:9 v/v dilution m distilled water) 5.1.2. Procedures

5.1.2.1. TISSUE PREPARATION Mice are stunned and decapitated and the brains are dissected out and placed on ice The samples are homogenized m 2 mL ice-cold O.lN HCl using a cooled glass homogenizer and the homogenate is placed m polypropylene or cellulose nitrate centrifuge tubes Ice-cold distilled water (1 mL) is used to wash the homogenizer, and these washings are added to the original homogenates. Each homogenate is frozen in liquid nltrogen and stored at 4°C until homogenization of all samples has been completed. Homogenates are thawed at room temperature and concentrated perchloric acid (0.12 mL) is added to each. After addition of excess solid potassium chloride, samples are centrifuged at 0°C (lS,OOO~, 5 mm) to precipitate protein and remove the potassium perchlorate precipitate. Supernatants are transferred to glass-stoppered tubes, a few crystals of potassium chloride are added to ensure saturation, and ti-butyl acetate (10 mL) IS added to each tube The tubes are shaken by hand (5 mm) and centrifuged (1 min) at room temperature. Two portions (4.5. mL each) of the butyl acetate layer are retained. Tris solution (2 mL) is added to one portion and 1,2-diammoethane reagent (2.2 mL) 1s added to the other. The tubes are cooled m ice, shaken (3 mm), and centrifuged (1 mm) Followmg these procedures, the butyl acetate layers are discarded. 5.1.2.2. ESTIMATION OF HVA. Three portions (0.6 mL each) of the Tris extract are used for analysis. A known amount of HVA (usually 0.1 kg) is added to one portion to act as an internal standard To another portion cysteine solution (0.2 mL) is added Potassium ferricyanide m ammonium hydroxide (1 mL) is added to each of the three tubes. After letting the tubes stand exactly 4 mm, cysteme solution (0.2 mL) is added to the two tubes not containing cysteine. Fluorescence IS measured at X,,, 315 nm; A,,,,, 430 nm. 5.1.2.3. ESTIMATION 1,2-diaminoethane

Two samples (1 mL each) of the extract are employed for analysis. Authentic

OF DOPAC

24

Baker,

Butterworth,

and Dewhurst

DOPAC (0.1 kg) IS added to one of the samples to act as an mternal standard. After heating both tubes in the dark in a water bath (60°C for 20 mm), they are cooled m ice. Hydrochloric acid solution (0.3 mL) is added to each. The samples are left on ice for a further 10 min and are then neutralized by adding 1,2-diaminoethane solution (0.3 mL). Fluorescence IS measured at A,,, 385 nm; h,,, 450 nm. The overall procedure is summarized in the flow diagram m Fig. 7.

Tissue drssectlon on ice

Homogemzatron Addrtlon

III 0.W HCI

of perchlorlc

acrd and solid KC1 15,OOOg,

Extraction

Addltron

5

mm

wrth n-butyl acetate

I

I

of Tris solution

Add&on

I

of 1,2-diammo-

ethane reagent

I FerncyanIde oxldatlon I Read HVA fluorescence A,, 315 nm A,, 430 nm

I Heat m the dark (6O”C, 20 min) I Cool on ice and a;d HCl I

I

Neutralize with 1,2-dlaammoethane ! Read DOPAC fluorescence A,, 385 nm A,, 450 nm Fig. 7.

Flow diagram for the analysis of HVA and DOPAC

Fluoresence Analysis ofAmines and TheirMetabolites

25

5.1.3. Recovery The recoveries of authentrc HVA and DOPAC are reported to be 70 -C 2.3% and 65 2 1.6%, respectively.

5.2. Simultaneous

Determination of DA, lY4, and 5-HT

The following procedure is representative of combined alumina absorption-spectrofluorimetric assays for the simultaneous measurement of nanogram quantities of DA, NA, and 5-HT in small samples (< 50 mg) of nervous &sue. Catecholamines are separated by alumina adsorption chromatography (DA and NA are adsorbed on alumma at pH 8.4-9.0). Subsequent elutron with acetic acid and oxrdatlon with iodine produces trihydroxymdole derivatives. 5-Hydroxytryptamine is measured by application of the OPT reaction. The method 1sa modification and extension of previously published procedures (Chang, 1964, Cox and Perhach, 1973; Metcalf, 1974; Butterworth et al., 1975).

5.2.1. Materials 5.2.1.1. CHEMICALS, SOLVENTS All chemicals and solvents used for the procedures described are analytical reagent grade unless otherwise specified. Water: Double glass-distilled, deionized water was used throughout both for preparation of standard solutions and buffers. Alumma: Acid-washed alummum oxide IS prepared according to the method of Udenfrrend (1962) as follows. 200-g batches of aluminum oxide (BDH, for chromatographic adsorption) are boiled under reflux for 30 min in 1 L HCl(2N). Following removal of the supernatant, the alumina is mixed with 1 L distilled water, shaken gently, allowed to settle (5 mm), and the supernatant decanted This process 1s repeated 8-10 times until washings are clear and the pH is 4-5. The alumina IS filtered under suction, allowed to dry in a desiccator overnight at room temperature, and then dried m an oven at 200 “C for 2 h. Aczdzfiedn-butanol containing 0.1% sodium metabisulfite and 0.01% EDTA* Freshly glass-distilled n-butanol (500 mL, Fisher Scientific Co.) IS washed successively with 50 mL NaOH (PI), 50 mL HCl (WI), and 4 x 50 mL distilled water, and 1s then saturated with NaCl (Ansell and Beeson, 1968). This saturated n-butanol IS then strrred vigorously with 0.43 mL concentrated HCl; 0.5 g sodium

26

Baker,

Butterworth,

and Dewhurst

metabisulfite is added, followed by 0.05 g sodium EDTA and the mixture is again stirred vigorously. Borate buffer (0 35M) pH 11.O: Boric acid (3.14 g) is dlssolved m 100 mL distilled water, 5.5 mL NaOH (1OiV) is added, the solution is saturated with n-butanol and sodium chloride and adjusted to pH 11.0 as required. EDTA reagent (O.ZM), pH 6.5. Sodium EDTA (9.30 g) IS dissolved m 225 mL sodium acetate (IM), the pH is adlusted to 6.5 with NaOH (lON), and the solution made up to 250 mL with sodmm acetate (1M) (Ansell and Beeson, 1968). lodzne reagent (0.W: Iodine (1.27 g) is dissolved m 100 mL freshly distilled absolute ethanol (Chang, 1964). o-Phfhalaldehyde reagent: o-Phthalaldehyde (1 mg) is dissolved in 100 mL HCI (1ON) This solution is freshly prepared for each series of determmations Alkulzne sulfite reagent Sodium sulfite (1.25 g) is dissolved m 5 mL drstrlled water; 0.5 mL of this solution is added to 4.5 mL NaOH (5N) lust prior to use. 5.2.2. Procedures

Catecholamme content of the brain decreases durmg the postmortem perrod For thus reason, following sacrifice, tissue is rapidly removed and dissected on dry ice, wrapped In preweighed alummum forl, and frozen on dry ice. Trssue is stored at -70°C until time of assay. 5.2.2.1. EXTRACTION Each weighed fragment of nervous tissue is separately homogenized (teflonglass homogenizer, 10 passes, 2700 rpm) on ice m 3.3 mL acidified n-butanollsodium metabisulfite/EDTA. Tissue samples weighing over 300 mg are homogenized m 10 vol acidifed n-butanol mixture and 3.3 mL of this homogenate is then used for the extraction procedure. Homogenates are shaken (10 mm) with 0.5 mL HCl (O.OlN). Standard solutions of ammes are prepared m 0 5 mL HCl (O.OlN), 3.3 mL of acidified n-butanol mixture is added, and the mixture shaken 10 min. Followmg centnfugation (lOOOg, 5 min), 3 mL of organic phase is removed, shaken with 4.5 mL heptane, and 1.0 mL distilled water for 5 mm and centrifuged (lOOOg, 5 mm). The upper (organic) phase is discarded and 1 0 mL of aqueous phase added to 10 mL polypropylene tubes containing 100 mg alumma in 1.3 mL sodium acetate (2M) containing 0 2% EDTA. The tightly capped tubes are gently shaken horizontally for 5 mm and centrifuged (15OOg, 5 min)

Fluoresence Analysis ofAmlnes and Their Metabobtes

27

5.2.2.2. ISOLATION AND FLUORESCENCEOF~-HT To 2.0 mL of SUpernatant from the alumma adsorption step, NaCl(2.Og), 0.70 mL borate buffer (0 35M, pH 11 0), and 3 0 mL acidified n-butanol containing 0.1% sodium metabisulflte and 0.01% EDTA are added. The mixture is shaken (10 min), centrifuged (lOOOg, 5 min), 2.5 mL of organic phase removed, and 0.25 mL HCl (O.lN) and 3 0 mL n-heptane added. The mixture 1s shaken (5 mm), centrifuged (10008, 5 mm), and the organic layer IS dlscarded. To 0.2 mL of the acid extract 1sadded 0.3 mL OPT reagent. The mixture IS heated (lOO“C, 10 mm) and 5-HT fluorescence read (X,,, 365 nm, At.?m,480 nm, uncorrected) 5.2.2.3. ISOLATION AND FLUORESCENCE OF DA AND NA. Any SUpernatant remaining from the alumina separation IS removed. Distilled water (2.0 mL) IS added to the alumma and the mixture IS shaken 5 min. Followmg centrifugatlon (15OOg, 5 mm), the supernatant is discarded, 0.6 mL acetic acid (1N) added, and the mixture shaken gently m the horizontal position for 15 mm to elute catecholamines Alternatively, 0 6 mL sodium phosphate buffer (0 5M, pH 6.0) containing 0 75% EDTA may be used for elution (Metcalf, 1974) The mixture 1s centrifuged (ZSOOg,5 mm), 0.5 mL acid extract IS removed, and to this extract IS added 0.5 mL EDTA (O.lM) and 2 0 mL sodium acetate (1M) To this mixture, 0.25 mL lodme reagent IS added, followed precisely 2 mm later by 0.50 mL alkaline sulfite reagent. Exactly 2 mm later, 0 50 mL acetlc acid (5N) is added and the mixture is heated (lOO”C, 2 mm). The fluorescence (from NA) of the cooled solution IS read (h,,, 390 nm; Al3tlr 375 nm) A flow diagram summarizing the extraction and separation procedures 1s shown in Fig 8. 5.2.3. Reiovery Recoveries of ammes added to the initial homogenate prior to the extraction procedure have been found to be DA, 7270, NA, 75%; 5-HT, 65%. 5.2.4. Sensltwty The sensltlvity of the assay, defined as the amount of each amme that must be added to an extract in order to produce a final fluorescence reading of twice that of the tissue blank (Metcalf, 1974) 1s 50 ng for DA and 5-HT and 20 ng for NA. 5.2.5. interference of Various Agents in the Assay Previous studies have shown that the use of nmhydrm for formation of the fluorescent product of 5-HT may be accompanied by

28

Baker, Butterworth,

and Dewhurst

Raprd drssectron of nervous trssue on dry ice

Homogemzatlon

in acidified

I n-butanol/metablsulfrte/EDTA IOOOg,

Extraction

(water, n-heptane) ! lOOOg,

Alumina

absorptron

5 mm

chromatography, /

Elutron

5 mm

15008,

pH 8 4-9.0

5 mm

(acetic acid 0 1N) I

Acid extract ,

15OOg, 5 mm

I Trrhydroxymdole reaction (rodme, alkaline sulfite)

I

OPT reaction

i

Read fluorescence I

Fig. 8. Flow diagram DA, and 5-HT

Read fluorescence I

of extractron-separation

procedure

for NA,

formation of a contammatmg fluorophore that interferes with the 5-HT assay (Ansell and Beeson, 1968). In addmon, the 5-HTdepleting drug, p-chlorophenylalanme, routmely used m 5-HT turnover studies, reportedly reacts with nmhydrm to form a fluorescent product that may also cause interference (Metcalf, 1974). Such drffrculties appear to be circumvented to a malor degree using OPT as described in the present procedure. Neither p-chlorophenylalanine (Cox and Perhach, 1973) nor the ammedepleting drugs reserpme or ol-MPT (Butterworth et al , 1975) cause sigrufrcant interference using this method for the assay of 5-HT However, a-MPT reportedly interferes with the fluori-

Fluoresence

Analysis ofAmlnes

and ThelrMetabobtes

29

metric analysis of catecholamines (Dolphin et al., 1975). Results of this latter study suggested that, when using the CY-MPT technique for turnover studies, the most reliable results for fluorlmetric estlmation of catecholammes are to be obtained using methods involving Dowex-50 ion-exchange column separation The use of certam buffers can reportedly lead to difficulties in the fluorimetrlc estimation of catecholamines. It has been reported that dibasic potassium phosphate, for example, has a deleterious effect on the linearity of DA standard curves (Greenland and Mlchaelson, 1974). As a result of these findings, it 1s essential, when studying the effects of drugs on amme distrrbution by the method described, to include m the experimental design appropriate control experiments m which the drug (and known metabolites) are investigated for possible interference m the assay system. 5.2.6. Some Representative Applica tlons Procedures similar to the one described have been used extensively for simultaneous estimation of DA, NA, and 5-HT m small quantities of nervous tissue. Studies of the effect of drugs and of discrete 6-hydroxydopamme lesions on cerebral amine distnbution have been described using the above technique (Izumi et al , 1978, Butterworth et al., 1978). Concentrations of DA, NA, and 5-HT m rat and mouse brain regions are shown m Table 1 These values are in good general agreement with those in the current literature obtamed usmg alternative techniques Cerebral amme turnover studies involving the use of ammedepleting drugs have been used as an aid to the elucidation of the mechanisms of action of drugs affecting the central nervous system. The method described is applicable to such studies. For ex-

Amme

ContenP

TABLE 1 of Some Rat and Mouse Brain Regions Amme

Brain region (species) Cerebral cortex (rat) Caudate nucleus (rat) Hypothalamus (rat) Hypothalamus (mouse) Values

represent

mean values (tSEM)

k * _’ +

bglg wet wt

NA

DA 0.33 6.33 0 27 0.54

concentration,

0 03 0 56 0 12 0.20

0.22 0.22 1 52 1.32

t 2 + k

5-HT 0.02 0 04 0.17 0 12

of five determmatlons

0 26 0 52 0.95 1 56

k ? * 2

0.02 0 09 0.10 0.17

30

Baker,

Buttenvorth,

and Dewhurst

ample, amme content of striatum followmg reserpme depletion of cerebral monoamine stores is shown m Fig. 9.

6. Summary As discussed above, fluorescence methods have been used for measurement of a variety of biogemc ammes and then metabolites. Such methods offer sufficient sensitivity and speciflclty to be useful in many types of neurochemical and neuropharmacologlcal experiments. The recent development of HPLC technology has often mcreased both the sensitivity and specificity for a particular fluorescence technique so that many of these combined procedures may be the method of choice for specific compounds. Fluorescence assays can be performed without a large capital outlay, and this property as well as the fact that a large number of samples can be processed by relatrvely mexperrenced personnel have made this type of assay attractive to many laboratories. The large number of studies reported m the literature utllizmg fluorescence methods indicate that this popularity will continue.

0 Saline Qj Reserpine (5mgperkg,ip)

NA

5HT

Fig. 9. Concentrations of catecholammes and 5-HT in strlatum of rats InJected with salme or reserpme 4 h before sacnfrce. Values shown represent mean + SEM (n = 5).

Fluoresence

Analysjs of Amlnes

and Their Metaboktes

31

Acknowledgments Funding from the Alberta Mental Health Research Fund, the Alberta Heritage Foundation for Medical Research, and the Medical Research Council of Canada 1s gratefully acknowledged.

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Noah J W and Brand A (1961) A fluorometrlc method to determine levels of histamine m human plasma. 1 Allergy 32, 236-240 Noah J W and Brand A. (1963) Slmplifled mlcromethod for measuring hlstamme m human plasma J, Lab Clin Med. 62, 506-510. Oates J. A. (1961) Measurement of urinary tryptamme, tyramme and serotonm, in Methods zn Medzcal Research (Quastel J H., ed ). Year Book Medical Publisher Ltd., Chicago pp. 169-174. Oberman Z , Chayen R., and Herzberg M (1970) A rapid fluorlmetrlc method for the determmatlon of dopamme m urine. Clan Chvn Acta 29, 391-394. Osborne N. N. (1974) Mlcrochemlcal analysis of nervous tissue, m Methods ZMLzfe Saences, Pergamon Press, Oxford Vol 1, 225 pp Palmer J F. (1963) The use of p-thtoproplonlc acid for stabilizing the fluorescence of adrenolutme and noradrenolutme J Pharm Pharmacol

15, 777-778

Parkm J V , Lorenz W , Barth H , Rohde H., Ohmann Ch., Thon K., Weber D , and Chrombach M (1982) Assay and ldentlflcation of m human gastric aspirate by a fluorometnchistamine fluoroenzymatlc technique Its appllcatlon m patients with chronic duodenal ulcer. Agents Actzons 12, 17-25. and Glbb J W (1983) High-performance liquid Peat M chromatographlc determmatlon of mdoleammes, dopamme, and norpinephrme m rat brain with fluorometrlc detection Anal Blochem 128, 275280.

Prasad A. L N and Fahn S. (1974) 3-O-Methyl metabolites of catecholammes automated fluorometrlc assay and their plasma levels m patients receiving levodopa and carbldopa Bzochem Med. 9, 136-147 Price H. L and Price M L (1957) The chemical estimation of epinephrme and norepmephrme m human and canine plasma II A critique of the trlhydroxymdole method J Lab Clm Med 50, 769-777. Prozlaleck W C , Boehme D H , and Vogel W H. (1978) The fluorlmetrlc determmatlon of 5-methoxytryptamme m mammalian tissues and fluids J Neuvochem 30, 1471-1477 Quay W. 8. (1963) Differential extractions for the spectrophotofluorometrlc measurement of diverse 5-hydroxy- and 5-methoxymdolcs AnaI Blochem 5, 51-59. Quay W. (1968) Speclflclty of fluorometry of 5-hydroxytryptamme by means of products with nmhydrm. J Pharm Scz 57, 1568-1572 Recasens M , Zwlller J , Mack G , Zanetta J. P , and Mandel I’. (1977) Douple isotope method for the determination of catecholammes, serotonm, and other ammes m the plcomole range as their dansyl derivatives Anal Blochem 82, 8-17. Remders E J , Chlu H C , and Yoo T J (1980) Human leukocyte hlstamine release assays with whole ragweed and alternarla antigens using Techmcon Autoanalyser II Allergy 35, 391-398. Rohde H., Lorenz W , Troldl H , Relmann H -J , Hafner G., and Weber D (1980) Hlstamme and peptic ulcer influence of sample-taking on

Fluoresence

Analysis ofAmlnes

and ThelrMetabolltes

41

the preclslon and accuracy of fluorometrlc histamine assay 1n biopsies of human gastric mucosa Agents Acfmns 10, 175-185 Rosengren E. (1960) On the role of monoamine oxldase for the inactivation of dopamine in brain Acta pkyslol stand. 49, 370-375 Saller C F and Kop1n I. J (1980) O-Methyl catecholam1ne metabollte analysis using H-dansyl chloride. Sac. Nettvosc1. Abstr 6, 444. Seiler N. (1977) Assay procedures for polyam1nes 1n urine, serum, and cerebrosplnal fluid. Clrn. Ckem 23, 1519-1526. Seller N. and Demlsch L (1978) Fluorescent denvatlves, 1n Handbook of Derwatwes for Chromatography (Blau K. and King G. S., eds.). Heyden, London. pp. 346-390 Seller N., Schmidt-Glenewlnkel T., and Schneider H H (1973) 5-D1-nbutylamlnonaphthalene-l-sulphonyl chloride-a new reagent for fluorescence labelrng of amInes, amino aads, and peptIdes. I Ckromatogr

84, 95-107.

Seiler N. and Wiechmann M (1970) TLC analysis of am1nes as their DANS-denvatlves, 1n Progress rn Tkm-Layer Chromatography and Related Methods, Ann Arbor-Humphrey. Ann Arbor, MI. Vol. 1, pp. 95-144. Seki T and Hamal M (1979) Method for the fluorlmetrlc estimation of dopamine J Ckromutogr 162, 388-393 Sharman D F. (1963) A fluorlmetrlc method for the estimation of 4-hydroxy3-methoxyphenylacenc acid (homovan1111c aad) and 1ts ldentiflcatlon 1n brain tissue Brat ] Pkarmacol 20, 204-213 Sharman D F (1971) Methods of determlnatlon of catecholamlnes and their metabohtes, 1n Methods of Neurockemzstvy (Fried R., ed.). Marcel Dekker, New York Vol 1, pp. 83-128 Sharman D. F., Po1r1er L. J , Murphy G F , and Sourkes T L (1967) Homovan1111c acid and dlhydroxyphenylacetlc acid 1n the strlatum of monkeys with brain lesions. Can. J Pkyslol Pkarmacol 45, 57-62 Shellenberger M K. and Gordon J. H (1971) A rapid s1mphf1ed procedure for simultaneous assay of norepinephrine, dopamme, and 5-hydroxytryptamme from discrete brain areas. Anal Bzochem 39, 3.56-372 Shelley W. B and Juhl1n L (1966) Histamine chromatography and electrophoresls The o-phthalaldehyde fluorogram J Ckromatogr. 22, 13c-138 Shore I’. A., Burkhalter A , and Cohn V H (1959) A method for the fluorometrlc assay of histamine in tissues 1 Pkarmacol Exp Ther 127, 182-186

Shore I’ and Olin J. (1958) Identlf1catlon and chemical assay of norepinephrlne 1n brain and other tissues 1 Pkarmacol. Exp Tker 122, 295-300. S1ragan1an R. I’ (1975) Refinements 1n the automated fluorometrlc histamine analysis system. J Immunol Methods 7, 283-290 Sloerdsma A., Oates J. A , Zaltzman I’., and Udenfrlend S. (1959) Identlf1cat1on and assay of urinary tryptamme application as an in-

42

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dex of monoamme

oxldase mhlbltlon

m man

and Dewhurst

J Phurmacol

Exp

Ther. 126, 217-222.

Skofltsch G , Sarla A , Holzer I’ , and Llmbeck F (1981) Hlstamme m tissue. determination by high-performance liquid chromatography after condensation with o-phthaldlaldehyde. J. Chromatogr 226, 53-59. Smith E. and Well-Malherbe H (1961) Estimation of metanephrme and normetanephrme m urine Fed. PYOC 20, 182 Snyder S. H., Axelrod J , and Zwelg M (1965) A sensitive and specific fluorescence assay for tissue serotonm. Btochem Pharmacol 14, 831-835. Snyder S H., Baldessanm R. J , and Axelrod J. (1966) A sensltlve and specific enzymatic lsotoplc assay for tissue histamine. 1 Pharmacol Exp. Ther 153, 544-549. Sourkes T L. and Murphy G F (1961) Determmatlon of catecholammes and catecholammo acids by differential spectrophotofluorlmetry, m Methods zn Medrcal Research (Quastel J. H., ed). Year Book Medical Publishers Ltd , Chicago pp 147-152 Spatz H. and Spatz N (1972) Spectrophotofluorometnc determmatlon of beta-phenylethylamme m blood and urme Bzochem Med 6, l-6. Spector S , Melman K , Lovenberg W., and Sloerdsma A (1963) The presence and dlstrlbutlon of tyramme m mammalian tissues 1 Pharmacol. Exp

Ther 140, 229-235

Suzuki S and Yagl K (1976) A fluorometrlc assay of P-phenylethylamme m rat brain Anal Blochem 75, 192-200 Szabo G , Kovacs G L , and Telegdy G (1983) A modlfled screening method for rapid simultaneous determmatlon of dopamme, noradrenalme, and serotonm m the same brain region Acfa Physlol Hung.

61, 51-57

Tamguchl K., Kaklmoto Y., and Armstrong M D (1964) Quantltatlve determination of metanephrme and normetanephrme m urine. 1 Lab Clm

Med 64, 469484.

Thompson W L and Walton R I’ (1964) Elevation of plasma histamine levels m the dog followmg admmlstratlon of muscle relaxants, oplates, and macromolecular polymers I Pharmacol Exp Ther 143, 131-136. Todorlkl H , Hayashl T , Nasuse H , and Hlrakawa A Y (1983) Sensltlve high-performance liquid chromatographlc determmatlon of catecholammes m rat brain using a laser fluorlmetrlc detection system ] Chromatogr 276, 45-54. Udenfrlend S. (1962) Fluorescence Assay IM Biology and Medzcm Academic Press, New York, p 144. Uuspaa V J (1963) A new method for the determination of dopamme (3-hydroxytyramme) Ann Med Exptl Bzol. Fenmae (HelsmW 41, 194201

Valk A DeT Jr and Price H L (1956) The chemical estlmatlon of eplnephrme and norepmephrme m human and canine plasma I A cn-

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Analysis ofAmines

and The/rMetabobtes

43

tique of the ethylene diamme condensation method. I Clan. Invest. 35, 837-841 Vanable J (1963) A nmhydrm reaction givmg a sensitive quantltatlve fluorescence assay for 5-hydroxytryptamme Anal Blochem 6, 393403 Vendsalu A. (1960) Studies on adrenaline and noradrenalme m human plasma. Acta PhysioI. Scand 49 (suppl 173), 23-32. von Euler C , von Euler U. S , and Flodmg I (1955) Biologically inactive catechol derivatives m urme. Acta Pkysml Scund 33, (suppl 118), 32-38. von Euler U. S. and Flodmg I. (1955) A fluorimetric micromethod for differential estimation of adrenaline and noradrenalme. Acfa Pkyszol Stand 33, (suppl 118), 45-56. von Euler U. and Lishajko F. (1959) The estimation of catechol ammes m urine. Acta Physrol Stand 45, 122-132 von Euler U. S. and Lishalko F (1961) Improved techmque for the fluorimetric estimation of catecholammes. Acfa Physzol Stand 51, 348-356. von Redlich D and Glick D (1965) Studies in histochemistry LXXVI Fluorometric determmation of histamine m microgram samples of tissue or microliter volumes of body fluids Anal Bmchem 10, 459467. von Redlich D and Glick D (1969) Improvements m fluorometric microdetermmation of histamine and serotonm Anal Bmhenz 29, 167-171 Warren K., Dyer J Merlin S , and Kalmer M (1983) Measurement of urinary histamine comparison of fluorometric and radioisotopic-enzymatic assay procedures / Aller(yy C/III lmmunol 71, 206-211. Well-Malherbe H (1960) The condensation of catechols with ethylenediamme Bmckfm Bmphys. Acfn 40, 351-353 Well-Malherbe H (1961) The fluorimetric estimation of catecholammes, in Methods m MedlcaI Research(Quastel J H., ed ), Year Book Medical Publishers Ltd., Chicago. Vol 9, pp 130-146. Well-Malherbe H. and Bigelow L B. (1968) The fluorometric estimation of epmephrme and norepmephrme an improved modification of the trihydroxymdole method. Anal, Bzochem. 22, 321-334 Weil-Malherbe H. and Bone A D. (1952) The chemical estimation of adrenaline-like substance m blood. Blochem. \ 51, 311-318 Well-Malherbe H and Smith E R B (1966) The estimation of metanephrme, normetanephrme, and 3,4-dihydroxymandelic acid m urine Pharmacol Rev 18, 331-341 Weissbach H , Kmg W , Sloerdsma A , and Udenfriend S (1959) Formation of mdole-3-acetic acid and tryptamme m animals J Biol Chem 234, 81-86 Welch K M A., Meyers J. S , and Kwant S (1972) Estimation of levels of serotomn and 5-hydroxymdoles m whole blood by an

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autoanalytrcal

Butterworth,

and Dewhurst

procedure observatron on the blood-brain barrier J 19, 1079-1087 Westermk B H C (1982) Correlation between high-performance liquid chromatography and automated fluorrmetrrc methods for the determmatron of dopamme, 3,4-drhydroxyphenylacetlc acid, homovarullrc acid, and 5-hydroxymdoleacetrc acid m nervous tissue and cerebrospmal fluid J Chromafogr 233, 69-77 Westermk B. H C and Korf J (1977) Rapid concurrent automated fluorometrrc assay of noradrenalme, dopamme, 3,kdrhydroxyphenylacetrc acid, homovanlllrc acid, and 3-methoxytyramme u-r millrgram amounts of nervous tissue after rsolatron on Sephadex G-10 J Neurochem 29, 697-706 Wllhelms 0. -H. (1980) An improved automated fluorlmetrrc method for determination of histamine J lmmunol Methods 36, 221-226 Wolf W A and Kuhn D M (1983) Simultaneous determmatlon of 5-hydroxytryptamme, Its ammo acid precursors and acid metabohte m discrete brain regions by high-performance liquid chromatography with fluorescence detection J Chromafogr 275, l-9. Yamada J , Sugrmoto Y , and Honsaka K (1983) Simultaneous determrnatron of tryptophan and its metabolrtes m mouse brain by hrghperformance hqurd chromatography with fluorometrrc detection Anal Btochem 129, 460-463 Yamada K. and Arzawa Y (1983) Determmatron of catecholammes m rat tissue by precolumn dansylatron using micro high-performance lrqurd chromatography with fluorescence detection. J, Pharmacol Methods 9, l-6. Yamatodaru A., Sekr T., Taneda M , and Wada H (1977) Determination of hrstamme and methylhrstammes by dansylatron and its applrcatron to brologrcal specrmens. J Chromafogr 144, 141-145. Young S. N. and Anderson G M. (1982) Factors mfluencmg melatonm, 5-hydroxytryptophol, 5-hydroxymdoleacetlc acid, 5-hydroxytryptamme, and tryptophan m rat pmeal glands. Neuroendocrtnol 35, 464-468 Young S. N , Anderson G M., Gauthrer S , and Purdy W, C. (1980) The origm of mdoleacetrc acid m rat and human cerebrospmal fluid. ] Neurochem 34, 1087-1092 Neurochem

Chapter 2

Gas Chromatography of Amines and Their Metabolites in Tissues and Body Fluids RONALDT.COUTTS,GLEN B. BAKER,AND ADIL J. NIIZPWU 1. Introduction Gas chromatography (CC) 1s a technique used to separate and quantify the components of a mixture of volatile compounds by partitioning them as vapors between two phases-a stationary and a mobile phase. The stationary phase IS contained in a narrow tube (the column) through which the mixture of compounds to be separated IS percolated m a stream of gas (the mobile phase or carrler gas). Commonly used carrier gases are helium, nitrogen, and hydrogen. Although the stationary phase can be a solid (hence the term “gas-solid partition chromatography”), rt IS most often a high-boiling, virtually nonvolatile liquid. When this IS so, a more precise, but rarely employed, term for the separation technique IS “gas-1rquld partition chromatography ” The shorter expression, “gas-liquid chromatography” (GLC), IS stall encountered; however, the technique IS now most commonly referred to as “gas chromatography” or GC. Gas chromatography 1s performed m a gas chromatograph, which has various components inlets to permit entry of both the mixture to be separated and the inert carrier gas, an oven m which the column IS placed, with a variable temperature, normally over a 20-300°C range; and a detector that detects each component as it elutes from the column. The detector is connected to a recorder that provides a chart with each component of the mix45

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ture displayed as a peak. The time interval between the point of inlection of the mixture and the apex of the recorded peak is termed the retention time of the elutmg compound. This value 1s characteristic of, but not unique to, the eluting compound. Retention times vary with changes m oven temperature, nature and quantity of stationary phase, carrier gas-flow rate, column length and diameter, and other factors They are reasonably reproducible, however, if the GC condltlons used are kept constant. Chromatographic separations are performed under isothermal conditions (constant column temperature), or by temperature programming m which the column temperature 1s increased at preselected rates during the analysis. A mixture contammg constituents of a limited boiling range can usually be separated adequately using isothermal operating condltlons. Temperature programming 1s necessary when mixtures contammg a wide range of components are investigated This results in improved resolution and a great reduction m analysis time Space does not allow a comprehensive discussion of the fundamentals of CC, but the reader 1s referred to a number of useful reference books (e.g , Dal Nogare and Juvet, 1962; Purnell, 1962; Ettre, 1965; Harris and Habgood, 1966, Ettre and Zlatkls, 1967; Gudzinowlcz, 1967; McNau- and Bonelh, 1968, Schupp, 1968, Jones, 1970, Lltllewood, 1970, Grant, 1971; Leathard and Shurlock, 1971; Walker et al., 1972; Ettre, 1973; Sevlck, 1976; Jennings, 1980, Freeman, 1981).

1.1. GC Columns Columns have been made from numerous materials including glass, stainless steel, nickel, copper, aluminum, and nylon. Glass-lined metal columns are also available. In blologlcal studies, however, it is necessary to mmimlze “on-column” degradations, which occur more often on heated metal columns They are therefore generally avoided Because of their inertness, glass columns are most often employed. There are two major types Conventional glass columns are typically l-2 m m length, 24 mm in internal diameter, and coiled. Capillary columns are generally made of borosllicate glass or flexible fused silica They are typically lo-100 m m length (coiled) and 0 25-0.50 mm m internal dlameter Conventional columns (packed columns) contain the stationary phase coated onto an inert solld support material.

1.2. Support Materials Most support materials (diatomlte, diatomaceous

are prepared from diatomaceous slhca, kleselguhr). They are inert,

earth regu-

Gas Chromatography

ofAmrnes

and Their Metabolites

47

larly shaped particles, uniform in size, highly porous, and have a large surface area. There are varrous forms, differing in density and mesh size. Low-density supports can be loaded with more stationary phase than supports of high density. The most commonly available supports are Chromosorb P and Chromosorb W, produced by the Johns Manvrlle Company Chromosorb P IS pink in color and is made from C-22 Firebrick. Its surface is adsorptive and it has a high surface area. Chromosorb W is white m color. It has a nonadsorptrve surface and a lower surface area than Chromosorb I’. Chromosorb P is the preferred support for the separation of nonpolar substances, especially hydrocarbons, Chromosorb W IS commonly used for the separation of polar substances. Support surfaces are covered with srlanol ($--OH) and siloxane (Si-0-Si) groups, which hydrogen bond with solvents and the solutes that are to be separated. Even when coated with a stationary phase, the solrd support can still hydrogen bond with solutes, causmg broadening of peaks and tailing. Thus a common practice IS to treat the packed column, containing the stationary phase, with a srlylatmg agent, such as drmethyldichlorosilane (DMCS) or hexamethyldisrlizane (HMDS). These reagents react with and neutrahze the srlanol groups and greatly improve column efficiency Polymer beads (Porapak@) have also been employed as packing material (Hollis, 1966) and serve as both stationary liquid phase and solid support. Separation of components of a mixture occurs on Porapak by a combination of partitron and adsorptron (Zwerg and Sherma, 1972).

1.3. Stationaly Phases Numerous liquid stationary phases are available and selectron of the best liquid phase IS dictated by the nature of the compounds that are to be separated. Those commonly used by the authors are Carbowax 20M, SE-30, OV-1, OV-17, OV-101, Apiezon-L, and Porapak Q, but these are simply personal preferences; other hqurd phases are also effective. Although the amount of a stationary phase used to coat a solid support may vary greatly, usually l-5% wt/wt of the stationary phase IS used. Stationary liquid phases can be classrfred on the basis of their polarity Polymers of polyethylene glycol (Carbowax) are polar, while polymers of drmethylsilrcone (e.g., SE-30, OV-101) are nonpolar. Semipolar phenylmethylsrlrcone polymers (e.g., OV-17) are also used extensively As a general rule, separations are best achieved through matching solute and liquid types, i.e., polar liquid phases retain polar solutes, whereas nonpolar liquid phases retam nonpolar solutes (Mitchard, 1978)

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1.4. Capillary Columns Two types of capillary, or open tubular, columns are in common use. In the wall-coated open tubular (WCOT) type, the liquid phase is deposited directly onto the inner glass surface of the column without the mclusion of a solid support. In support-coated open tubular (SCOT) columns, the inner surface of the capillary column is covered with a thin layer of a solid support that is coated with the liquid phase. Capillary columns provide much better resolution of solutes in a mixture than can be attained using packed columns. Although it is the common practice for mvestigators to prepare their own packing (stationary phase or solid support) for conventional glass columns, capillary columns containing the stationary phase are generally purchased from a commercial source. Numerous liquid phases are used in capillary columns, although relatively few have been coated successfully on Those used by the authors include glass WCOT columns Carbowax 20M, OV-1, OV-101, and SP 2100, but many other excellent liquid phases are available Fused silica capillary columns are popular because of then mertness, flexibility, and durability. In addition, the necessity for time-consuming column straightening is eliminated. Because of these features, these columns are now used almost exclusively by the authors. Metal (stainless steel or nickel) capillary columns are also now available. Potential users of GC will fmd that most of the catalogues for the commercial companies contain comprehensive mformation about column dimensions and typical uses for particular columns. The novice practitioner (or the experienced user who is changing applications) is encouraged to read these sources for mformation such as temperatures for optimum operation of columns, advantages and disadvantages of SCOT columns vs WCOT columns, and the polarity of certain liquid phases. In the case of certain compounds that are assayed by many laboratories, detailed protocols and relevant references may also be given m the catalog or m newsletters published by companies providmg chromatographic supplies. 1.5.

Detectors

The separated components tected and quantified using ferent detectors have been ity detector (TCD), the electron-capture detector

emerging from the GC column are dea chromatographlc detector Four difused routinely. the thermal conductivflame iomzatlon detector (FID), the (ECD), and the nitrogen-phosphorus

Gas Chromatography ofAmines and The/r Metabobtes

49

(alkali flame) detector (NPD) A mass spectrometer (MS) may also be used as a detector In addition to its ability to detect and quantify, the MS detector also provides information on the identities of unknown compounds elutmg from the column. 1.5.1. Thermal Conductivity Detector The TCD operates on the principle that a heated filament will lose heat upon contact with the gaseous solute emerging from the column. The degree of heat loss 1s proportional to the quantity of solute molecules that collides with the filament. The change in filament temperature 1s amplified and recorded as a peak on the recorder. The TCD is nondestructive, elutmg compounds can be collected for further investigation The TCD method has advantages: It is an inexpensive, robust detector that produces a linear response over a wide range of sample amounts (104); it 1s nonselective; and it responds to all compounds. Its malor dlsadvantage IS the lack of sensitivity (minimum detectable quantity 1s about 10 kg). 1.5.2. Flame ionization Detector The FID is widely used for the routine analysis of biological samples. The effluent from the GC column 1s mlxed with hydrogen and burned m air or oxygen. Ions and electrons form m the flame and collect on a charged electrode, producing an electric current whose strength 1s directly proportional to the amount of compound cornbusted. The advantages of the FID are detector selectivity and sensitivity. The FID responds to all compounds that combust with ionization m a hydrogen/air flame. This includes all organic compounds, but excludes helium, argon, oxygen, nitrogen, carbon dioxide, water, and other morgamc gases. The detector produces a linear response over a wide sample range (107) and has a minimum detectable quantity of about 1 ng for many organic compounds, and around 20-50 pg for alkanes. 1.5.3. Electron Capture Detector The ECD measures the reduction m strength of a standing current when organic compounds capable of absorbing (capturmg) electrons enter the detector (Lovecock and Lipsky, 1960; Sevcik, 1976). The detector contains a radloactlve source, usually 3H or ‘j3N1, which emits p-particles. These particles collide with molecules of the carrier gas (95% argon&10% methane or nitrogen), producing slow electrons that are collected at electrodes to give a small current (standing current). When a sample capable of capturing electrons emerges from the GC column, the strength of

50

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the standing current is reduced. The reduction m current is amplified and recorded as a peak The ECD is a selective detector that 1s extremely sensitive to electronegative compounds, i.e., compounds that form negative ions by capturing electrons Sensitive compounds include those that contam halogen atoms, conjugated ketones, mtro compounds, nitriles, and organometals Other compounds, e.g., hydrocarbons and alcohols, are not detected. This detector IS extremely sensltlve. Subplcogram quantities of electronegative compounds, e.g., pesticides, can be detected and quantified, and, with the newer ECDs, the detector response 1s linear over a range of sample amounts of lo4 (Maggs et al., 1971). 1.5 4. Nitrogen-Phosphorus

Detector

The NPD 1s a modified flame detector conslstmg essentially of an electrically heated chamber into which 1s fed hydrogen, au-, and the eluant from the GC column. Located near the heater (lgmtor) 1s a small alkali salt pellet (often a cesium or rubidium salt) When the mixture 1s ignited in the chamber, a low-temperature plasma, rather than a flame, IS formed and emits a minute current that 1s amplified. If the GC eluant contains a phosphorusor nitrogencontaining compound, the current produced by the plasma 1s greatly enhanced. Precise control of hydrogen and air flow are necessary for stable baselines and optimal responses. The NPD is a relatively selective detector. Its sensitivity to N- and P-contammg compounds 1s remarkable, mimmum detectable quantities of compounds containing these elements are about 5 and 200 pg, respectively. The detection method is destructive. The NPD has a relatively narrow linear detectlon response (103). 1.5.5, Mass

Spectrometrx

DetectIon

A mass spectrometer 1s an excellent, sensitive, and specific detector for GC, but its cost often precludes routine use. Most of the carrier gas 1s removed from the eluant from the gas chromatograph before it 1s passed into the mass spectrometer, where the eluted compound is bombarded with high energy electrons. This results m ionization and fragmentation of the compound Into positlve, negative, and neutral fragments Commonly, the positively charged fragments are passed into an electron multlpher that produces an electric current proportional m magnitude to the number of positive ions formed. This current (total ion current) 1s amplified and recorded. The trace obtamed (total Ion current vs time) is comparable to the typical GC trace (detector response vs time). Instead of measuring total ion current, the current pro-

Gas Chromatography

ofAmines

and Ther Metabolltes

51

duced by a positively charged ion of a particular mass (m/z) can be selected and amplified. The current produced is then referred to as a single (or selected) ion current and a plot of selected ion current vs time is recorded. In this instance, the mass spectrometer is a very selective detector. Only compounds that produce an ion of the selected mass will be detected. Thus, by ludiciously selecting an appropriate ion, a single compound can be detected and quantified even though it coelutes with other compounds. The practical detection limit of the mass spectrometer 1s in the high picogram range, a linear detector response over a wide range of sample amounts (104) is attainable. Further details on mass spectrometric methods of detection are provided in the chapters by Karoum and Durden m this volume

1.6. Injection Systems A variety of inlection systems is now available. The use of a splitless injection system, in which the sample is introduced and vaporized in a glass-lined tube extending from the septum cap to the column, is recommended for analysis of very dilute and wide boiling-range samples. This is the system used m our laboratories for analysis of amines and metabolites. In a splitter system, the carrier gas stream IS split and only a small proportion of the injected sample actually enters the column. This eliminates overloading the column with large amounts of sample, and ensures deposition of the sample on the column in a very narrow bandwidth. Such a system 1s recommended for analysis of concentrated samples (e.g., essential oils, petroleum). The use of a concentrator/headspace 1s recommended when it is desired to concentrate samples from aqueous solutions. Volatile materials are adsorbed onto a porous polymer of graphitized carbon, but water is not adsorbed to any significant degree. This adsorbent can also be used to sample large volumes of au for organic pollutants. Grob-type splitless injection is employed m trace analysis of very dilute solutions containing samples of mediumto highmolecular weight This would be used when it 1s necessary to inlect large amounts (up to 10 pL), because volatile components would be lost during concentration of the sample by solvent removal procedures The sample is condensed m the first one or two turns of the capillary column by maintaining the oven temperature at 20-30°C below the boiling point of the solvent The mlector is then flushed clean by a high flow of carrier gas, thus removing solvent that would otherwise cause tailing. Following this procedure, the column temperature is raised.

52

Coutts, Baker, and IYazarab

2. Isolation of Amines and Their Metabolites From Tissues and Body Fluids and Derivatization for Gas Chromatography After precipitation of protein (e.g., with perchlorrc acid, trichloroacetic acid, hydrochloric acid, formic acid, or zmc sulfate-barium hydroxide), where necessary, and centrifugation, supernatants are retained for analysis. Many acids can be extracted with relatively high efficiency with organic solvents after adlustment of aqueous supernatants to a low pH value Similarly, some ammes (e.g , 2-phenylethylamme and tryptamme) can be extracted efficiently with organic solvents after basification of the aqueous supernatant However, solvent extraction of amphoteric ammes (e.g., tyramme) is often inefficient and gives poor reproducibility. For this reason, and also to reduce contammation of extracts of ammes and their metabolites by other naturally occurrmg substances, a variety of resins, adsorbents, and molecular sieves have been utilized for isolation of the compounds of interest (Kakimoto and Armstrong, 1962; Temple and Gillespie, 1966; Sharman, 1971, Martin and Ansell, 1973; Snodgrass and Horn, 1973; Karasawa et al., 1975, Boulton et al., 1976; Holman et al., 1976; Higa et al., 1977; Westermk and Korf, 1977; Martin and Baker, 1977; Warsh et al , 1977, Earley and Leonard, 1978, Marmi et al., 1979; Ogasahara, 1979; Nelson et al., 1979, Artigas and Gelpi, 1979, Baker et al , 1980; McQuade et al., 1981). In addition, as described m more detail later in this chapter, some reagents such as acetic anhydride (Chattaway, 1931; Welsh, 1955, Goldstein et al., 1959, Hagopian et al., 1961, Brooks and Hornmg, 1964, Laverty and Sharman, 1965; Roder and Merzhauser, 1974; Martin and Baker, 1976), 2,6-dmitro-4-trifluoromethylbenzenesulfomc acid (Doshi and Edwards, 1979), and pentafluorobenzoyl chloride (Mabta et al , 1975, Cole et al , 1977; Cristofoli et al , 1982) can react with amme and phenol functions under aqueous conditions, often producing relatively nonpolar compounds that can be readily extracted mto organic solvents. Much of the work involving gas chromatographic analysis of ammes and their metabohtes m tissues and body fluids requires that the substances of interest be derivatized. In most cases, derivatization is employed to increase sensitivity and selectivity, but derivatization can also provide other advantages, including (a) increased volatility, (b) increased stability, (c) reduced polarity to improve chromatographic properties, and (d) improved efficiency m extraction from aqueous media

Gas Chromatography

ofAmines

and TherrMetabohtes

53

Derivative formatron usually mvolves replacement of the active hydrogen atom of polar compounds (e.g., NH, OH, SH) by chemical procedures such as acylation, alkylatlon, silylatron or condensation. A number of comprehensive book chapters, review papers, and articles describing derrvatrzatron techniques for GC are available (Clarke et al., 1966, Gudzinowrcz, 1967; Anggard and Sedvall, 1969; Moffat and Horning, 1970; Karoum et al.‘, 1972; Matm and Rowland, 1972; Moffat et al., 1972; Arnold and Ford, 1973; Franken and Trrjbels, 1974; Gelpi et al. 1974; Sugiura and Hirano, 1974; Poole and Morgan, 1975, Ahuja, 1976, Blau and King, 1978; Perry and Freit, 1978; Knapp, 1979; Baker et al , 1981, Baker et al., 1982; Martin et al., 1984). As mentioned above, derivatives are chosen to impart propertres such as sensitrvity and volatility. However, the potential user of GC should also keep in mind that certain substances are unstable in the presence of acid or base and that this property may dictate to some extent the choice of derrvatrzmg agent. For example, reagents such as perfluoroimidazoles (Sugiura and Hirano, 1974) or trrazoles (Mryazaki et al., 1974), which produce basic leaving groups, may be utilized for derivatrzation of acid-labile substances

3. Analysis of Specific Types of Amines and Their Metabolites Using Gas Chromatography 3.1. Catecholamines and Their Me tabofites 3.1.1. Dopamme,

NoradrenalIne,

and Adrenaline

Because of its high sensitivity, GC-ECD has been used most frequently for gas chromatographic analysis of catecholamines in tissues. The amines are reacted with perfluoroacylating reagents such as trifluoracetrc anhydride (TFAA), pentafluoropropiomc anhydride (PFPA), or heptafluorobutyric anhydrrde (HFBA) following then rsolatron from tissue or body fluids using alumina, ion-exchange resin or boric acid gel. A major problem with analysis of catecholammes IS their inherent mstabihty m solutron, particularly under basic condrtions. This factor, combined with the amphoteric nature of these phenolic amines, means that solvent extraction of the catecholamines directly from body fluids or ussue homogenates IS not utilized. Perfluoroacylation of catecholamines followmg surtable extraction has been utihzed by a number of researchers for gas chromatographlc quantrtatron of catecholamines in tissues (Kawai

Coutts, Baker, and Nazarali

54

and Tamura, 1968; Imal et al., 1971, Martin and Ansell, 1973, Bigdeli and Collms, 1975; Kawano et al., 1978; Arnold and Ford, 1973; Lhuguenot and Maume, 1974) and body fluids (Kawai and Tamura, 1968; Imai et al., 1971; Wong et al., 1973; Bertam et al., 1970; Imai et al., 1973; Wang et al., 1975; Kawano et al, 1978) Some of these assays have mvolved the formation of mixed derivatives, i.e., the compound of interest was reacted with more than one derivatizmg reagent. Arnold and Ford (1973) etherified the alcohol function of noradrenalme (NA) and subsequently perfluoroacylated the amine and phenol groups. Lhuguenot and Maume (1974) formed the pentafluorobenzylidine-trimethylsilyl (TMS) derivatives of NA and dopamine (DA) for analysis of these ammes in extracts from rat adrenals. Doshi and Edwards (1981) reported a sensitive method for analysis of catecholamines in rat brain: N-2,6-dinitro-4-tr~~uoromethylphenyl-O-tr~methylsilyl derivatrves were prepared in aqueous medium. Mrxed derivatives may offer a number of advantages in GC analysis, mcludmg increased sensitivrty, improved peak shape (i.e., reduced “tailing”), and altered retentron time to provide separation from another compound that would otherwise interfere with analysis However, such techniques usually increase analysis time and may lead to formation of multiple side products. In later sections of this chapter, we discuss the use of pentafluorobenzoyl chloride (PFBC) under aqueous conditions for derivatization of trace ammes. Bock and Waser (1981) have employed this reagent for analysis of catecholammes, but carried out the derivatrzation reaction under anhydrous conditions (m acetomtrile containing pyridme) Some workers have employed GC-FID quantitation of the catecholammes. In the procedure of Maruyama and Takemori (1972), TMS-imidazole was employed as the derivatizmg reagent, and NA and DA were estimated m brain Kawai and Tamura (1968) analyzed catecholammes in bovine adrenal medulla by GCFID; hydroxyl groups were converted into trimethylsilyl ethers and the primary amines into Schiff bases (secondary ammes did not react). Mixed TFA-TMS derivatives were utilized by Haeffner et al. (1976) to identify DA and NA m urine by GC-FID. 3.1.2.

3-0-Mefhy’afed

Amine Mefabohfes

of Cafech’ammes

Unlike the parent catecholammes, the 3-0-methylated metabolites 3-methoxytyramine (3-MTA), normetanephrine (NMN), and metanephrine (MN) are relatrvely stable in basic solution. However, because they are still amphoteric substances, solvent extrac-

Gas Chromatography

ofAmines

and Their Metabobtes

55

tion from aqueous solution often results m low and inconsistent recoveries. Greer et al. (1968) measured 3-MTA, NMN, and MN in the urine of patients suffering from neuroblastoma or pheochromocytoma. The amines were converted to trrfluoroacetyl derivatives, and GC-FID was used for quantrtatlon. Perfluoroacylation combined with GC-ECD has been utilized for analysis of the 3-0methylated ammes in urine (Bertam et al., 1970) and plasma (Wang et al., 1975). Mixed derivatives have also been employed m the gas chromatographlc analysis of the 3-0-methylated amrnes. The procedure of Haeffner et al. (1976) mentioned in the previous section (TFATMS derlvatlves and CC-FD were used) provided for the identifrcation of NMN and MN as well as NA and DA. Nelson et al (1979) developed an assay for simultaneous measurement of 3-MTA, NMN, and MN: followmg etherlfrcatlon of alcohol moleties, amme and phenol groups were derivatized by perfluoroacylation. LeGatt et al. (1981) modified a procedure that had originally been developed for analysis of m- and p-tyramine (m- and p-TA) (Coutts et al., 1980) in urine and used rt to quantltate NMN and 3-MTA in rat whole bram. The ammes were acetylated m aqueous solution and extracted with ethyl acetate. Acetylated phenol groups were selectively hydrolyzed with ammonium hydroxide solution, and, after removal of the ethyl acetate under a stream of nitrogen, the resultant N-acetylated compounds were reacted with TFAA under anhydrous conditions to produce the denvatives shown in Fig. 1. This procedure has now been modified to provide for simultaneous analysis of 2-phenylethylamine (PEA), m- and p-TA, 3-MTA, and NMN m tissues and body fluids (Coutts et al., 1981; Hopkmson et al., 1982), and will be discussed m greater detail later in this chapter. 3.1.3. Alcohol

and Acid Metabolites

of Catecholamines

Alcohol and acid metabolltes of catecholammes contain alcoholic (and sometimes phenohc) and/or carboxyhc acid groups, and these functions are usually derlvatlzed preceding GC analysis. 3-Methoxy-4-hydroxyphenylethylene glycol (MOPEG) and 3,4-dihydroxyphenylethylene glycol (DOPEG), metabolrtes of NA, have been analyzed by GC-ECD followmg perfluoroacylatlon (Wilk et al., 1967, 1970; Deklrmenllan and Maas, 1970) Some workers have acetylated the phenol groups of MOPEG and DOPEG m aqueous medium to improve extractron of these compounds before derivatizmg the alcohol groups with a

Coutts,

56

Baker, and Nazarah

COCH, CH,CH,N’ ‘COCF,

a

,COCH3 CHCH,N, I OCOCF,

COCF,

b Fig. 1 Derivatives normetanephrme (b) after acetylated phenols wrth tnfluoroacetrc anhydride

formed from 3-methoxytyramme (a) and from acetylatlon m aqueous medium, hydrolysis of ammonmm hydroxide, and reaction with under anhydrous condrtions (LeGatt et al ,

1981)

perfluoroacylating reagent (Sharman, 1969; Kahane et al., 1976; Tang et al., 1978; Warsh et al., 1980 (see Fig. 2) Analysis of acidic metabolrtes of blogemc ammes usually involves extractron from an acidified sample of trssue homogenate or body fluid, derlvatlzation of carboxyllc acid moieties with halogenated or nonhalogenated alcohols, and preparation of different derlvatlves of any phenollc or alcoholic groups present on the molecule (Sloquist and Anggard, 1972, Wiesel et al., 1974, Watson et al., 1974; Dzledzlc et al., 1973). The conversion of

CHCH,0COCF3 I OCOCF, Fig.

2. Structure of the denvatrve formed by acetylatron of m aqueous solution followed by reaction wrth TFAA under anhydrous condrtrons MOPEG

Gas Chromatography

of Amines

and Their Metabolites

57

vanillylmandelic acid (VMA) to vamllm or vamllyl alcohol and subsequent reaction with TFAA has been utilized for analysis by CC-ECD (Wilk et al., 1965; Dekirmenlian and Maas, 1971). In a similar type of assay, van de Calseyde et al. (1971) employed OXIdation with sodium perrodate at different pH values to convert MN and VMA separately to vamllin The TMS derivative of the van&n was formed and used for analysis on GC-FID. Gas chromatographic analysis of acid metabolites of phenylethylammes and indolethylammes has been applied to urine and brain samples after formation of TMS derivatives (Karoum et al., 1968, Sprinkle et al., 1969; Addanki et al., 1976). Many of the acid metabolites of ammes are present in very high concentrations in urine, and Sandler et al (1979) assayed such metabolites of several phenylethylamines using GC-FID and a capillary column. Muskiet et al (1977) extracted a number of catecholamine metabolites from urine and analyzed them using GC-FID. Values for VMA, homovanlllic acid (HVA), and MOPEG were determined in normal sublects, and excretion patterns for VMA, HVA, MOPEG, 3,4-dihydroxyphenylacetic acid (DOPAC), vanillactic acid, DOPEG, and vamlethanol were studied m patients with neurogenic tumors. In a later paper, Muskret et al (1981) described a method for simultaneous quantitative determmation of HVA, VMA, MOPEG, and DOPAC and for estimation of 5-hydroxymdole-3-acetic acid (5-HIAA) 1x-rurine. terl-butyldimethylsilyl derivatives were formed, and analysis was on a GC equipped with a capillary column and an FID. Chauhan and Dakshmamurtr (1982) described a technique in which boronate derivatives were utilized for analysis of alcohol metabolites of catecholammes by GC-FID. Urine extracts were treated with dlazoethane and n-butylboronic acid, resulting m the formatron of ethyl esters of HVA and DOPAC and cyclic boronate derivatives of MOPEG and DOPEG. The structures of the derivatives are shown in Fig. 3. Watson and Wilk (1974) described a GC-ECD procedure that was used successfully for determination of VMA and HVA in CSF. The method employed a combmation of PFPA and a halogenated alcohol to derivatize carboxyl groups, followed by reaction with PFPA to derivatize phenollc functions. Another procedure for determination of urinary acids by GC-ECD involved formation of trrfluoracetyl, hexafluoroisopropyl esters, and separation of the derivatives on a glass capillary column (Chauhan and Darbre, 1980). In an application of analysis of acid metabolites of ammes to brain tissues, Pearson and Sharman (1974) reacted the extracted acids with TFAA and hexafluoroisopropanol. The procedure was used for estimation of DOPAC and HVA m

58

Coutts, Baker,

and Nazaralr

CH,COOC2H,

a

CH-CH, I I 0 0

Fig 3 Derivatives tlon with dlazoethane Dakshmamurtl, 1982)

of HVA (a) and of MOPEG (b) formed by reacand n-butylboromc acid (Chauhan and

brain regrons, superror cervical ganglia, mors, retinal tissue, and CSF.

3.2. 5Hydroxytryptamine

aqueous

and vitreous

hu-

(5HT; Serotonin)

The technique of Martin and Ansell (1973) mentioned m the above section on catecholammes also provided for the analysrs of 5-HT. The tn-TFA derivative was formed and quantitation was by GC-ECD. Baker et al. (1980) employed a double derrvatizatron procedure for analysis of 5-HT m rat brain extracts The method involved extraction with the liquid ion-pairing compound dr-(2ethylhexyl)phosphorrc acid (DEHPA), back-extraction with HCl, basification, acetylation with acetic anhydrrde, extraction wrth ethyl acetate, and reactron with PFPA under anhydrous condrtrans. Under these conditions, the final derivative formed is a sprrocyclic compound (Blau et al., 1977) that has excellent chromatographrc properties and high sensitivity on the ECD. Thus method has now been adapted for urine and has the added advantage of allowing simultaneous quantitation of tryptamme (Baker et al., 1979, Calverley et al., 1980). This procedure 1s described m detail later m this chapter.

Gas Chromatography

ofAmlnes

and ThenMetabolites

59

3.2.1. Metabolites of 5Hydroxytfyptamine As mentioned above, Muskiet et al (1981) employed a GC equipped with a capillary column and an FID for separation and measurement of 5-HIAA and several acidic and alcoholic metabolites of catecholamines. The procedure, which involved formation of terf-butyldimethylsilyl derivatives, was applied to urme extracts Urinary 5-HIAA was analyzed by Goodwin et al. (1975b) followmg esterification with ethanolic HCl and reaction with PFPA. Degen et al. (1972) were able to quantrtate melatonin in extracts from pmeal gland by utilizmg reaction with HFBA and separation and quantitation by GC-ECD

3.3. Trace Amines and Their Acid Metabolites 3.3 1. Trace Amlnes These substances, termed “trace” ammes because of their low concentrations m brain relative to the classical biogemc ammes such as the catecholammes and 5-HT (Usdin and Sandler, 1976), have been mvestigated by GC using a variety of detectors, (I) GC-FID Borison et al. (1974) analyzed 2-phenylethylamme (PE) m rabbit brain after formation of the dmltrophenylsulfonic acid derivative of the amme Subsequent analysis of rabbit brain PE levels by other workers using high-resolution mass spectrometry (Boulton et al., 1975) gave a value much lower than that obtained by Borison et al (1974). Haeffner et al (1976) identified p-tyramme (p-TA) m urine after preparation of a mixed TFA-TMS derivative, and Reynolds and Grey (1976, 1978) analyzed PE and detected N-methyl-PE m urme after conversion of these amines to their tnfluoroacetylated derivatives. Analysis of PE and tryptamme (T) m putrefymg human tissue (Oliver et al., 1977) and estimation of I’E m plasma followmg mfusion of this amine m dogs (Cone et al , 1978) have also been reported (ii) GC-ECD Perfluoroacylation and GC-ECD have proved useful for analysis of trace amines Edwards and Blau (1972a,b, 1973) prepared mtro-containing derivatives for analysis of PE and related ammes extracted from brain and liver. Martin et al. (1974) identified T m perfusates m dog brain after extraction and perfluoroacylation. Schweitzer et al. (1975) derivatized PE from a urine extract with HFBA prior to GC analysis. Martin and Baker (1977) formed a mixed derivative of PE (N-acetyl, N-PFP) and were able to utilize this method for measurement of this amine in control rat brain This procedure was also applicable to analysis of T and 5-HT

60

Couth,

Baker,

and Nazarali

(Baker et al., 1979, 1980; Calverley et al., 1980), and was further modified to provide simultaneous extraction and quantitation of PE, m- and p-TA, NMN, and 3-MTA (Coutts et al., 1980, Baker et al., 1981; LeGatt et al., 1981). Further details of these analytical procedures are given later in this chapter. Pentafluorobenzoyl chloride (PFBC), a reagent known to impart high sensitivity on ECD to phenylethylamine-like compounds (Cummmgs, 1971, Moffat et al., 1972; Matm and Rowland, 1972; Midha et al., 1979) has been employed for quantitation of PE m human and sheep brain and human urine (Reynolds et al., 1978, 1980; Blau et al., 1979). Pentafluorobenzoyl chloride will react with amines and phenols under anhydrous and aqueous conditions, and in our laboratories we have used this reagent under both conditions for analysis of trace amines. In one procedure for analysis of PE m brain &sue, the amme is reacted with acetic anhydride under slrghtly basic aqueous conditions. After extraction of the N-acetyl-PE with ethyl acetate and subsequent removal of the organic solvent under a stream of nitrogen, the residue is reacted with PFBC in the presence of toluene. The resultant N-acetyl, N-PFB-PE (see Fig. 4) has hrgh sensitivity on GC-ECD and has been utilized for analysis of PEA in rat brain and urine (Hampson et al., 1984). Reaction with PFBC under basic aqueous conditions has been employed for analysis of amphetamme and related compounds in bram tissue (Cristofoli et al , 1982; Nazarali et al., unpublished) and attempts are bemg made in our laboratories to adapt the method to analysis of I’E in bram and urine. However, pentafluorobenzoylatron has been employed successfully by us for srmultaneous measurement of PE, m- and p-TA, histamme, and tele-methylhlstamme m food products (Wong et al., 1974). The pentafluorobenzoyl derivative of PE IS shown in Fig 4. A recent finding in our laboratories IS that reaction of trichloracetic anhydride with PE occurs readily under aqueous conditions. This reagent has been employed to measure PE in extracts from bram tissue (Baker et al., 1984a). Another reagent that has proven useful for quantrtatron of PE m urine samples is pentafluorobenzenesulfonyl chlorrde (Baker et al., unpublished). The reaction occurs in aqueous solutron under basic conditions, and the resultant derivative 1s shown in Fig. 4. (iii) GC-API) Narasimhachari and Friedel (1981) utrllzed GC-NPD for analysis of PE and T m tissues (including bram) and body fluids. The ammes were converted to isothiocyanate derivatives by reaction

Gas Chromatography

ofAmlnes

and ThewMetabolkes

CH,CH*N

61

,COCH, ‘COC,F,

a

CH,CH,NHCOC6F5

b

CH,CH,NHCOCCI

3

C

0\ /

x

CH,CH,NH-S-C& ::

d Fig 4 Derrvatlves of PE formed by (a) aqueous acetylatron followed by anhydrous pentafluorobenzoylatlon, (b) aqueous pentaflurobenzoylatlon (Hampson et al , 1984), (c) reaction wrth trlchloroacetlc anhydride under aqueous condmons (Baker et al., 1984), and (d) reaction with pentafluorobenzenesulfonyl chloride under aqueous condmons In those cases m whrch derrvatrzatron takes place m aqueous medium, the solution IS adjusted to a basic pH with solld sodium carbonate or sodium bicarbonate before addltlon of the derivatrzmg reagent m the absence or presence of organic solvent

with carbon dlsulflde prior to GC analysis Oon and Rodmght (1977) used trlfluoroacetylatlon for measurement of urinary levels of N-methyl-T using CC-NPD. This selective GC detector has also been utlllzed for quantitatlon of tranylcypromme, an antldepres-

62

Coutts,

Baker,

and

fYazara/l

sant closely related structurally to PE, after extraction from plasma samples and reaction with HFBA (Bailey and Barron, 1980) It is also possible that reagents such as dimethylthiophosphmic chloride (Jacob et al., 1978) and diethylchlorophosphate (Deo and Howard, 1978) may prove useful for future analyses of a number of biologically important ammes. 3.3.2. Acid Metabobtes

of Trace Amlnes

Goodwin et al (1975a) extracted phenylacetic acid (PAA) from urme, esterrfied it with n-propanol, and performed analysis by GC-FID The procedure was employed for measurement of free and total PAA m human urme. Naruse et al. (1977) applied a GCFID procedure to analysis of mdole-3-acetic acid (IAA) m urine from normal sublects and from patients with leukemia, gastric cancer, and phenylketonuria After extraction of IAA from urine, it was methylated with diazomethane and then reacted with TFAA Capillary column GC-FID was reported by Sandler et al (1979) for measurement of PAA, p-hydroxyphenylacetic acid (pHPAA), and p-hydroxymandelic acid, metabolites of PE, p-TA, and p-OA, respectively, m human urine GC-ECD has been used with increasing regularity for quantitation of these acid metabolites m bran-t (Davis et al., 1977, McQuade et al., 1981) and urme (Davis and Boulton, 1981a,b) These procedures mvolve reaction with halogenated anhydrides (for derivatization of phenol and alcohol moieties) and halogenated or nonhalogenated alcohols (to derivatize carboxylic acid groups) and will be discussed m greater detail later in this chapter.

3.4. Histamine, tele-Methylhistamine, Putrescine, and Cadaverine

Spermidine, Spermine,

These polyammes have proved to be difficult to analyze by gas chromatography (Mita et al , 1979, Seiler, 1980), but several procedures are now available 3.4.1. Hstamine

(HA) and tele-Methyhstamme

(MeHA)

Mlta et al. (1979) reacted HA sequentially with HFBA and ethyl chloroformate. The technique was utilized for analysrs of HA and MeHA in urine, whole blood, and leukocytes by GC-MS (Mita et al., 1980a,b), although the derivatives were reported to have a sensitivity of 20 ng on GC-FID Mahy and Gelpi (1978) reported a GC-FID sensitivity of lo-30 ng for pentafluoroacylated derivatives of HA, histidine, and their tele-methyl metabolites. Doshi and Edwards (1979) employed reaction with 2,6-dmitro-4-t&u-

Gas Chromatography

ofAmines

and Their Metabobtes

63

oromethylbenzenesulfonic acid (DNTS) (under aqueous conditions) and CC-ECD analysis for measurement of HA and MeHA in urine. Reports of quantitation of HA and its metabolites usmg GC-NPD have also appeared in the literature Navert and Wollin (1980) employed extraction with an ion-exchange resin and subsequent reaction with HFBA for GC-NPD analysis. The method was employed for quantitation of HA and MeHA m urine samples from depressed patients (Gagne et al., 1982). Keyzer et al. (1982) isolated te2e-methylimidazoleacetic acid from urine by ionexchange chromatography. This step was followed by esterification with 2-propanol and analysis on a GC equipped with a capillary column and an NPD. As mentioned m previous sections, we have used acetylatron followed by perfluoroacylation for measurement of a number of biogenic amines in our laboratories. However, we were unable to derivatrze HA or MeHA using those procedures. A sensitive alternate procedure developed in our laboratories (LeGatt et al., 1981) has proved to be particularly useful for extraction and derivatization of these imrdazolealkylamines, and is potentially applicable to analysis of a number of other brogenic ammes and amine-containmg drugs m tissues and body fluids (Cristofoli et al., 1982). Briefly, the technique mvolves extraction of the ammes from brain homogenate or body fluid samples with DEHPA, back-extraction with HCl, basification of the HCI phase with solid and shaking of the mixture wrth a solution of NaCb pentafluorobenzoyl chloride (PFBC, 2 FL) in ethyl acetate/ acetomtrile (9/l). The organic phase is retained, taken to dryness under a stream of nitrogen, and the residue is taken up in toluene for mlection onto a GC-ECD. The method is relatively rapid and provides for analysis of HA and MeHA in a smgle hypothalamus. 3.4.2. Spermidine,

Spermine,

Putrescine,

and Cadavenne

Benmati et al. (1978) measured underivatized polyammes by GCFID following extraction from a variety of organs with 1-butanol. Derivatives used for CC-FID analysis of polyamines extracted from urme Include trifluoroacetyl (Denton et al., 1973; McGregor et al., 1976) and isobutyloxycarbonyl (Makita et al., 1978) derivatives. Makita et al (1975) combined pentafluorobenzoylation under aqueous conditrons and CC-ECD for quantitation of urinary polyamines. Bakowskr et al. (1981) reported the use of GC-NPD for analysis of spermidine, spermine, and putrescine in plasma; isobutyloxycarbonyl derivatives of the ammes were prepared.

64

3.5. Acetykholine

Coutts, Baker, and Razarab

(ACh) and Choline (Ch)

Lack of volatrlity presents a mayor obstacle n-r the measurement of these compounds by GC. Because of this difficulty, hydrolysis products (ethanol and acetic acid) (Cranmer, 1968; Stavmoha and Ryan, 1965) or the demethylatron product (dimethylammoethyl acetate) (Harm-r and Jenden, 1969, Harm-r et al, 1970, 1972, Szrlagyi et al., 1972) of acetylcholine have been utilized for analysis. Chemical means (Handen and Jenden, 1969, Hanin et al., 1970; Harm-r, 1974) and pyrolysis (Szrlagyr et al., 1968, 1972; to cause Schmidt et al , 1970) have both been employed demethylatron of ACh. These demethylation procedures have also been combined with acylatlon techniques (e g , propronylatlon, butyrylatron) to provide for simultaneous quantrtatlon of Ach and Ch (Schmidt and Peth, 1975; Jenden et al., 1972; Maruyama et al., 1979, Kosh et al., 1979). The above analyses were conducted using GC-FID, but the use of other detectors has also been reported Kilbmger (1973) used GC-NPD for analysis of ACh in a rabbit heart tissue extract followmg chemical demethylatron Harm-r et al (1972) modified an existing procedure (Hanm et al., 1970) to permit simultaneous assay of Ch and ACh actlvrties m tissue extracts from rats which had received phosphoryl (Me-r4C) cholme. Specific actrvitles were determined using a GC-radloactrvrty momtormg (CC-RAM) system, and the analysis was applied to extracts from rat submaxillary and sublmgual salivary glands

4. Examples of Some Protocols Used for Analysis of Amines and Their Metabolites 4.1. Simultaneous Analysis of PEA, m-TA, p-TA, NMN, and 3-MTA 4.1.1. Preparation of Samples This procedure 1s based on previously reported methods (Baker et al , 1981, Coutts et al , 1981). Bram samples are homogenized in five volumes of ice-cold 0 1N HC104 contammg 10 mg% EDTA and one or more of the followmg internal standards: benzylamine, 3-phenylpropylamme, or 2-(4-chlorophenyl)ethylamine. This is performed with an electric homogenizer consrstmg of a teflon pestle and a cyhndrrcal glass mortar After centrifugation at 10,000~ for 5 mm, the supernatant is retained and 4 mL is used for the analysis

Gas Chromatography ofAmines and TherMetabobtes

65

If urine samples are being analyzed, they are used directly (4 mL) after addition of one of the internal standards mentioned above. Standard curves are run on a routine basis and are prepared by carrying a series of tubes containmg varying known amounts of standards of the ammes of interest and a fixed amount of mternal standard through the procedure, in parallel with the samples (to which the same amount of internal standard is added) Standard curves are prepared by measuring the peak heights of the responses for each amme and the internal standard and dividing the peak height of the ammes by that of the internal standard to obtain a peak height ratio for each amine in each tube These ratios are plotted against the known amounts of each amine in the sample. It is our fmdmg that standard curves may vary slightly from day to day on borosihcate glass capillary columns, but are more reproducible on packed columns and fused silica columns, We routinely prepare 12-18 samples plus a standard curve and blank per analysis, but this can be increased substantially if the GC mvolved has an automatic sampler to mlect samples and can therefore be run almost continuously 4.12 Extraction of Amlnes From Samples The brain homogenate supernatants or urine samples are made slightly basic by addition of solid KHC03 Sodium phosphate buffer (0.25&l, pH 7 8) (!/ICI the volume) is added The addition of KHC03 to the brain supernatant results m formation of a precipitate of KClO*, which is removed by a brief centrifugation. The basifled samples are shaken with 5 mL of the liquid ionpairing compound di-(2-ethylhexyl)phosphoric acid (DEHPA, 2.5% v/v in chloroform) for 10 min After a brief centrifugation, the top layer is removed by aspiration and discarded The bottom layer is shaken (5 min) with 3 mL of 0.5N HCl to elute the ammes. After centrifuging (5 min), the top layer is retained and removed to another set of tubes. The HCl layer is made slightly basic by the addition of solid NaHC03, and the ammes (and any phenols present) are acetylated by the addition of acetic anhydride (3OOk.L). This reaction is allowed to proceed until effervescence ceases (usually 15-20 min). Small amounts of solid NaHC03 are added during this time to maintain a small excess on the bottoms of the tubes. At this time, the supernatants are transferred to another set of tubes (leavmg excess NaHC03 behind m the original tubes) The acetylated amines (and phenohc ammes) are extracted by shaking with 4 mL of ethyl acetate (5 mm). The tubes are centrifuged, and the top layers are transferred to another set of tubes.

66 To the tubes containing the N-acetylated compounds (N- and 0-acetylated in the case of phenollc amines) is added 10N ammonium hydroxide solution (400 ILL) This mixture is shaken for 40 mm, which results m selective hydrolysis of acetylated phenol groups. After neutralization of the ammomum hydroxide layer with 6N HCl, the top layer is retained and taken to dryness under a stream of nitrogen. The residue is reacted with ethyl acetate (25 kL) and TFAA (75 I.LL) at room temperature for 30 min. Cyclohexane (300 kL) and saturated sodium tetraborate buffer (3.0 mL) are added and the mixture is shaken for 15 s. Excess reagent goes mto the aqueous phase, whereas the derivatized ammes are retained in the cyclohexane layer. An aliquot (OS-l.0 ~.LL)of the cyclohexane layer is mlected onto a gas chromatograph equipped with an ECD. The derivatives formed during this procedure are similar to those shown for 3-MTA and NMN m Fig 1. They have good peak shape and high sensitivity. The hydrolysis procedure frees the phenols of the acetylated phenolic amines for subsequent reaction with TFAA, resulting m increased volatility and sensitivity over the derivatives that would otherwise be formed (LeGatt et al , 1981) In addition, this hydrolysis step results m separation of derivatized p-TA from another mterfermg substance present m brain tissue. Since PE has no phenol group, it forms the same derivative whether or not the hydrolysis step is Included. The method described above IS rather lengthy, but it does provide for simultaneous measurement of PEA, m-TA, JPTA, NMN, and 3-MTA The assay normally requires two days, one for extraction and derivatization, and another for mlection of samples on the GC However, this can be combined into a single day if an automatic sampler is available to provide unattended overnight mlection Suitable capillary columns Include a WCOT SP 2100 glass column (10 m) and 3% OV-1 or 3% OV-101 fused silica columns (12 m m each case). In a typical experiment, an initial oven temperature of 80°C is mamtained for 0.6 min, and increased at 30”/mm to 120°C. After 9-10 mm, the oven is programmed to increase to 160°C. Under these conditions, peaks for the derivatized amines of interest all appear m less than 15 min

4.2. Simultaneous Analysis of TIyptamine (T) and 5Hydroxytlyptamine (W-IT) This protocol is a modification of previously reported procedures (Calverley et al , 1980; Baker et al , 1981, Coutts et al., 1981; Baker et al, 1982) An abbreviated version of the procedure described in

GaS Chromatography

ofAm/nes

and Their Metabohtes

67

section 4.1 immediately above is employed for analysis of T and 5-HT. With the exception of the addition of a different internal standard (5-methyltryptamme), all steps are identical up to the extraction of the acetylated amines into ethyl acetate. Hydrolysis with ammonium hydroxide is not carried out. Rather, the ethyl acetate layer is taken directly to dryness under a stream of mtrogen. The resultant residue IS reacted with ethyl acetate (25 PL) and PFPA (75 IJ,L) for 30 mm at 60°C. Cyclohexane (300 ~.LL)and saturated sodium tetraborate buffer (3.0 mL) are added, and after mixing and centrifugmg briefly, the top layer is retained for GCECD analysis. We have found that the most suitable columns for this analysis are a 6-ft glass column (4 mm id) packed with 3% OV-17 on Gas-Chrom Q, a 10-m WCOT OV-17 glass capillary column (0.25 mm id), and an HP fused silica capillary column (crosslmked 5% phenyl methyl silicone, 0.31 mm id, 25 m). For the packed column, the oven temperature utilized is 220°C isothermal, whereas with the capillary columns, an initial temperature of 80°C is mamtamed for 0.5 mm, and the oven programmed to heat at 20”/mm to 220°C and 27O”C, respectively Under these conditions, derivatives of both ammes appear before 10 min. The packed column can be utilrzed for analysis of both amines in urine samples, but capillary columns must be employed to quantitate tryptamine m brain tissue. If the researcher wishes to analyze one or more of the five ammes described m section 4.1 in addition to T and 5-HT, the procedures in 4 1 and 4.2 can be combmed (Baker et al., 1982; Baker et al., 1984), the ethyl acetate phase (containing all the acetylated amines) being split into two portions, one to be hydrolyzed and reacted with TFAA, and the other to be taken to dryness immediately and reacted with PFPA. 4.3. Simultaneous Measurement of Noradrenaline (NA), Dopamine (DA), and 5HT in Brain Tissue This protocol is based on the procedure described by Martin and Ansell (1973) The samples are homogenized m 7 5 vol of acid-butanol at 4°C using a Potter-Elvelhem homogenizer Preparation of the acid-butanol: batches of n-butanol are washed by shaking with 4 X 250 mL of water m a separatory funnel. The butanol is then saturated with sodium chloride and a 1-L volume is shaken with 0 85 mL cone HCl Potassium metabisulfite (1 g) and disodium EDTA (0 1 g) are added and the mixture is shaken thoroughly.

68

Coutts, Baker, and Nazarali

After homogenlzatron of the tissue m the acid-butanol, the extraction and analysis are carried out m three stages. Stage 1 The homogenates are centrifuged for 10 mm, and 4 mL of the clear supernatant are retained and shaken with 2,2,4-trimethylpentane (10 mL) and water (5 mL) for 5 mm After centrrfugation, the lower aqueous phase 1s retained and 2M sodrum acetate (0.2 mL) and alumma (0.2 g) are added. The mixture IS shaken (5 mm) and centrifuged (5 min) The supernatant IS retamed and transferred to another tube The alumma IS washed by shaking with water (2 mL) for 5 mm and centrrfugmg. The washmgs are pooled wrth the prevrously retamed supernatant, and thus 1s stored at -17°C for analysis of 5-HT the next day. Stage 2. The alumina is shaken with 0.05M HC104 (2 mL) for 15 mm to elute NA and DA After centrrfugatron, the supernatant 1s transferred to another tube. Sodium phosphate buffer (0.5M, pH 8 0, 0 2 mL) and 0 5M sodium brcarbonate (0.2 mL) are added to bring the pH of the solutron to 8.0. This solutron is shaken with 2 0 mL of the Ion-pairing compound DEHPA (2.5% v/v m chloroform) for 5 mm Followmg centrrfugatron, the lower organic layer IS removed and shaken for 5 mm with 2.0 mL of 0.5N formic acid to elute the catecholamines. Subsequent to a brief centrrfugatron, the aqueous phase IS retained and evaporated to dryness at 35°C under vacuum. Methyl cyanide (0.5 mL) and TFAA (50 PL) are added to each tube, and the solutron IS reacted at room temperature for 5 mm. An ahquot of the solutron (1 pL) is injected onto a CC-ECD. Stage 3. To the supernatant retained for the 5-W assay 1s added 5 mL of a 20% v/v solutron of n-butanol m diethyl ether. Borate buffer (0.5M, pH 10, 0.2 mL) is added, and the mixture is shaken for 5 mm and centrifuged The upper organic layer is retamed and the ether IS evaporated off at 35°C under a stream of au The extraction IS repeated twice more, and the remaining butanol 1s taken to dryness at 35°C under vacuum The residue 1s reacted wrth TFAA (30 FL) m methyl cyanide (0.5 mL) for 5 min at room temperature. A 1 ~.LL alrquot of this mixture is Injected onto the GC-ECD, 4.3.1. Column and GC Oven Conditions The column is a 1 5 m, 4-mm bore glass column packed with 5% SE-52 on 100-120 mesh Gas Chrom Q Oxygen-free nitrogen at a flow rate of 45 mL/mm 1s used as the carrier gas. For the catecholammes, the mrtral oven temperature of 115°C IS maintained for 11 mm, at which time the temperature IS increased at a rate of 1 5”lmm until the derivatives have been eluted. Retention

Gas Chromatography of Amlnes and Their Metabobtes

69

trmes for the NA and DA derivatives are 23.2 and 26.9 min, respectively. The oven IS heated to 180°C for 10 min to clean the column for the next mJectron. For the GC analysis of the derrvatrve of 5-HT, the initial oven temperature of 180°C IS maintained for 6 min. This 1s followed by an increase at a rate of 0 75O/mm until the derivative IS eluted (13.9 mm) The oven temperature IS raised to 205°C for 10 mm to clean the column for the next mlection. Under the derivatrzatlon conditions utilized m this assay, the phenol, alcohol, and amine functions of the compounds mvolved react with TFAA. Rather than add an internal standard to the samples and run a standard curve, the authors of this assay procedure add 250 ng of each of the mines to be assayed (NA, DA, and 5-HT) to a brain sample and carry It in parallel through the procedure. By finding the difference m peak areas between this spiked sample and the identical sample without the added amine standards, the peak area correspondmg to 250 ng can be determined and the amounts of amines in the samples determined.

4.4. Determination of m- and p-Hydroxyphenylacetic in Mouse Brain

Acids

The protocol described here has been published by McQuade et al. (1981) Trssue IS homogenized m 6 vol of 0 1M zinc sulfate, and p-hydroxyphenylproplonic acid (250 ng) IS added as internal standard. An equimolar amount of barium hydroxide solutron IS added. Followmg centrifugatron, the resultant protein pellet is washed with 2 mL of drstrlled water, and this IS added to the supernatant. This mixture IS percolated through a 0.6 x 2.0 cm DEAE Sephadex column that has been previously washed wrth the followmg: 2M pyrrdinium acetate buffer (200 mL), distilled water (1000 mL), 0.2M HCl (500 mL), distilled water (1000 mL), 0.2M NaOH (500 mL), and distilled water (1000 mL). After adding the sample to the column, the column IS washed with distilled water An acidic fractron is then eluted using 8 mL of pyrrdmmm acetate (1.5M, pH 5.0). The eluant IS acidified to pH 1 with cone HCl(1 mL), saturated with NaCl, and extracted with ethyl acetate (3 X 5 mL). Th is organic extract IS taken to dryness under a stream of nitrogen. The residue IS redissolved m ethyl acetate, transferred to a clean test tube, and taken to dryness again The residue IS redissolved in ethyl acetate and transferred to a reaction vial to which IS added PFPA (25 FL) and 1,1,1,3,3,3-hexafluororsopropanol(l50 pL). The reactron mixture IS heated at 55°C for 90 min. After cooling, the mrxture IS taken almost to dryness

70

Coutts, Baker, and Nazarali

under a stream of nitrogen Hexane (400 FL) is added, and the solution transferred to another tube, where it IS washed (4 x 8 mL) with 1M sodmm phosphate buffer (pH 6 0). The organic layer 1s retained and an alrquot mlected onto a GC-ECD equipped with a 60-m SCOT capillary column coated with a methyl srhcone (SP 2100 or SE-30). 4.4 3. Gas Chromatographlc Conditions Carrier gas, argon/methane, 95/5 at 3 kg/cm2, mlection port temperature, 200°C; detector temperature, 300°C; oven program, 115°C initially, increasing to 150°C at lo/mm. Under these condrtions, the derrvatrves formed (structures shown m Frg 5) from p- and m-hydroxyphenylacetic acid have retention times of 38 and 35 min, respectively, whereas that of the derrvatized internal standard IS 57 mm.

5. Advantages and Disadvantages of Gas Chromatography Relative to Other Methods Available for Analysis of Biogenic Amines and Their Metabolites The apparatus required for GC is relatively mexpensrve when compared to mass spectrometry. After the inmal investment in the GC and a suitable printer/integrator, operating costs are reasonably low With increasing technology, sophistrcated gas chromatographs and integrators are now becoming available at costs that can be considered by many research laboratorres Fused silica capillary columns are relatrvely expensive, but we have found that they are very durable and can be used routmely for many months with a mmrmum of maintenance Gas chromatography can be crrtrcrzed on the basis of specificity when compared wrth mass spectrometry We agree that It 1s necessary to confu-m results obtained by GC by comparmg them with those obtained with mass spectrometrrc techmques. In addmon, when developing new analytical techniques with GC, rt IS desirable to confirm structures of the derrvatrves using GC-MS Once these criteria have been met, however, the GC techniques can be used as routine, relatively inexpensive means of analysis. Sensrtivrty of GC with ECD or NPD can be excellent, but on a practrcal basis IS often not as good as that found with HPLC with electrochemrcal detection (HPLC-EC) or with many radioenzymatic techniques However, compounds lackmg readily oxr-

Gas Chromatography

ofAmlnes

and TheirMetabobtes

71

0

II0 hC *c /

\

8

CH,C-

OCH(CF,),

oa

F&!Oo

CH,C-OCH(CF,), ’

Fig 5 Derwatwes formed from m- and p-hydroxyphenylacetlc acid (a and b, respectively) using the procedure of McQuade et al (1981). dizable groups (e. g , PE) cannot be assayed satisfactorily by HPLC-EC In addition, CC can be interfaced readily wrth MS. Through HPLC-MS capability is possible and will probably become mcreasingly important, such technology is still at an early stage of development. Thermolability of derivatives is a potential difficulty with GC, but does not pose a malor problem with HPLC. However, we have not found thermolability problems with the derivatives we use for analysis of biogemc amines and metabolites. In general, more sample tubes can be analyzed m a typical radioenzymatic procedure than m a GC procedure. However, while a radioenzymatic method often provides quantitation of only one or two compounds per sample tube, we are able to analyze simultaneously up to seven ammes per tube (see section on trace ammes) The throughput of samples m GC analysis can be increased markedly by employing an automatic sampler that can inlect samples unattended on a continuous basis The reported radioenzymatic procedure for TA does not provide for separation of the meta and pana isomers of this amme (Tallman et al., 1976), but our GC techniques do (Coutts et al , 1980, Baker et al , 1981) In addition, m radioenzymatic procedures enzymes

72

Coutts,

Baker, and NazaraO

must must

be prepared or purchased, and solutions of radiochemicals be disposed of Capillary columns now provide for high resolution GC As mentioned above, GC, particularly when combined with these columns and selective detectors, provides a relatively inexpensive technique for analysis of amines and their metabolites in tissue and body fluids The potential user should be aware that GC can also be used for analysis of a number of other blologlcally lmportant compounds (e g., amino acids) and drugs.

Acknowledgments The authors are grateful to the Alberta Mental Health Advisory Council, the Alberta Heritage Foundation for Medical Research, the Unlverslty of Alberta Hospitals Special Services and Research Committee, and the Medical Research Council of Canada for continuing support

References Addankl, S., Kmnenkamp E. R., and Sotos J. F (1976) Simultaneous quantltatlon of 4-hydroxy-3-methoxymandellc (vamlmandellc) and 4-hydroxy-3-methoxyphenylacetlc (homovamlhc) acids m human urine. Clan Chem 22, 310-314 Ahula, S. (1976) Derlvatlzatlon m gas chromatography / Pharm Su 65, 163-182. Anggard, E. and Sedvall G (1969) Gas chromatography of catecholamme metabohtes using electron capture detection and mass spectrometry Anal Chem 41, 1250-1256 Arnold E L. and Ford R (1973) Determmatlon of catechol-contammg compounds m &sue samples by gas-liquid chromatography Anal Chem 45, 85-89

Artlgas F and Gelpl E (1979) A new mass fragmentographlc method for the simultaneous analysis of tryptophan, tryptamme, mdole-3acetic acid, serotonm, and 5-hydroxymdole-3-acetic acid m the same sample of rat brain Anal Bmchem 92, 233-242 Bailey E and Barron E J (1980) Determmatlon of tranylcypromme m human plasma and urine usmg high-resolution gas-liquid chromatography with nitrogen-sensltlve detection. 1 Chronzatogr Bzomed. Apjd. 183, 25-31 Baker, G. B., Calverley D G., Dewhurst W G., and Martin I L (1979) A sensitive gas chromatographlc technique for quantlflcatlon of urlnary

Baker

G

tryptamme

B , Kuefler

Brzf

D

]

Phaumacol 67, 469P-470P

L

W , Coutts

R

T

and

Rao T

S

Gas Chromatography ofAmlnes and TheirMetabobtes

73

Pentafluorobenzoylsulfonyl chloride for analysis of 2-phenylethylamine m tissues and body fluids by gas chromatography (manuscript in preparation) Baker G. B , Coutts R T , and LeGatt D F. (1982) Gas chromatographic analysis of ammes n-t biological systems, m Analysis of Bzogen~c Ammes (Baker G. B and Coutts R. T., eds.) pp. 109-128, Elsevrer, Amsterdam Baker G. B., Coutts R. T , and Martin I. L (1981) Analysis of ammes m the central nervous system by gas chromatography with electroncapture detection Progr. Neurobiol 17, 1-24 Baker G. B , Coutts R T., and Martin I L. (1984) Gas chromatography with electron-capture detection for anaIysls of trace ammes in ussues and body fluids, in Neuroblolocgy of the Trace Ammes (Boulton A A , Baker G. B , Dewhurst W G., and Sandler M., eds ) pp. 57-68 Humana Press, Clifton, N.J. Baker G. B , LeGatt D. F., and Coutts R T. (1982) A gas chromatographic procedure for quantification of pava-tyramme m rat bram. ] Neuroscr. Methods 5, 181-188. Baker G. B., Martin I. L., Coutts R T., and Benderly A (1980) Determination of 5-hydroxytryptamme m rat brain regions by gas-Irquid chromatography with electron-capture detection J. Pharmacol Methods 3, 173-179. Baker G B , Nazarall A. J., and Coutts R T (1984a) Aqueous trlchloroacetylatlon and electron-capture gas chromatography for the analysis of 2-phenylethylamme and tranylcypromme m bram tissue Proc Meet Int Unton Pharmacology, 1222~. Bakowskl M. T , Toseland I’. A , Wicks J. F , and Trounce J. R. (1981) A rapid gas chromatographic method for the determmation of plasma polyammes and its application to the prediction of tumour response to chemotherapy Clm Chum Acfa 110, 273-286 Benmati S., Piacentmi M., and Ceru M I’ (1978) A gas chromatographic method for the determmation of di- and polyammes m human urine. lfal ] Blochem 27, 156-167. Bertam L M , Dzledzic S. W., Clarke D. D , and Gitlow S. E. (1970) A gas-liquid chromatographic method for the separation and quantitatlon of normetanephrine and metanephrme m human urme Clm Chum Acfa 30, 227-233 Bhargava H. N and Way E L (1975) Brain acetylcholme and cholme following acute and chronic morphme treatment and during wlthdrawal. ] Pharmacol Exp Ther 194, 65-73 Bigdeli M G and Collins M. A. (1975) Tissue catecholamines and potential tetrahydrolsoqumolme alkaloid metabolites a gas chromatographic assay method with electron-capture detectron. Bzochem Med 12, 55-65.

Blau K , Claxton I M , Ismahan G., and Sandler M (1979) Urmary phenylethylamme excretion: Gas chromatographic assay with electron-capture detection of the pentafluorobenzoyl derivative. ] Chromafogr Blamed Appl 163, 135-142

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Blau K and King G S. (1978) Acylatlon, III Handbook of Devlvatlves for Chromatography (Blau K and King G S , eds ). pp. lOP151. Heyden, London. Blau K., King G S , and Sandler M (1977) Mass spectrometrlc and nuclear magnetic resonance conflrmatlon of a 3,3-splrocychc mdole derivative formed from melatonm and related acyl tryptammes Btomed. Mass Specfrom 4, 232-236 Bock U E G. and Waser I’ G (1981) Gas chromatographlc determmatlon of some blogenlc ammes as their pentafluorobenzoyl derivatives m the picogram range and its appllcablllty to blologlcal matenals 1 Chromatogr 213, 415428 Borlson R. L., Mosnalm A D , and Sabelh H. C. (1974) Biosynthesis of brain 2-phenylethylamme. Influence of decarboxylase mhlbltors and D-amphetamme Life Scl 15, 1837-1848. Boulton A A , Juorlo A V , PhilIps S R , and Wu P. H (1975) Some arylalkylammes m rabbit brain Bram Res 96, 212-216 Boulton A A , Philips S R , Durden D A , Davis B A , and Baker G B. (1976) The tissue and cerebral subcellular dlstrlbutlon of some arylalkylammes m the rat and the effect of certain drug treatments on these dlstrlbutlons Adv Mass Spec. Brochem. Med 1, 193-205 Brooks C J W and Hornmg E C (1964) Gas chromatographlc studies of catecholammes, tryptammes, and other blologlcal ammes. Part 1. Catecholammes and related compounds. Anal Chem 36,1540-1545 Calverley D. G , Baker G. B., McKlm H R., and Dewhurst W G. (1980) A gas chromatograph technique using electron-capture detection for simultaneous estlmatlon of tryptamme and 5-hydroxytryptamme m blologlcal tissue. Can. 1. Neural SCI. 7, 237. Chattaway F. D. (1931) Acetylatlon m aqueous alkaline solution J Chem. Sot (London), Part 1, 2495-2496. Chauhan J and Darbre A (1980) Determination of homovamlhc, lsohomovamlhc, and vanlllylmandehc acids m human urine by means of glass capillary gas-liquid chromatography with temperature-programmed electron-capture detection J Chromatogr 183, 391401 Chauhan M. S. and Dakshmamurtl K (1982) Gas-chromatographlc method for the simultaneous determination of dopamme and norepmephrme metabohtes ] Chromatogr 227, 323-330. Clarke D. D., Walk S , and Gltlow S E (1966) Electron-capture properties of halogenated amme denvatlves 1, Gas Chromato~r 4,310-315 Cole W J , Parkhouse J , and Yousef Y Y (1977) Appllcatlon of the extractlve alkylatlon technique to the pentafluorobenzylatlon of morphine (a heroin metabollte) and surrogates, with special reference to the quantitative determination of plasma morphine levels using mass fragmentography J Chromafoxr 136, 409-416 Cone E J , Rlsner M. E , and Neldert G L (1978) Concentrations of phenethylamme m dog followmg smgle doses and during mtravenous self-admmlstratlon Res Common Chcr?~ Path Pharmacol 22, 211-232.

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Coutts R. T and Baker G. B (1982) Gas chromatography, m Handbook of Neurochemzstry (Laltha A , ed ) 2nd ed , pp 429448. Plenum Press, New York Coutts R T , Baker G. B , and Calverley D G (1980) A rapid, sensitive method of measurmg meta- and parcl-tyramme levels m urine usmg electron-capture gas chromatography Res Commun Chem Path Pharmacol 28, 177-184. Coutts R. T , Baker G. B., LeGatt D. F., McIntosh G. J., Hopkmson G , and Dewhurst W G. (1981) Screening for ammes of psychiatric mterest m urine using gas chromatography with electron-capture detection. Progr Neuro-Psychopharmacol 5, 565-568 Cranmer M. F. (1968) Estimation of the acetylcholme levels m bram tissue by gas chromatography of acetic acid trfc Scz 7, 995-1000 Crlstofoh W. A., Baker G B., Coutts R T and Benderly A. (1982) Analysis of a monofluormated analogue of amphetamine m brain tissue using gas chromatography with electron-capture detection. Progr Neuropsychopharamcol

& Btol. Psychzat. 6, 37%376.

Cummings L. M (1971), in Recent Advances m Gas Chromatography (Dombsky I and Perry J , eds.). p 313. Marcel Dekker, New York Dal Nogare S. and Juvet R S (1962) Gas Chromatography. Theory and Pracfree Interscience, John Wiley & Sons, New York Davis B. A and Boulton A A. (1981a) Excretion of nz-hydroxymandehc acid in human urine ] Chromatogr Blamed Appl 222, 271-275 Davis B A. and Boulton A. A (1981b) Longitudmal urinary excretion of some “trace” acids m a human male ] Chronzatogr Blamed Appl 222, 161-169

Davis B A., Durden D A , Pun-L1 I’ , and Boulton A. A (1977) Gas chromatographic procedure for the determmation of meta- and parahydroxyphenylacetic acids 1 Chromatogr 142, 517-522 Degen I’. H , Do Amaral J R., and Barchas J. D. (1972) A gas-liquid chromatographic assay of melatonm and mdoleammes usmg heptafluorobutyryl derivatives. Anal Bzochem 45, 634644 Dekirmenlian H and Maas J W. (1970) An improved procedure for 3-methoxy-4-hydroxyphenylethylene glycol determmation by gas-liquid chromatography Anal Blochem. 35, 113-122 Dekirmenlian H. and Maas J W (1971) Determmation of urinary 3-methoxy-4-hydroxymandelic acid by gas-liquid chromatography as vamllyl alcohol Clm Chum Acta 32, 310-312 Denton M. D., Glazer H S., Zellner D C , and Smith F. G. (1973) Gaschromatographic measurement of urinary polyamines m cancer patients Clm Chem 19, 904-907 Deo I’ G. and Howard P H (1978) Phosphorylatlon of alcohols/phenols for gas-liquid chromatographic separation and flame photometric detection 1. Of% Anal Chem 61, 210-213 Doshi I’. S. and Edwards D. J, (1980) Determmation of urmary methylhistamme m male and female rats by gas chromatography with electron-capture detection. L$e Scr 26, 1947-1953 Doshi I’ S. and Edwards D J (1981) Effects of L-DOPA on dopamme

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and norepmephrme concentrations m rat bram assessed by gas chromatography J. Chromatogr 210, 505-511. Doshi I’. S. and Edwards D J. (1979) Use of 2,6-dmitro-4trifluoromethylbenzenesulfonlc acid as a novel derivatizmg reagent for the analysis of catecholammes, histammes and related ammes by gas chromatography with electron-capture detection. 1, Chromatogr 176, 359-366. Drozd J. (1975) Chemical derivatization in gas chromatography. I Chromatogr 113, 303-356. Durden D A., Philips S R., and Boulton A. A (1973) Identification and distribution of J3-phenylethylamme m the rat Can J. Blochem. 51, 995-1002 Dziedzic S W., Bertam-Dziedzic L , and Gltlow S. E (1973) Separation and determmation of urinary homovamllic acid and isohomovamllic acid by gas-liquid chromatography and electroncapture detection ] Lab Clm Med 82, 829-835 Earley C J and Leonard B E (1978) Isolation and assay of noradrenalme, dopamme, 5-hydroxytryptamme, and several metabolites from brain tissue using disposable Bio-rad columns packed with Sephadex G-10 J Pharmacol. Methods 1, 67-79. Edwards D J and Blau K. (1972a) Analysrs of phenylethylammes m biological tissues by gas-lrquid chromatography with electron-capture detection Anal Blochem. 45, 387402 Edwards D J and Blau K (1972b) The in viva formation of p-chloro-pphenylethylamme m young rats mlected with p-chlorophenylalanme. ] Neurochem 19, 1829-1832. Edwards D J. and Blau K (1973) Phenylethylammes m bram and liver of rats with experimentally induced phenylketonuria-like characteristics Biochem J 132, 9E+lOO. Ettre L. S (1965) Open Tubular CoIumns m Gas Chromatography Plenum Press, New York, 164 pp Ettre L S (1973) Practical Gas Chromatography. Perkm Elmer Corp., Norwalk, Conn , 151 pp. Ettre L S. and Zlatkis A (eds ) (1967) The Practice of Gas Chromatography Interscience, John Wiley & Sons, New York, 591 pp Franken J J, and Trilbels M M. F (1974) Prelimmary studies m the analysis of biological ammes by means of glass capillary columns I Studies with model compounds J. Chromatogr 91, 425-431. Freeman R R (1981) High-Resolution Gas Chromatography, 2nd ed , Hewlett Packard, Palo Alto Gagne M. -A , Wollm A , Navert H., and Pinard G (1982) Anomaly of histamine methylation m endogenous depression. Progr NeuroPsychopharmacol. & Blol Psychzat 6, 483-486. Gelpi E., Paralta E., and Segura J. (1974) Gas chromatography-mass spectrometry of catecholammes and tryptammes Determmation of gas chromatographic profiles of the ammes, their precursors, and their metabolites J Chromatogr Set 12, 701-709.

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Goldstein M., Friedhoff A J., and Simmons C (1959) A method for the separation and elimination of catecholammes m urine Experlenfla 15, 80-m

Goodwm B. L., Ruthven C R , and Sandler M (1975a) Gas chromatographic assay of phenylacetic acid m biologrcal fluids. Clan Chmz. Acfa 62, 445446.

Goodwin 8. L , Ruthven C. R., Weg M. W., and Sandler M (1975b) A specific assay for urinary 5-hydroxymdole-3-acetic acid by gas chromatography CIUZ Chrm Acta 62, 439442. Grant D W (1971) Gas-Lquzd Chromatography Van Nostrand Reinhold Co., London. Greer M J., Sprinkle T J , and Wrlliams C M (1968) Determmatron of urinary 3-methoxytyramme, normetanephrme, and metanephrme m pheochromocytoma and neuroblastoma by gas chromatography. Clzn Chlm. Acfa 21, 247-253. B. J. (1967) Gas Chromafographlc Analyszs Pesfuzldes Marcel Dekker, New York, 605 pp

Gudzmowlcz

of Drugs

and

Haeffner L J , Magen J , and Kowlessar 0 D (1976) The gas-liqurd of selected catecholammes on chromatographrc separation polyamide A103 J Chromatogr 118, 425-428 Hagopran M , Dorfman R I , and Gut M. (1961) A method for the rsolatron and separation of catecholammes and their transformation products from biologrcal media. Anal Blochem 2, 387-390 Hampson D. R , Baker G B , and Coutts R. T (1984) A rapid and sensrtive gas chromatographrc method for quantltatron of 2-phenylethylamme m brain tissue and urine Res. Commun Chem Pafhol. Pharmacol 43, 169-172.

Hanrn I. (ed.) (1974) Cholmeand Acetylcholme: Handbook of ChemicalAssay Methods Raven Press, New York Hanm I and Jenden D J (1969) Estunatron of choline esters n-t bram by a new gas chromatographic procedure Blochem Pharmacol 18, 837-845

Harm-r I,, Massarelll R., and Costa E. (1970) Acetylcholme concentrations in rat brain. diurnal oscrllatron Science170, 341-342 Hanm I., Massarellr R., and Costa E (1972) An approach to the m vrvo study of acetylcholme turnover m rat salivary glands by radio gas chromatography. J Pharmacol Exp Ther 181, 10-M. Harris W E and Habgood H W (1966) Programmed Temperature Gas Chromatography. John Wiley & Sons, New York, 305 pp Hrga S., Suzuki T , Hayashl A , Tsuge I , and Yamamura Y (1977) Isolation of catecholammes m brological fluids by boric acid gel Anal Blochem 77, N-24.

Hollrs 0 L. (1966) Separation of gaseous mixtures using porous polyaromatrc polymer beads Anal Chem 38, 309-316 Holman R. B , Angwm I’ , and Barchas J. D (1976) Simultaneous determmatron of mdole- and catecholammes m small bram regrons m the rat using a weak-cation exchange resin Neuroscl. 1, 147-150

78

Coutts, Baker, and Nazarab

Hopkmson G , Baker G. B., Douglass A. B , McKim H R., and Dewhurst W G (1982) Analysis of urinary excretion patterns of bioactive ammes and their metabolites m normal control subIects Progr. Neuro-Psychopharmacol

& Bzol. Psychzaf

6, 495-498

Imai K , Sugiura M , and Tamura Z (1971) Catecholammes m rat tissues and serum determined by gas chromatographic method. Chem Pharm. Bull (Tokyo) 19, 409411 Imai K , Wang M. T , Yoshme S., and Tamura Z (1973) Determmation of catecholamines in the plasma of patients with essential hypertension and of normal persons Clan. Chun Acta, 43, 145-149. Jacob K., Falkner C , and Vogt W (1978) Derivatlzation method for the high-sensitive determmation of ammes and ammo acids as dimethylthiophosphimc amides with the alkali flame-iomzation detector 1 Chromafogr. 167, 67-75 Javaid J. I and Davis J M (1981) GLC analysis of phenylalkyl primary ammes using a nitrogen detector ] Pharm Scz 70, 813-815 Jenden D J , Booth R A , and Roth M (1972) Simultaneous microestimatlon of choline and acetylcholme by gas chromatography. Anal Chem 44, 1879-1881 Jennings W. (1980) Gas Chromatography wrfh Glass Capdlnry Columns Academic Press, New York, 320 pp Johnston G A., Lloyd H J , and de March1 W. J. (1970) Gas chromatographic estimations of compounds derived from acetylcholme J Chromafogr. 47, 482485. Jones R. A. (1970) An lnfroductlon to Gas-Lqtltd Chromatography. Academic Press, London, 202 pp Kahane Z , Jmdal S I’ , and Vestergaard I’. (1976) Gas chromatographic estimation of 3,4-drhydroxyphenylglycol m urme as the dlacetylphenyl-bis(trimethyl)silyl ether Clan. Chum Acta 73, 203-206 Kakimoto Y and Armstrong M D. (1962) The phenohc ammes of human urine I Bzol Chem 237, 208-214. Karasawa T , Furukawa K , Yoshida K , and Shimizu M (1975) A double column procedure for the simultaneous estimation of norepmephrme, normetanephrme, dopamme, 3-methoxytyramme, and 5-hydroxytryptamine in brain tissue ]pn ] Phnrmacol. 25, 727-736 Karoum F , Cattabem F., Costa E , Ruthven C. R J,, and Sandler M (1972) Gas chromatographic assay of picomole concentrations of biogemc ammes And Blochem 47, 550-561. Karoum F , Nasrallah H , Potkm S , Chuang L , Moyer-Schwmg J, Phillips I , and Wyatt R J (1979) Mass fragmentography of phenylethylamme, m- and p-tyramme and related ammes m plasma, cerebrospmal fluid, urine, and bram. I. Neurochem 33, 201-212 C. R J , and Sandler M (1968) Gas Karoum F , Ruthven chromatographic measurement of phenolic acids and alcohols m human urine Clan Chvn Acta 20, 427437

Gas Chromatography

ofAm/nes

and Their Metabolites

79

Kawar S. and Tamura Z. (1968) Gas chromatography of catecholammes as their trrfluoroacetates m urine and tumour. Chem. Pharm. Bull. Uokyo) 16, 1091-1094. Kawano T., Niwa M., FuJrta Y , Ozakr M., and Man K. (1978) An rmproved method for analysis of catecholammes-gas-liquid chromatography (GLC) equipped with electron-capture detector. Jpn 1, Pharmacol 28, 168-171. Keyzer J. J , Wolthers B. G., Breukelman H., Kauffman H. F , and Demonchy J G. R. (1982) Determination of N-TAU-methylrmrdazoleacetic acid (a histamine metabolrte) m urine by gaschromatography using nitrogen-phosphorus detection Clan. Chum Acta 121, 379-387. Krlbmger H. (1973) Gas chromatographic estrmatron of acetylcholme u-r the rabbit heart using a nitrogen selective detector. 1. Neurochem 21, 421429. Knapp D R. (1979) Handbook of Analytical Dcnvatzatlon Reactlvns, John Wiley & Sons, New York. pp. 65-145. Kosh J W , Smith M. B., Sowell J. W , and Freeman J. J (1979) Improvements m the gas chromatographrc analysis of acetylcholme and choline 1 Chrvmafogr 163, 206211. Laverty R. and Sharman D. F. (1965) The estlmatlon of small quantmes of 3,4-drhydroxyphenylethylamme m tissues Brzf J Pharmacol 24, 538-548. Leathard D. A. and Shurlock B. C. (1971) ldentrficatron Technzqueszn Gas Chromatography John Wiley & Sons, New York, 282 pp. LeGatt D F., Baker G B , and Coutts R T. (1981) Srmultaneous extraction and separatron of trace amines of brologrcal interest J Chromatogr Boomed Appl. 225, 301-308. LeGatt D. F., Baker G. B , Coutts R. T , and Crlstofoli W. A (1982) Electron-capture gas chromatographic procedure for concomrtant analysis of hrstamme and l-methylhistamme u-r brain tissue. Proc Can. Fed Blol. Svc 25, 142

Lhuguenot J, -C and Maume B. F. (1974) Improvements m quantltatlve gas phase analysis of catecholamines u-r the prcomole range by electron-capture detection and mass fragmentography of theu pentafluorobenzylrmmetrimethylsilyl derrvatrves \ Chromatogr Scz. 12, 411418. Lrttlewood A. B. (1970) Gas Chromatography* Prmclples, Techniques, and Applzcatzons, 2nd Ed. Academic Press, New York, 546 pp Lovecock J E and Lrpsky S R. (1960) Electron-affinity spectroscopy. A new method for the rdentrfrcatron of functronal groups m chemrcal compounds separated by gas chromatography. I Am Chem Svc 82, 431-441 Maggs R. J., Joynes I’ L., Davies A. J., and Lovecock J E. (1971) The electron-capture detector-a new mode of operation. Anal Chem 43, 1966-1971, Mahy N. and Gelpl E. (1978) A simple gas chromatographrc-mass spec-

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trometric method for the concurrent determmation of novel pentafluoropropionated derivatives of histidme, histamine, and their l-methyl metabolites Ckromatograpkm 11, 573-577 Makita M , Yamamoto S., and Kono M (1975) Rapid determmation of di- and polyammes m human urme by electron-capture gas chromatography. Clan Cklm Acta 61, 403405. Makita M , Yamamoto S., Miyake M , and Masamoto K (1978) Practical gas chromatographic method for the determination of urinary polyammes ] Ckromafogr. 156, 340-345 Marml J L , Williams S P , and Sheard M H (1979) Simultaneous assay for L-tryptophan, serotonm, 5-hydroxymdoleacetic acid, norepinephrme, and dopamme m brain PkarmacoI Blockem Bekav. 11, 185187 Martin I. L. and Ansell G B (1973) A sensitive gas chromatographic procedure for the estimation of noradrenalme, dopamme, and 5-hydroxytryptamme m rat bram &o&em Pkarmacol 22, 521-533 Martin I L and Baker G B (1976) Procedural difficulties m the gas-liquid chromatographic assay of the arylalkylammes 1 Ckromafogr. 123, 45-50 Martin I L and Baker G B (1977) A gas-liquid chromatographic method for the estimation of 2-phenylethylamme m rat brain tissue Bzockem Pkarmacol 26, 1513-1516 Martin I L., Baker G B , and Coutts R T (1984) Gas chromatography with electron-capture detection for measurement of bioactive ammes m tissues and body fluids, m Metkodologzcal Surveys ~JZ Blockemzstry and Analyst (Reid E , ed.), vol. 14. Plenum Press, New York Martin W R , Sloan J W , Buchwald W F , and Bridges S. R. (1974) The demonstration of tryptamme u-r regional perfusates of the dog brain Psychopharmacofo~X2a (BerIm) 37, 189-198 Maruyama Y., Kusaka M., Man J , Horikawa A , and Hasegawa Y (1979) Simple method for the determination of choline and acetylcholine by pyrolysis gas chromatography ] Ckromafogr 164, 121-127 Maruyama Y and Takemorl A E (1972) New gas chromatographx method for estlmatlon of norepmephrme and dopamme m brain And Bzockern 49, 240-247 Matm S B. and Rowland M (1972) Electron-capture sensitivity comparison of various derivatives of primary and secondary ammes / Pkarm Scl 61, 12351240 McGregor R F , Sharon M S , Atkinson M , and Johnson D E (1976) An improved isolation procedure for the gas chromatographlc analysis of urinary polyammes Prep Bzockem 6, 405419 McNalr H M and Bone111 E J (1968) Bnslc Gas CIlronlato~~rapl2y. Varlan Instrument Dlvlslon, Palo Alto, 306 pp McQuade P L , Juorlo A V , and Boulton A A (1981) Estlmatlon of F’and m-isomers of hydroxyphenylacetic acid m mouse bram by a gas chromatographlc procedure Their regional dlstrlbutron and the effects of some drugs ] Neurochu 37, 735739

Gas Chromatography

ofAmmes

and Thew Metabobtes

81

Midha K K., McGrlveray I J , and Cooper J K. (1979) A GLC-ECD assay for simultaneous determmation of fenfluramme and norfenfluramme m human plasma and urme Cau ] Pharm. Scr 14, 18-21. Mlta H , Yasueda H , and Shlda T. (1979) Hrstamme denvatlve for quantltatlve determination by gas chromatography ] Chromatogr. 175, 339-342 Mlta H , Yasueda H , and Shlda T. (1980a) Quantltatrve analysrs of hrstamme m biological samples by gas chromatography-mass spectrometry J Chromatogr 181, 153-159 Mlta H , Yasueda H , and Shlda T. (1980b) Simultaneous determmatton of histamine and N-methylhlstamme m human plasma and urine by gas chromatography-mass spectrometry 1 Chromafogr &owed Appl 221, 3-7

Mltchard M (1978) Chromatographlc methods m the study of drug metabolism m man, m Drug Metabolsm ln Man (Gorrod J W. and Beckett A H., eds ). Taylor and Francis, London pp. 175-191 Mlyazakr H , Hashrmoto Y , Iwanaga M , and Kubodera T (1974) Analamines and their metabolrtes by gas ys1s of brogemc ionization mass spectrometry. chromatography-chemrcal J Chromatog 99, 575-586

Moffat A C and Hornmg E C (1970) A comparrson of some derlvatlves of primary ammes for gas chromatography usmg electron capture detection. Anal Leff 3, 205-216. Moffat A C , Hornmg E C., Matin S. B , and Rowland M. (1972) Perfluorobenzene derlvatlves as derrvatrzmg agents for the gas chromatography of primary and secondary ammes usmg electron capture detection 1. Chromato~r 66, 255-260 Morr A , Yasaka Y , Masamoto K., and Hlramatsu M (1978) Gas chromatography of 5-hydroxy-3-methylmdole in human urine. Urn Chun Acta 84, 63-68

Musklet F. A , Fremouw-Ottevangers D C , Wolthers B G , and Vrles J. A (1977) Gas-chromatographrc profrlmg of urinary acrdlc and alcoholic catecholamme metabolltes. Clrn Chem 23, 863-867 Musklet F A , Stratmgh M C , Stob G J., and Wolthers B G (1981) Simultaneous determmatlon of the four major catecholamme metabohtes and estlmatlon of a serotonm metabohte m urine by caprllary gas chromatography of their tert-butyldrmethylsllyl derrvatrves. Cllrz Chum. 27, 223-227 Naraslmhacharr N. and Fnedel R 0. (1981) Quantltatron of blologlcally important prrmary ammes as then isothlocyanate derlvatlves by gas chromatography usmg nitrogen detection and validation by selected ion momtormg Clm Chum Acfa 110, 235-243 Naruse H , Kato N , Nasu E , Kawar S , Hashrmoto K , Masada Y , and Ohno T (1977) Gas chromatographrc determmatron of urinary mdole-3-acetlc acid Ckem Pharm Bull (Tokyo) 25, 2032-2034 Navert H and Wollm A. (1980) Determmatlon quantltatlve slmultanee de l’hlstamme et de ses metabolltes baslques methyles. Unwon Medxale dtr Crrnada, Part 2, 109, 1507.

82

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Nazaralr A. J., Baker G B , Coutts R T , Pasutto F M , and Cristofoh W. A (1985) Simultaneous analysis of N-(2-cyanoethyl)amphetamme (fenproporex), amphetamme, and yaru-hydroxyamphetamme m the mammalian central nervous system: Res. Commtln Subs Abtlse 317-320. Nelson L. M., Bubb F. A., Lax I’ M., Weg M W., and Sandler M (1979) An improved method for the differential assay of 3-0-methylated catecholammes m human urine using ion-pair extraction and gas chromatography electron-capture detection Clm Ckrm. Actu 92, 235-240. Ogasahara S , Mandai T., Yamatodani A., Watanabe T., Wada H., and Sekr T. (1979) Simple method for the simultaneous determmatlon of dopamine, and serotonm by stepwlse elutron from a short column of weak-cation exchange resin J Ckromutogr 180, 119-126 Ohver J S , Smith H , and Williams D. J (1977) The detection, rdentrfrcatron, and measurement of mdole, tryptamine, and 2-phenylethylamme m putrefymg human tissue. Forenszc Set 9, 195-203 Oon M C. H and Rodmght R. (1977) A gas chromatographlc procedure for determmmg N,N-drmethyltryptamme and N-monomethyltryptamme m urine using a nitrogen. Blockem Med 18, 41B-419 Pearson J. D. and Sharman D. F (1974) A gas-liquid chromatographrc method for the estrmation of the acidic metabolites of dopamme m cerebrospmal flurd and brain tissues. Bvft 1 Pkarmacol 51, 114P. Perry J, A and Freit C. A (1978) Derivatlzatron techniques m gas-liquid chromatography, m GLC and HIXC Defermrnatlon of Tkerapeutlc Agents, Part 2 (TSUJ~ K. and Morozowlch W , eds ) pp 137-208, Marcel Dekker, New York Phlhps S. R., Durden D. A , and Boulton A A (1974a) Identrfrcatton and distribution of p-tyramme u-t the rat. Can J. Brockem 52, 336-373 Philips S R , Durden D A , and Boulton A A (1974b) Identification and dlstributlon of tryptamme in the rat Can 1, Blochem 52, 447451 Poole C E and Morgan E D. (1975) Structural requirements for the electron-capturing properties of ecdysones / Ckromatogr 115, 587-590 Purnell H (1962) Gus Chromatography John Wrley & Sons, New York, 441 pp. Reynolds G P and Gray D. 0. (1976) A method for the estimation of 2-phenylethylamme m human urme by gas chromatography CIzn Chum Actu 70, 213-217 Reynolds G. I’. and Gray D 0. (1978) Gas chromatographic detection of N-methyl-2-phenylethylamme a new component of human urine ] Chromatogr. Boomed Appl 145, 137-140. Reynolds G. P , Rrederer I’., Sandler M , Jellmger K , and Seeman D (1978) Amphetamine and 2-phenylethylamme m post-mortem Parkmsoman brain after (-)-deprenyl admmistratlon 1. Net& Trans 43, 271-277

Gas Chromatography

ofAmlnes

and TheJr MetabolJtes

83

Reynolds G. I’., Sandler M., Hardy J., and Bradford H. (1980) The determinatlon and distribution of 2-phenylethylamme in sheep bram 1 Neurochern. 34, 1123-1125 Roder E. and Merzhauser J. (1974) Determination of blogemc ammes by high-pressure liquid chromatography Anal. Chem 34, 272-277 Sandler M., Ruthven C R. J, Goodwin B. L., and Reynolds G. I’. (1979) Deficient production of tyramme and octopamme m cases of depresslon. Nature (London) 278, 357-358 Schmidt D E and Peth R C (1975) Simultaneous analysis of choline and acetylcholme levels m rat brain by pyrolysis gas chromatography. Anal Blochem. 67, 353-357. Schmidt D E , Szllagyl P I., Alkon D. L , and Green J. I’. (1970) A method for measurmg nanogram quantities of acetylcholme by pyrolysis-gas chromatography The demonstration of acetylcholme m effluents from the rat phrenic nerve-diaphragm preparation. 1, Pharmacol, Exp

Ther 174, 337-345

Schupp 0 E (1968) Gas Chromatography. John Wiley & Sons, New York, 441 pp. Schweitzer J W., Frledhoff A. J., and Schwartz R. (1975) Phenethylamme m normal urine Failure to verify high values Blol Psychzat 10, 277-285 Seller N (1980) Assay of polyamines m tissues and body flulds, m Polyamznes zn Bromedrcal Research (Gaugas J M, ed.) John Wiley & Sons, New York, pp 435461 Sevcik J. (1976) Detectors m Gas Chromatography (Journal of Chromatography Library, vol. 4) Elsevler Sclentlflc Publlshmg Co , Amsterdam, 192 PP Sharman D. F. (1969) Glycol metabolltes of noradrenaline m bram tissue. Brrt

1 Pharmacol

36, 523-534.

Sharman D. F (1971) Methods of determmatlon of catecholammes and their metabolltes, m Methods of Neurochemzstry, vol 1 (Fried R , ed). Marcel Dekker, New York. pp. 83-128 SIoqulst B. and Anggard E (1972) Gas chromatographlc determination of homovanilhc acid m human cerebrospmal fluld by electroncapture detection and by mass fragmentography with a deuterated internal standard. Anal. Chem. 44, 2297-2301. Slmgsby J M and Boulton A A. (1976) Separation and quantltatlon of some urinary arylalkylammes. J. Chromatogr. 123, 51-56. Snodgrass S R and Horn A. S. (1973) An assay procedure for tryptamme m bram and spmal cord using its [3H]-dansyl derlvatlve 1. Neurochem. 21, 687-696. Sprinkle T. J , Porter A H , Greer M , and Wllllams C. M (1969) An improved method for the determmatlon of homovamlhc acid and vamlmandehc acid by gas chromatography Cfln Chum Acta 25, 409411. Stavmoha W B. and Ryan L C (1965) Estlmatlon of the acetylcholine content of rat bram by gas chromatography. I Pharmacol. Exp Ther. 150, 231-235.

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Sugmra M and Hirano K (1974) Determmatlon of prostaglandm Fi alpha and F2 alpha by gas-lrqurd chromatography J Chromafogr 90, 169-177 Szilagyi P I., Green J I’ , Brown 0 M , and Margolts S. (1972) The measurement of nanogram amounts of acetylcholme m tissues by pyrolysis gas chromatography. 1, Neurochenl. 19, 2555-2566 Szilagyi I?. I., Schmrdt D E , and Green J I’ (1968) Microanalytical determination of acetylcholme, other choline esters, and cholme by pyrolysis-gas chromatography. Anal Chem 40, 2009-2013 Tallman J. F , Saavedra J M , and Axelrod J (1976) A sensltrve enzymatic-lsotoplc method for the analysis of tyramme m brain and other tissues 1 Neurochem 27, 465469. Tang S. W., Helmeste D. M., and Stancer H. C (1978) The effect of acute and chronic desrpramme and amltriptylme treatment on rat brain total 3-methoxy-4-hydroxyphenylglycol Naunyn-Schmledeberg’s Arch. Pharmacol 305, 207-211 Temple D. M and Gillespie R (1966) Llqurd ion-exchange extraction of some physiologically active ammes Nature LLondonJ 209, 714715 Usdm E. and Sandler M (eds ) (1976) Trace Amznes and the Brazn Marcel Dekker, New York, 301 pp van de Calseyde J F , Scholtrs R J , Schmidt N A., and Leyten C J (1971) Gas chromatography m the estimation of urmary metanephrines and VMA. Clrn Chnn Acta 32, 361-366 Walker J 0 , Jackson M. T Jr., and Maynard J. B. (1972) Chromatographzc Systems. Marntenance and Troubleshootzng Academic Press, New York, 359 pp. Wang M. T , Imai K., Yoshioka M., and Tamura Z (1975) Gas-liquid chromatographic and mass fragmentographic determmation of catecholammes m human plasma. CIzn Chim. Acfa 63, 13-19 Warsh J. J., Godse D. D , Lr P , and Cheung S. (1980) Sigmfrcance of 3,4-dihydroxyphenylethylene glycol (DHPG) formation m CNS norepmephrme metabolism. Can ] Neural Scl 7, 230. Warsh J J, Godse D. D., Stancer H C , Chan P W , and Coscma D V (1977) Bram tryptamme m rats by a new gas chromatography-mass fragmentographic method Blochem. Med. 18, lo-20 Watson E , Travis B., and Wilk S. (1974) Simultaneous determmahon of 3,4-dihydroxyphenylacetic acid and homovamllic acid m m&gram amounts of rat striatal tissue by gas-liquid chromatography. Lrfe Scl 15, 2167-2178 Watson E. and Walk S. (1974) Derlvatlzatlon and gas chromatographic determmatron of some biologrcally important acids m cerebrospmal fluid. Anal. Blochem 59, 441451 Welsh L. H (1955) The analysis of solutions of epmephrme and norepmephrme ] Am Pharm. Assoc 44, 507-514. Westermk B. H. C and Korf J. (1977) Rapid concurrent automated fluorlmetrlc assay of noradrenalme, dopamme, 3,4-drhydroxyphenylalanme, 3,4-dihydroxyphenylacetlc acid, homovanlllic acid,

Gas Chromatography

ofAmines

and Ther Met&o&es

85

and 3-methoxytyramme m mrllrgram amounts of nervous trssue after rsolatron on Sephadex G-10. J Neurochem 29, 697-706 Wresel F -A , Frr C -G., and Sedvall G. (1974) Srmultaneous mass fragmentographlc determmatron of 3,4-drhydroxyphenylacetrc acid and 4-hydroxy-3-methoxyphenylacetrc acid m brain tissue. J Neural Trans?n 35, 319-326 Walk S., Davrs K L , and Thacker S. B (1970) Determmatron of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) m cerebrosprnal fluid Anal Blochem 39, 49%504. Walk S , Grtlow S E , Clarke D. D , and Paley D. H (1967) Determmatron of urinary 3-methoxy-4-hydroxyphenylethylene glycol by gas-lrqurd chromatography and electron-capture detection Clan. Chum. Acta 16, 403-408. Wilk S., Gitlow S E , Mendlowrtz M , Franklm M J , Carr H. E , and Clarke D. D (1965) A quantrtatrve assay for vamllylmandelrc acid (VMA) by gas-lrqurd chromatography. Anal Blochem 13, 544-551. Wong J., Baker G B., Coutts R. T, and Pasutto F M (1984) A gas chromatographrc procedure for srmultaneous analysis of several bioactive ammes m cheese products. Proc Can Fed Bzol. Sot. 27,63 Wong K. I’., Ruthven C R., and Sandler M. (1973) Gas chromatographrc measurement of urinary catecholammes by an electron-capture detection procedure Clan Chwn Actlz 47, 21S222 Yamamoto S , Kakuno K., Okahara S., Kataoka H , and Makrta M (1980) Gas chromatography of phenolrc ammes, 3-methoxycatecholammes, mdoleammes, and related ammes as then N,Oethyloxycarbonyl derivatives ] Chromatogr 194, 399403 Zweig G. and Sherma J. (1972), m Handbook of Chromatography, vol 2 Chemical Rubber Corp. Press, Cleveland pp l-23

Chapter 3

Radioenzymatic Micromethods for the Quantitation of Biogenic Amines in Brain JUAN M. %WEDRA 1. Introduction The biogenic ammes 5-hydroxytryptamme (5-HT; serotonm), dopamine (DA), noradrenaline (NA), adrenaline (A), and histamme (HA) are present in mammalian brain, u-t which they play fundamental roles as neurotransmitters. Concentrated in a few brain nuclei and tracts (Dahlstrdm and Fuxe, 1964; Fuxe, 1965), they are mvolved in the regulation of specific brain functions. Their study requires selective dissection techniques to isolate discrete brain areas and sensitive quantitative microtechniques for the determination of amine levels in submilligram amounts of tissue. This is now possible with the combmatron of microanatomical dissection procedures (the “punch” technique) and the development of radioenzymatic micromethods. The high sensitivity of the radioenzymatic micromethods also allows for the quantitation of biogemc ammes present in bram in exceedingly low concentrations. @phenylethylamme (PE), phenylethanolamine (PEOH), tyramme (TA), and octopamine (OA). These amines have high turnover rates m brain, and their physiological importance could be greater than that assumed by their low steady state levels. The radioenzymatic micromethods described here are based on the incubation of a brain extract with specific methyltransferases and the natural donor of methyl groups, S-adenosyl-Lmethionine (SAME) In the test tube as well as normally in tis87

88

Saavedra

sues, the methyltransferases catalyze the transfer of the methyl group from SAME to the 0- or N-termmal positrons of the ammes, resulting m the formatron of the corresponding 0- or N-methyl derrvatrves. We use a donor of radroactrvely labeled methyl groups, [3H]-methyl-SAME, of high specific activity, and we utilize selective organic extraction and chromatographrc procedures to separate and quantitate the radioactive 0 or N derrvatrves formed The micromethods developed are speclfrc, most of them with a sensitivity at the low picogram level (Table 1). Such a high sensrtrvity is necessary to accurately detect low levels of “mmor” biogemc amines m brain and to study brogemc amme metabohsm in selected areas of the rat bram, werghmg less than 1 mg A precise selection of brain areas for quantrtatron 1s now possible with the use of standardized dlssectlon techniques, such as the “punch” technique that allows the localrzatron and drssectron of single nuclei of the rat brain. When compared to other quantltatrve techmques for the study of brain amines, the radioenzymatic methods have advanTABLE

Sensltlvlty

of the Enzymatrc nation

1

Isotopic

of Blogemc

Methods

Ammes

for the Determlm Tissues

Amme

Enzyme

Sensitivity,

Indoleammes N-Acetylserotonm Serotonin

HIOMT NAT”-HIOMT

Hlstamme

HMT’

5

Catecholammes Dopamme Noradrenalme Adrenaline

COMT’ COMT COMT

5

Phenylethanolamme Octopamine P-Phenylethylamme Tyramine

PNMT PNMT DBHVNMT DBH-PNMT

pg

50 10

2 2 25 25 200 200

“HIOMT Hydroxymdole 0-methyltransferase (EC 2 1 1 4) “NAT N-acetyltransferase (EC 2 3 1 5) ‘PNMT Phenylethanolamme N-methyltransferase (EC 2 1 1 28) “DBH Dopamme-p-hydroxylase (EC 1 14 17 1) ‘HMT Hlstamme N-methyltransferase (EC 2 1 1 8) ‘COMT Catechol 0-methyltransferase (EC 2 1 1 7)

Radioenzymatic

Micromethods

for Brain Amines

89

tages based on their specificity, sensitivity, scope of application, cost, and skills needed for adequate performance (Figs. 1 and 2). At the present time, the radioenzymatic methods utilizing Nor 0-methyltransferases are the methods of choice for the quantification of small amounts of N-acetylserotonin, serotonin, and other indoles, the catecholamines DA, NA, and A, histamine (HA), and the “trace” amines PE, PEOH, OA, and TA. Assays based on similar principles are used to study the activity of enzymes associated with the synthesis and degradation of biogenic amines.

2. General Procedure for Radioenzymatic

Methods

A preliminary, simple tissue extraction procedure is necessary to separate the endogenous amine from contaminating materials. These procedures generally involve acid precipitation and centrifugation with subsequent analysis of the supernatant, or organic extraction with evaporation of the organic phase prior to the enzymatic assay. The extracted material is incubated with [3H]-methyl-SAME of high specific activity, about 5-15 Ci/mmol, and partially purified 0- or N-methyltransferases. In some cases, a doubleQUANTITATIVE

’ 2 3 4

Additional steps sometimes necessary. One order of magnitude less than MS or RE. Some compounds cannot be measured. Position Also high for determination of enzyme activities.

METHODS

isomers

FOR BIOGENIC

difficult

AMINES

to separate.

Fig. 1. Comparison of different quantitative termination of biogenic amines.

methods

for the de-

Saavedra

90

1.

Sensitive

(low

2.

Easy

3.

Fast

4.

Reliable

5.

Specific procedures

6.

Inexpensive

7.

No Sophisticated Necessarv

8.

Srmrlar Prtncrple for Determrnatron

picogram

level)

1.

Some Compounds Measured

2.

Position

Cannot

Be

to Perform

Fig. 2.

Isomers

Difficult

to Separate.

(additional chromatographic sometimes necessary)

Equipment

and Technology Used of Enzyme Activrty

Advantages

of radioenzymatic

assays for biogenic amines.

enzyme incubation is performed to convert the amine to a derivative with higher affinity for the methylation process. A very simple procedure is usually necessary to purify the methyltransferases. The use of partially purified enzymes is sufficient for selectivity of methylation and allows the storage of relatively stable enzyme preparations for months. The use of specific methyltransferases results in the selective methylation of relatively small groups of endogenous compounds. The specificity and sensitivity of the assays are dependent on the separation of the resultant methylated derivatives from other possible products, as well as from the unreacted labeled SAME and its labeled metabolites. These procedures are carried out by a preliminary extraction of the labeled product into specific organic solvents, taking advantage of the favorable partition coefficient of the N- or 0-methylated amine derivatives. This is followed by chromatographic procedures and sometimes by the evaporation of the organic phase to eliminate labeled volatile contaminants. Internal standards are carried through the assay to correct for losses of material (Table 2). In most assays, there is a linear relationship between the amount of amine present in the tissues (up to 2000 pg) and the

RadIoenzymatIc

Muomethods

General Procedure 1 2 3 4 5

91

for BraIn AmInes

TABLE 2 for the Enzymatic-Isotopic Assay of Blogemc Ammes and Related Substances

Extract the amme from the tissue or body fluid (acid or buffer extraction) Incubate with the correspondmg N- or 0-methyltransferase and radioactive methyl donor ([lH]methyl-SAME) Extract the radioactive N- or 0-methylated products formed m the reaction with specific organic solvents Eliminate the radioactive contammants by selective drying or chromatographlc procedures Count the radloactlvlty by lquld scmtlllatlon spectroscopy

number of dpm obtained. Intra-assay variability 1s less than 5% Such characterlstlcs allow the quantltation of catecholammes, HA, and serotonm m areas of the rat brain weighing less than 1 mg, measured

m duplicate

samples

from smgle

animals

(Table 3)

(Saavedra, 1979).

3. The “Punch” Dissecting Technique A “punch” dlssectmg techmque was first described by Elk-Nes and Brlzzee (1965). These authors studied the dlstrlbutlon of radloactive cortlcold metabolites m the dog brain by dlssectmg brain “nuclei” from frozen sections with the use of a hollow needle. This method was hardly noticed until 1973, when Schlumpf described m her doctoral thesis the “punch” microdlssecting techniques for the rat and mouse brain in combmation with fluorometnc analysis of serotonin and catecholamines (Schlumpf, 1973). At this time, the development of sensitive radloenzymatlc techniques for serotonm (Saavedra et al., 1973), catecholammes (Coyle and Henry, 1973), HA (Snyder et al., 1966), and related enzymes (Molmoff et al., 1971) made possible the study of amme metabolism m submilllgram amounts of brain tissue. The “punch” technique was then “rediscovered” (Palkovlts, 1973), and applied to the study of amine content of individual rat brain nuclei This method 1s now routme in many laboratones around the world In brief, rat brains are frozen, cut in 300 km-thick sections in a microtome cryostat at -PC, and the brain nuclei are dissected

of Serotonm,

N suprachzasmatls Area retrochrasmatrca N perlventrzcularzs N supraoptus N parauentrmdar~s N hypothalamzcus anterior N arcuatus N ventromedlalzs N dorsomedlalls N perlformcalls

Area or nuclei

Levels

90220 15.1 k 1 1 7.1 t 07 37r06 loo*15 50-‘09 20Ot30 7OklO loo-to7 60-r-11

Dopamme” 250 -c 40 480+79 335-+33 236r50 510264 162?46 250 -t 40 327521 2192 70 179k38

Noradrenaline”

Amme

TABLE 3 Noradrenalme, Adrenaline, and Histamine Nuclei of the Rat Hypothalamus

25.4 2 8 0 159 t 23 109+41 95?28 135?31 102 2 23 364 t 98 85 A 39 13 6 t 5.3 300 -c 109

Serotonin”

Dopamme,

ND ND 27504 ND 06kOl 17203 ND 19+02 24kO5 ND

Adrenalme

li

m Mlcrodlssected

422 03 ND 44-06 30-06 24 2 05 33? 08 55204 67208 50204 ND

Histamine’

110~15 650+61

307k80

153+32

of 610

43210 39512 34 2 09 61 ?09

245k76 22.9 + 8 8 183288 216t58

‘From Saavedra et al , 1973 ‘From Palkovlts et al , 1974b ‘From Saavedra and Alexander, 1983 “From Kvetnansky et al , 1978
N hypothalamxus posterzor N premammdlarzs dorsalzs N premamm~llar~s ventralzs Medial forebram bundle (antenor) Medzal forebram bundle Cposterzor) Medlart emrrzerrce

rats

295

z!I 40

20 2 k 2.7

139222 142242 163-62 169t19

07~005

ND

ND ND ND ND

178+22

ND

46kO7 39505 81 -08 ND

Saavedra

94

from the frozen, unstained sections with hollow needles of internal diameter from 0.2 to 1 mm, according to the area studied (Fig. 3) (Schlumpf et al., 1974). The combination of neuroanatomical and microbiochemical methods, which occurred by chance when researchers in the respective fields met at the Laboratory of Clinical Science at the National Institutes of Health in 1973, provided a new dimension to the biochemical study of specific amine systems within the central nervous system.

4. General Procedures for Enzyme Purification A partial purification of the methylating enzymes is necessary to provide adequate preparations for their use in radioenzymatic as-

Fig. 3. The “punch” dissecting technique. Photograph of a coronal rat brain section at the level of the yedunculus cerebellaris supcrioris. The section was “punched” while frozen. The nucleus raphe dorsalis (upper) and nucleus ccwtralis superioris (lower) have been dissected (from Schlumpf, 1973)

Radioenzymatic

Micromethods

Enzymes

for Brain Amines

95

TABLE 4 Used m the Detection of Brogemc Ammes Purification procedures’

Amme

Enzyme

Source

Histamine N-methyltransferase (EC 2 0.8) Catechol O-methyltransferase (EC 2 1.1 7) Dopamme-B-hydroxylase (EC 1.14.2.1)

Gumea pug brain

12

Hlstamme

Rat liver

12

Bovme adrenal medulla

123

Bovme pmeal

12

Dopamme Noradrenalme Adrenaline B-Phenylethylamine Tyramme N-acetylserotonm Serotonin

Rat liver

12

Serotonm

Bovme adrenal medulla

1,2/J

Phenylethanolamine

Hydroxyindole O-methyltransferase (EC 2.1.1.4) N-acetyltransferase (EC 2.3 1.5) Phenylethanolamme N-methyltransferase (EC 2.1 1.28)

measured

Octopamine B-Phenylethylamine Tyramme “Purrfrcatron procedures 1, ammomum sulfate fractronatlon, canavalm-A sepharose column, and 4, acid preclpltatlon

2,

dralysrs,

3,

says These procedures generally involve ammonmm sulfate precipitatron and dialyses and result in the production of highly stable enzyme preparations (Table 4, Fig. 4). All purlfrcatlon procedures are conducted at 4°C and are completed within 24 h Homogemzatron m specrfrc buffers IS performed with the use of a Waring blender or Teflon-glass homogenizers. Homogenates are centrifuged first at low speed (up to 5OOOg)to remove cell debris and nuclei and consequently at hrgh speed (up to 78,000g) to obtain active enzymes soluble m the supernatant. Ammonmm sulfate fractionation of the supernatant is performed by slowly adding solid ammonium sulfate to the required saturation points, followed by centrrfugatron. Extensrve dialysis (against 200 vol of buffer) is performed overnight to remove the ammonium sulfate and contaminants of small molecu-

con-

Saavedra

96 TISSUE Homogenlzatlon c HOMOGENATE Centnfugatlon I SUPERNATANT Ammomum Sulfate and Centrdugatlon

I AMMONIUM

SULFATE

Preclpctatlon

PRECIPITATE

DIALYZATE Centnfugation 1 SUPERNATANT b FINAL

Fig

Analysis Storage ENZYME

4.

of Enzyme at - 20°C

Actwtty

PREPARATION

Flow

sheet for the partial purificatron

of methyltransferase

enzymes lar weight (Table 4) After dialysis, the resulting enzyme preparations are divided mto small ahquots and stored at -20°C until used. Most of the enzyme preparations obtained are stable for several months. Lyophilization of enzyme ahquots prior to storage increases enzyme stability. After purification, analysis of the enzyme activity is necessary to determine the optimum amount of enzyme to use, due to variations m the biologrcal activity of the different tissue preparations (Fig. 4)

5. Indoleamines 5.1.

General Procedure

for the Assay of Indoleamines

In the pmeal gland, serotonm is converted through acetylation by N-acetyltransferase (NAT) mto N-acetylserotonm, which is later 0-methylated by hydroxymdole O-methyl transferase (HIOMT) to form the pmeal hormone melatonm HIOMT methylates only the 5-hydroxy group of hydroxymdoles (Axelrod and Weissbach, 1961). Both serotonin and N-acetylserotonin are susceptible to 0-methylation, N-acetylserotonm being a much better substrate for HIOMT (Saavedra et al., 1973) 0-Methylation is the basis for the radioenzymatic assays of N-acetylserotonm, serotonm, and other indoleamines (Saavedra et al , 1973). N-Acetylserotonm is directly 0-methylated by HIOMT, serotonm is acetylated prior to its 0-methylation In both cases, radioactively labeled [3H]-methyl-melatonm is formed (Fig. 5). With this assay, endogenous levels of N-acetylserotonm

RadioenzymatlcMwomethods

for Bra/n Amines

97

and serotonin could be detected in a fraction of one rat pmeal gland, and serotonm could be measured m single nuclei of the rat brain (Saavedra et al., 1973, 1974d). In addition, the assay prmciple of serial N-acetylation and 0-methylation can be applied to the measurement of tryptophan hydroxylase activity (Saavedra, 1977) and to the determmation of 5-hydroxytryptophan levels and L-amino acid decarboxylase activity m brain nuclei (Saavedra, 1976). Simpler, modified techniques based on the same principle can be used for determination of NAT activity (by replacing serotonin with tryptamine as substrate) (Deguchi and Axelrod, 1972) and HIOMT activity (Fig. 5). 5.1.1.

Purifkatlon EC 2.1.1.4)

of Hydroxylndole

0-Methyltransferase

(HIOMT,

The enzyme is obtained from bovine pmeal glands purchased frozen from Pel-Freez Biologicals, Inc. (Rogers, AR). The pineals are homogenized in 5 vol of isotonic KC1 and centrifuged at high speed. Solid ammonium sulfate is added to the soluble supernatant fraction. The 36-55% precipitate is extensively dialyzed CH,-CH-COOH : ’ N ia”> 07 TRYPTOPHAN

Fig. 5. Radioenzymatic assay of serotonm, related mdoles, and associated enzymes

98

Saavedra

against 5 n-&l sodium phosphate buffer, pH 7.9. After dialysis the enzyme is divided mto ahquots, lyophihzed, and stored at -20°C until used. A typical enzyme preparation has a protein concentration of about 15-20 mg/mL and an activity of about 350-400 U/mL, using N-acetylserotonm as substrate. One unit of enzymatic activity 1s defined as the amount of enzyme that forms 1 nmol of product/h (Axelrod and Welssbach, 1961; Saavedra et al , 1973) 51.2. Purrhcatlon of N-Acetyhansferase (IVAT, EC. 2.3.1.5) The enzyme source IS the rat liver (Welssbach et al , 1961). Tissues are homogenized with 2 vol of O.lM sodium phosphate buffer, pH 7.2, and centlfuged at high speed. The supernatant IS saturated with ammomum sulfate and the 40-65% precipitate 1s dlalyzed against 0.02M sodium phosphate buffer, pH 7.2 After dialysls, the precipitate formed IS centrifuged and discarded. The clear supernatant, after removal by suction of a lipid layer, 1s dlvlded mto allquots and stored at -20°C Before storage, ethylene glycol (10% v/v) 1s mixed with the supernatant; ethylenedlamme tetraacetlc acid (EDTA) and mercaptoethanol are added m concentratlons of 0.1 and 1 mh4, respectively. Addition of these preservatives 1s necessary for enzyme stability beyond one week If the preparation 1s not lyophllized. Lyophlllzatlon prior to storage increases enzyme stability and makes addition of preservatives unnecessary. Under these conditions, the enzyme activity IS sfable for several months. A typical enzyme preparation has a protern concentration of 90-100 mg/mL and an activity of about 700-900 U/mL, usmg tryptamme as a substrate (Saavedra et al., 1973). 5.2. Assay of N-Acetylserotonin This assay depends on the enzymatic transfer of the [3H]-methyl group of [3H]-methyl-SAME to the hydroxyl group of Nacetylserotonm by HIOMT The enzymatlcally formed t3H]methyl-melatonm is separated by extractlon mto a nonpolar orgame solvent (Fig. 6). The use of methyl [3H]-labeied SAME of high specific activity makes it possible to measure as little as 50 pg of N-acetylserotonm (Table 1). For the assay of N-acetylserotonm in the rat pineal, rats are kllled by decapltatlon, the pineals are immediately removed, homogemzed in 0.4 mL of ice-cold O.lN HCl, and centlfuged at high speed. Aliquots (50 pL) of the supernatant are transferred to assay tubes contammg 5 FL of O.lN NaOH, 200 PL of 0 2&I sodium phosphate buffer, pH 7 9, 5 FL of [3H]-methyl-SAME, and 10 PL

99

Radioenzymatic Micromethods for Brain Amlnes HOMOGENIZE TISSUES (0 1 N H’J t SEPARATE ALICIUOT FOR PROTEIN DETERMINATION t CENTRIFUGE t SEPARATE DUPLICATE ALIQUOTS OF SUPERNATANT t ADD N-ACETYLSEROTONIN INTERNAL STANDARD t INCUBATE WITH PINEAL HIOMT AND SH-METHYL SAME t STOP WITH BORATE BUFFER AND MELATONIN

t

EXTRACT INTO TOLUENE t EVAPORATE TOLUENE AT 80°C t ADD PHOSPHOR t COUNT 3H-MELATONIN FORMED

F% 6. Flow N-acetylserotonm

sheet

for

the

radioenzymatic

assay

of

(about 4 U) of HIOMT. The final volume of the reaction mixture is 270 kL. N-Acetylserotonm (200 pg) is added to another ahquot as an internal standard. Blanks are obtamed by replacing the tissue m the reaction mixture with 50 PL of O.lN HCl and range from 150 to 250 cpm (Saavedra et al , 1973, Brownstem et al., 1973) The mixture is incubated for 20 mm at 37°C and the reaction is stopped by the addition of 0.5 mL of 0.5M borate buffer, pH 10 The radioactive products are extracted into 6 mL of toluene by mixmg for 5 s with a Vortex mixer, and the mixture is centifuged. A 5 mL aliquot of the organic phase is transferred to a countmg vial and allowed to dry overnight at 80°C m a chromatography oven. A 1 mL portion of ethanol is added to the vials, followed by 10 mL of counting solution (Liquifluor, New England Nuclear Carp,) /L of toluene. The radioactivrty of the samples is determined by liquid scmtillation spectrometry (Fig 6). The identification of [3H]-melatonm, the product of the reaction, is made by thm layer chromatography on Eastman chromagram sheets coated with silica gel, 100 km thickness The solvents used are (a) chloroform methanol:acetic acid (93 7 l), (b) methyl acetate : 2-propanol ammonmm hydroxide, 10% (45 35:20), (c ) acetone .ammonmm hydroxide (99: 1), and (d) toluene*acetic acid. ethyl acetate water (80.40 20.1) All sheets are activated by heating for 30 mm at 90°C m an oven immediately prior to use. Aliquots of rat pmeal supernatant are assayed as described above Radioactive standards are prepared by incubating 50 pg of authentic N-acetylserotonm with 5 PL of [“HI-methyl SAME, 10 PL of HIOMT, 200 PL of 0.2M sodium phosphate

100

Saavedra

buffer, pH 7 9,5 FL of O.lN NaOH, and 50 )IL of O.OlN HCl for 20 min at 37°C. Blanks are prepared m the same way without the addition of N-acetylserotonin. After the enzymatic reaction, the toluene extracts are dried under vacuum at 40°C for 1 h, and the resldue IS taken up m 50 FL of ethanol with the addition of 2 pg of nonradloactlve melatonm as carrier, and spotted on the chromatography sheets. Other nonradloactlve 5-hydroxymdoles are cochromatographed on the same sheet. After development, the sheets are stained with Ehrllch’s reagent (1 g of p-dlmethylaminobenzaldehyde m 10 mL of concentrated HCl plus 90 mL of acetone). The sheets are then marked m 1 cm sections and placed m vials containing 2 mL of ethanol and 20 mL of phosphor, and the radloactlvlty 1s measured In the pmeal, at least 90% of the radioactive product extracted has the same Xf values as authentlc melatonin and as the radioactive peaks obtamed from the radioactive standards (Saavedra et al., 1973, Brownstein et al , 1973) Although N-acetylserotonm is the best substrate for HIOMT, other hydroxyindoles, such as serotonin, N-methylated denvatlves of serotonm and 5-hydroxymdoleacetlc acid can also be methylated (Axelrod and Welssbach, 1961). However, when serotonm, N-methylserotonm, bufotenme, and 5-hydroxymdoleacetlc Speclflclty

Substrate”

Amount,

ng 1

N-Acetylserotonm Serotonin

1 100

1 100 1 100

Bufotenme N-Methylserotonm 5-Hydroxytryptophol 5-Hydroxymdoleacetlc

TABLE 5 of N-Acetylserotonm

1 100

acid

qThe substrates were carrled values were 230 cpm

1 100 through

Assay

Actlvlty obtained m assay, cpm

Actlvlty from 4.5 pm01 (1 ng) of N-acetylserotonm, %

6,300 0 50 50 1,650 150 180 120 10,000 0 0

100 0 25 07 25 2.5 27 2 160 0 0

the procedure

as described

m the text Blank

RadioenzymatrcMlcromethods

for Brain Amlnes

101

acid are carried through the entire assay procedure m equimolar concentrations, they produce less than 1% of the activity of N-acetylserotonm (Table 5). The reaction is linear with time up to 20 mm The amount of [3H]-melatonin formed in the reaction is proportional to the amount of N-acetylserotonin added to the reaction mixture up to 2000 pg. The recoveries of N-acetylserotonm added to the pmeal gland supernatant fractions are 95-100% and vary within a 5% range for duplicate samples. Internal standards of N-acetylserotonin are used to correct for recoveries. Several precautions should be observed. The sensitivity of the assay relies on the low blanks, between 150 and 250 cpm. An evaporation step is necessary to remove [3H]-methanol produced enzymatically from [3H]-methyl-SAME (Saavedra et al., 1973). It is essential that HIOMT be dialyzed to reduce the amount of endogenous substrates. After dialysis, the enzyme is divided into aliquots and stored at -20°C. Freezing and thawing of the enzyme should be avoided Lyophilization prior to storage increases stability.

5.3, Assay of Serotonin The assay 1s based on the conversion of serotonm to [3H]-melatonm by a two-step reaction involving the N-acetylation of serotonin to form N-acetylserotonm, followed by the 0-methylation of N-acetylserotonin to form melatonm (Fig. 5). [3H]-Melatonm is isolated by organic extraction, and is the only radioactive product present m detectable amounts. Other compounds and enzymes m the serotonin pathway can be measured by this principle (Saavedra et al., 1973, Saavedra, 1976; 1977; 1983) (Figs. 5 and 7). For the assay of brain serotonin, hypothalamic nuclei weighing less than 1 mg are dissected as described (Elk-Nes and Brizzee, 1965, Palkovits, 1973; Schlumpf, 1973, Schlumpf et al , 1974) and homogenized m 25 PL of O.lN HCl at 4°C. A 5 PL sample of the homogenate is removed for protein determmation (Lowry et al., 1951) and the homogenates are centrifuged at high speed. A 10 FL portion of the supernatant is transferred to assay tubes contammg 10 IJ,L of a solution made with 10 parts of 0.2M sodium phosphate buffer (pH 7.9) and 1 1 parts of 1N NaOH (final pH 7.9) (solution A). The first incubation step is the formation of N-acetylserotonm from serotonin. The reaction is carried out at 37”C, and is started by the addition of 5 ~.LLof a mixture contammg equal proportions

102

Saavedra HOMOGENIZE TISSUEStO 1 N HCI) t SEPARATE ALIGUOT FOR PROTEIN DETERMINATION t CENTRIFUGE t SEPARATE DUPLICATE ALIOUOTS OF SUPERNATANT t ADO SEROTONIN INTERNAL STANDARD t INCUBATE WITH LIVER NAT, ADO ACEl-YL COENZYME A t INCUBATE WITH PINEAL HIOMT AND 3H-METHYL SAME i STOP WITH BORATE BUFFER AND MELATONIN t EXTRACT t EVAPORATE

INTO TOLUENE TOLUENE

AT BO”C

t ADO PHOSPHOR t COUNT FOR 3H-MELATONIN

Fig

7

Flow

FORMED

sheet for the radioenzymatic

assay of serotonin

of partially purified rat liver N-acetyltransferase and acetylcoenzyme A (AcCoA), 4 mg/mL, diluted in 0.1 mM HCl. For the conversion of N-acetylserotonm to [3H]-melatonm, the mcubation is continued after 20 mm with the addition of 5 PL of a mixture containing 1 5 parts of partially purified HIOMT, 1 part of [3H]-methyl-SAME, and 2 5 parts of 0.2&l sodium phosphate buffer, pH 7.9 The reaction is allowed to proceed for 10 min and then stopped by the addition of 0.5 mL of 0.5M borate buffer, pH 10, and 10 FL of a 1 mg/mL solution of melatonm in 25% ethanol. The radioactrve product formed, [3H]-melatonin, is extracted by a procedure identical to the one used for the assay of N-acetylserotonm. The identity of the product formed is verified with thm layer chromatography by a procedure similar to the one outlmed for the assay of N-acetylserotonin More than 90% of the radioactive product extracted is found to have the same Rf values as authentic melatonm (Saavedra et al., 1973) The product is also isographic with the product obtained after carrying authentic serotonin through the assay. Internal standards are prepared by addition of 1 ng of serotonm to allquots of homogenate. Tissue blanks are prepared by replacing AcCoA by 0.1 mM HCI. If any radioactivity occurs m these samples, rt should represent endogenous N-acetylserotonm present m tissues. Although the amount of N-acetylserotonm m pmeal gland is high, no appreciable N-acetylserotonin levels have been found m any of the rat brain areas examined (Brownstein et al., 1983, Saavedra, unpublished observations). Figure 7 represents a flow sheet for the serotonm assay

Radloenzymatrc

MIcromethods

for Brain Amrnes

103

5-Hydroxytryptophan can be decarboxylated to serotonm by the ammo acid decarboxylase present m the liver preparation used. The addition of 5-hydroxytryptophan to the incubation mixture results in a significant formation of radioactive melatonin (Table 6). This property can be advantageously used for the sensitive determination of amino acid decarboxylase activity m rat brain nuclei (Saavedra, 1976). The formation of melatonin from 5-hydroxytryptophan during the serotonm assay can be prevented by the addition of the decarboxylase inhibitor MK-486 (5 x 10P”M) to the mcubation medium (Table 6). As described, both the serotonin and the N-acetylserotonm assays are sufficiently sensitive for most practical purposes, including the determination of endogenous levels of serotonin m duplicates obtained from mdividual nuclei dissected from single rat brains (Table 3). If necessary, further sensitivity can be achieved by routmely performing a thin layer chromatographic separation of the [“HI-melatonin formed, followed by extraction into an organic solvent before quantitation by liquid scintillation counting

6. Catecholamines

and Derivatives

6.1. General Procedure for the Assay of Catecholamines This assay IS based on the use of the partially purified catechol O-methyl transferase (COMT) to transfer a [ HI-labeled methyl group from [“HI-methyl-SAME to the catecholammes to form radioactive O-methyl derivatives (Da Prada and Zurcker, 1976, Coyle and Henry, 1973) (Fig. 8) COMT methylates all three catecholammes (DA, NA, and A) to approximately equal degrees In some brain areas, DA and A are only 5% of the total catecholamines, NA being the predominant amme (Palkovits et al., 197413;Saavedra, 1982) (Table 3). In other areas, such as the caudate nucleus, NA represents only a small percent of the total catecholammes, DA being the predominant amine Thus, complete separation of the 0-methylated derivatives formed (methoxytyramine, normetanephrine, and metanephrme), leading to absence of cross-contammation, is one of the essential conditions of the assay when this is used to specifically assess the levels of each amme m all different brain areas The development of adequate chromatographic procedures for the separation of the 0-methylated catechols after mcubation with 13H]-SAME was an essential step (Da Prada and Zurcker, 1976).

Saavedra

104

Speclfrcrty

TABLE 6 of the Serotonm

Assay Complete system + MK-486”

Complete system

Supernatant HCl and serotonm, 1 ng HCI and 5-HTP, 1 ng HCl and serotonm, 1 ng and 5-HTP, 1 ng Whole brain Whole hypothalamus Nucleus arcuatus Medial forebram bundle >MK-486 (5

x

cpm over blank

Serotonin, n@% protein

cpm over blank

Serotonm,

No AcCoA 230 235 240

4250 2970 7145

-

4230 4295

-

205 190 200 205

530 1050 1855 2820

6 98 36 30

550 1075 1820 2890

6 98 36 30

w mg protein

10-4M) was added at the beglnnmg of the lncubatron

6.1.1. Punficatlon of Catechol 0-Methyltransferase

(COMT, EC.

2.1.1.7)

COMT IS purified from rat liver. Tissues are homogenized m 5 vol of lsotomc KC1 and centrifuged at high speed. The supernatant IS decanted through glass wool and its pH adjusted slowly to 5.3 with the use of 1N acetic acid. This IS followed by centrifugation for 10 min at high speed. The resulting supernatant IS adlusted to pH 6.8 by addrtion of 0.5M sodmm phosphate buffer, pH 7.0 The supernatant is saturated with ammonmm sulfate, and the 30-55% preclprtate IS extensively dialyzed against 1 mM Tns-HCl buffer, pH 7.4, containing 0.1 miI4 dlthlothreitol After dialysis, the enzyme IS centrifuged at high speed. The supernatant 1s divided mto ahquots and stored at -20°C after addition of 10 mM reduced glutathlone and 1 mM 0-benzylhydroxylamme HCl. The enzyme preparation has an approximate protein concentratron of lo-20 mg/mL and an activity of 400-800 UimL, using NA as substrate (Saavedra, 1983)

6.2, Assay of Dopamine, IYoradrenaline, and Adrenaline All three catecholamines are measured m duplicate samples of single rat brain nuclei (Table 3). Brain tissue from one rat IS homogenized in 50 p,L of 0 1N perchlorrc acid and 5 FL are removed for protein determmatron After centrlfugation at 50,OOOgfor 50

Rad/oenzymat/c

Fig. 8

Mlcromethods

for Bra/n AmInes

105

Radroenzymatlc assays for catecholammes

mm, a 30 FL all&rot of the supernatant IS transferred to incubation tubes. A 10 ~.LLportion of 0 1N perchlorrc acid IS added, followed by addition of 40 PL of the mcubatron mixture contammg (a) 0.1 mg of dithrothrertol; (b) 0 4 FL of 1M M&l,; (c) 3 PL of [3H]-methyl-SAME, and (d) 3 FL of partially purified COMT After incubation at 37°C for 30 mm, the reaction is stopped by the addition of 100 PL of a freshly prepared mixture contammg 80 JJ,Lof 1M borate buffer, pH 8.0, and 20 PL of carrier (0.5 mg each of 3-methoxytyramme, normetanephrine, and metanephrine in O.OlN HCl). After adding 50 FL of 1.5% sodium tetraphenylborate, the 0-methylated radioactive products are extracted into 2.5 mL of an organic solvent (3 parts of toluene and 2 parts of rsoamyl alcohol, v/v). After shaking for 10 mm m a mechanical shaker and separatmg the phases by centrifugation, the aqueous phase is frozen m an acetone-dry ice bath The organic phase IS decanted mto another tube contammg 100 FL of O.lN HCl, and the methoxylated catecholammes are extracted back into the acid phase with shaking. Following centrifugation, the aqueous phase IS frozen m an acetone-dry ice bath, and the organic phase is aspirated and discarded. The acid phase is washed once with 1 mL of the toluene-rsoamyl mixture, and the organic phase 1s discarded. Methanol (100 t.r,L) IS added to the acid phase, and the total amount of liqurd (200 ~.LL)IS spotted on silica gel TLC plates

106

Saavedra

(LQDF, Quanta Gram). The plate IS developed with a solvent system containing chloroform: ethanol: 70% ethylamine (80: 15: 10, v/v). The methylated catecholamines are localrzed under UV hght and extracted. For the assay of DA, the product, 3-methoxytyramme, is scraped mto counting vials contammg 1 mL of 0 05M ammonium hydroxide. After shaking for 20 mm m an automatrc shaker, 10 mL of Aquasol (New England Nuclear) are added and the radioactivity IS counted m a lrqurd scintrllation counter For the assay of NA and A, the radioactive products, normetanephrine and metanephrme, are scraped mto separate countmg vials containing 1 mL of 0.05M ammonium hydroxide After shaking for 15 s, 50 FL of 4% sodium perrodate solutron IS added, and the cleavage reaction 1s stopped 5 min later by the addition of 50 FL of 10% glycerol The content of the vials 1s acidified with 100 PL of 1N acetic acid, and the radioactive products extracted into 10 mL of toluene containing 400 PL of Lrqurfluor by shaking for 15 s. After separation of the phases, the radroactrvrty IS counted m a liquid scintillation counter. Internal standards consrst of 30 FL ahquots of bran-r tissue homogenate plus 10 PL of O.lN perchlorrc acid containing 0 l-l ng of each catecholamine Blanks consist of 40 PL of O.lN perchlorrc acid (nontissue blanks) or bran-r tissue homogenates and the incubation mixture, which are incubated separately and combmed after addition of the borate buffer. Figure 9 depicts a flow sheet for the catecholamme assay.

6.3. Radioenzymatic Assays for Catecholamine Derivatives Radroenzymatrc methods echolamine derrvatrves, catecholamme precursor normetanephrme. 6.3. I. Acid Metabolites

are used to determme a number of catsuch as several acid metabolrtes, the L-DOPA, and the NA metabohte

of Catechofamlnes

The catechol group of several acid metabolrtes of catecholammes IS susceptible to 0-methylatron by COMT (Axelrod and Tomchick, 1958) The enzymatic reaction used 1s similar to that employed for the catecholamme assay, including the use of hrgh specific activity [3H]-SAME, and rt 1s possible to determine both catecholammes and metabolrtes srmultaneously Most assays require a thm layer chromatography purrfrcatlon step for product rsolatron, similar to that used for catecholammes The major DA metabolrte, 3,4-drhydroxyphenylacetrc acid (DOPAC), when methylated by COMT and [3H]-SAME results m

RadioenzymatlcMicromethods HOMOGENIZE

TISSUES

t SEPARATE

ALICIUOT

107

for Bfaln Amlnes (0 1 N PCA)

FOR PROTEIN

DETERMINATION

ALIQUOTS

OF SUPERNATANT

INTERNAL

STANDARDS

t CENTRIFUGE t SEPARATE ADD

DUPLICATE

t CATECHOLAMINE

t INCUBATE

WITH

t STOP WITH t EXTRACT t

BACK

DTT

BORATE

EGTA. BUFFER

INTO TOLUENE

EXTRACT

MgCI,,

COMT

AND

3H METHYL

METHOXYCATECHOLAMINE

ISOAMYL

ALCOHOL

SAME CARRIERS,

AND

TPB

(3 21

INTO 0 1 N HCI

t WASH

WITH

ADD

t METHANOL

TOLUENE

ISOAMYL

ALCOHOL

t SPOT

IN SILICA

t DEVELOP

GEL TLC PLATE

IN CHLOROFORM/

VISUALIZE

SPOTS

ETHANOL/ETHYLAMINE

70%

(12 3 21

BY U V LIGHT

t

t

NE

DA t SCRAPE t EXTRACT

c

OR

E

t INTO COUNTING WITH

AMMONIUM

SCRAPE

VIALS

SILICA

t EXTRACT HYDROXIDE

HYDROXIDE

t ADDAQUASOL

CLEAVE

INTO COUNTING

PRODUCTS

WITH

VIALS

AMMONIUM

t

WITH NA PERIODATE I STOP WITH GLYCEROL

t COUNT

ACIDIFY

t

t EXTRACT

t

WITH

ACETIC

ACID

INTO TOLUENE

ADDPHOSPHOR t COUNT

Fig. cholammes

9.

Flow

sheet

for

the

radloenzymatlc

assay

of

cate-

108

Saavedra

the formation of [‘H-methyl]-homovamllic acid (HVA). [3H-methyl]-HVA can be separated by thm layer chromatography, a procedure necessary when analyzing tissues containing relatively large amounts of NA, since the presence of the NA metabohte 3,4-dihydroxyphenylethyleneglycol (DOPEG) results m formation of 3-[3H-methoxy]-4-hydroxyphenylethyleneglycol (MOPEG) (Kebabian et al , 1977). In this case, both DOPAC and DOPEG can be analyzed m the same sample (Kebabian et al., 1977). A similar assay for DOPEG was recently developed. This method is useful for the determination of free and conlugated forms of DOPEG, and for their analysis m human fluids (Dennis and Scatton, 1982). A simplified procedure for purification of [3H-methyl]-HVA after enzymatic methylation involves the use of an ion-exchange resin (Dowex AG 50 W x 4, 100 x 200 mesh, H’ form) followed by solvent partition between an aqueous phase at pH 7.0 and ethyl acetate (Kebabian et al., 1977) This simple extraction procedure is sufficiently specific to allow measurement of endogenous DOPAC m brain areas such as the caudate nucleus, where DA represents most of the catecholamme present Several groups (Saller and Zigmond, 1978, Fekete et al., 1978) have reported methods for the measurement of all three catecholammes and several of their acid metabohtes, notably DOPAC and DOPEG, m the same sample Separation of [3H-methyl] derivatives by thm layer chromatography is always necessary. Other catecholamme acid derivatives, such as dihydroxymandelic acid (DOMA), can also be methylated by COMT and can be measured by radioenzymatic methods including thm layer chromatography (Saller and Zigmond, 1978, Vlachakis et al , 1979). There are several limitations to the study of catecholamme acid derivatives by radioenzymatic techniques. The sensitivity of the methods, although high (20-200 pg), is not sufficient for their determination m small tissue samples, with the exception of DOPAC levels m DA-rich brain areas (Kebabian et al , 1977) In addition, not all important catecholamme acid derivatives can be determined. Unfortunately, 0-methylated catecholamme derivatives such as MOPEG are not substrates for 0- or N-methylation and cannot be determined by the methylatmg radioenzymatic techniques

6.3.2 L-DOPA Heft1 and Lichtensteiger method for the quantitation

(1976) first reported a radioenzymatic of levodopa (L-DOPA) m small brain

Radioenzymatic

Micromethods

for Bra/n Amines

109

areas. This complicated method involved extraction of the 3-O-[3H]-methyl-DOPA by Ion exchange chromatography, adsorption on activated charcoal, elutron wrth phenol, and ionexchange chromatography (Heft1 and Lrchtensteiger, 1976). A somewhat simpler technique was developed by Zurcher and Da Prada (1979) and mvolves transformatron of 3-Oj3H]-methylDOPA to its 2,4-dmitrofluorobenzene (DNFB) derlvatlve before product purrfrcatlon A more sample technique (Argrolas and Gessa, 1981) involves purrfrcatron of 3-0-[“H-methyl]-DOPA by Sephadex GlO and Dowex 50W x 4 ion-exchange chromatography. These methods allow the determmatron of L-DOPA m small rat brain areas and can also be used to study L-DOPA accumulation after decarboxylase inhibition 6.3.3. /Yormetanephrine

The NA metabohte normetanephrme (NMN) can be quantrtated m plasma and in tissues by N-methylatlon with PNMT and 13H]-SAME and separation of the [3H]-metanephrine by thin layer chromatography (Vlachakis and De Quattro, 1977, Kobayashr et al , 1980).

7. HISTAMINE 7.1. General Procedure for the Histamine Assay Hrstamme is assayed by use of a modrfrcatron of the enzymatic isotopic method of Snyder et al. (1966). The assay depends on the transfer of the methyl group from [-7H]-methyl-SAME to HA by histamine N-methyltransferase (HMT), a specific lmrdazole Nmethyltransferase (Brown et al., 1959a; 1959b) (Fig. 10). 7.1.1, Punfka 2.1.1.8).

tion of Histamine

N -Methyltransferase

(HM T, E. C.

The enzyme source is the guinea pig brain The tissue IS homogenized m 10 vol of 0 25M sucrose, and this homogenate IS centrrfuged at high speed Ammonium sulfate IS added to the supernatant, and the 45-70% precipitate IS extensively dialyzed against 0 1M sodlum phosphate buffer, pH 7.4 The dialyzed preparation IS centrifuged at high speed, and the supernatant drvrded mto small allquots and stored at -20°C The enzyme preparation has a protein concentration of about 10 to 20 mg/mL, and an activrty of about 300-600 UlmL, using HA as substrate One unit of enzymatic actlvrty IS defined as the amount of enzyme that forms 1 nanomol of product (Taylor and Snyder, 1971; 1972).

Saavedra

110 ,CH

CH NH I HI SAME

/=-/ HISTAMINE

*

N METHYLTRANSFERASE

1‘HI METHYLHISTAMINE

HISTAMINE

Fig

10

Radloenzymatrc

assay for histamine

7.2. Assay of Histamine Rat brain nuclei are homogenized in 25 FL of a solution containing 10 parts of 0.2M sodium phosphate buffer, pH 7.9, and 1.1 parts of 1N NaOH (Solutron A). A 5 PL sample of the homogenate is removed for determination of protein and the rest 1s centrrfuged at hrgh speed. After centrifugation, 10 FL of the supernatant are removed and transferred to assay tubes on ice. A 10 FL portion of O.lN HCl IS immediately added to each tube to neutralize the NaOH. The final pH of the reaction should be 7.9 Blanks are prepared by adding 10 PL of O.lN HCl instead of tissue supernatant. Standard solutrons of HA are prepared m O.lN HCl immediately before use and added to duplicate tubes containing tissue extracts and solution A. The reaction IS inmated by the addition of 10 FL of a mixture containing 0.5 FL of 0.5M sodium phosphate buffer, pH 8.0, 7.5 PL of partrally purified HMT, and 2 FL of [3H]-methyl-SAME. Buffer and enzyme should be mixed together before the addition of SAME, since the methyl donor is preserved in a sulfuric acid solutron and sudden media acidlficatlon could result in loss of enzyme activity. The tubes are incubated at 37°C for 30 min. The reaction is terminated by the addition of 25 PL of 0 4N perchloric acid containing 25 kg of N-methylhistamme. To each tube, 0 5 mL of 3N NaOH is added, followed by 6 mL of chloroform. The product, [3H]-methylhistamine, is extracted mto the chloroform phase by shaking the tubes m an automatic shaker for 5 mm. The tubes are centrifuged at low speed, the aqueous phase is aspirated and discarded, and the organic phase washed with 1 mL of 1N NaOH. After shaking and centrifugation, the water phase IS aspirated and 5 mL of the organic phase are transferred to counting vials. The chloroform IS evaporated to dryness at room temperature under a stream of air, 1 mL of ethanol plus 10 mL of counting solution are added, and the radloactrvlty IS estimated by hquld scmtrl-

RadIoenzymatic

Micromethods

for Brain Amines

111

HOMOGENIZE TISSUES Itim phosphete buffer and NaOHJ t SEPARATE ALICIUOT FOR PROTEIN DETERMINATION t CENTRIFUGE t SEPARATE DUPLICATE ALIDIJOTS OF SUPERNATANT t ADD HISTAMINE INTERNAL STANDARDS t CHANGE pH T O 7 9 WITH HCI t INCUBATE WITH HMT AND +I METHYL- SAME t STOP WITH NaOH AND MFTHYLHISTAMINE CARRIER t EXTRACT WITH CHLOROFORM t ASPIRATE WATER PHASE t WASH WITH NaOH t DRY ORGANIC PHASE WITH AIR t COUNT

Fig 11

Flow

sheet for the

radroenzymatlc assay of histamine.

lation counting. Figure 11 is a flow sheet for the HA assay Several precautrons should be taken when performing the HA assay. Hrstamme standards must be prepared m scrupulously clean glassware or u-r polypropylene tubes to avoid losses. Failure to add the methylhrstamine carrier results m variable recovery of the labeled product Extraction of small amounts of HA from trssues is improved by use of strongly alkaline solutions. Care must be taken to evaporate the chloroform just to dryness and to dry all the tubes evenly For this purpose a lOO-outlet manifold was constructed, designed to fit over a tray of 100 counting vials. Uneven or protracted drying results in variable recovery of radiolabeled product. Drying at elevated temperatures, e.g., m a chromatography oven at BO”C, results in complete loss of methylhistamine. The product from tissue extracts is rsographic with radioactive, enzymatically synthesized methylhrstamine and with nonradioactive methylhrstamine (Taylor and Snyder, 1971, 1972). The amount of product formed bears a linear relationship to the amount of HA present u-r the sample up to 2 ng. Histamine content can be determined m duplicate samples from isolated brain nuclei (Correa and Saavedra, 1981; 1983) (Table 3). Hrstidine extracted from brain could theoretrcally undergo decarboxylation to yield HA in basic solutrons. However, a solution containing 100 ng/mL of hrstidme contained no measurable HA by our technique, even after storage for 24 h at 4°C (Brownstein et al., 1974).

112

Saavedra

8. Phenylethanolamine, f3-Phenylethylamine, Octopamine, and Tyramine 8.1. General Procedure for the Assay The biogenic ammes I’E, PEOH, TA, and OA, are present m the mammalian brain m exceedingly low quantmes (Durden et al , 1973, Philips et al , 1974, 1975, Boulton, 1979, Saavedra, 1974a, 1974b, 1984, Saavedra and Axelrod, 1973, Saavedra et al., 1974~) For this reason they were first called “minor,” and later, “trace” ammes Such denommations are not entirely adequate, for a number of reasons First, although their steady state levels m mammalian brain are low, their turnover rates are high, and much higher than those of catecholammes (Boulton, 1979, Molmoff and Axelrod, 1972, Tallman et al , 1976). Second, admmistration of centrally active drugs such as monoamine oxidase mhibitors often produces malor alterations m their levels, of a magnitude higher than changes m catechol- or mdoleamme systems (Saavedra, 1974a; Saavedra and Axelrod, 1973, Tallman et al , 1976) Third, “trace” ammes are only “trace” m mammalian brain, higher levels are present m peripheral tissues of mammals, and certainly very high levels occur m the brains of animals from lower species, such as invertebrates. In these animals, “trace” ammes should rather be considered as neurotransmitters m thenown right (Boulton, 1979, Saavedra, 1984) Quantitation of these amines m mammalian brain requires very sensitive and specific methods Of the techniques available, only the mass spectrometric and radioenzymatic techniques offer the sensitivity and specificity required for studies m relatively disCrete (whole hypothalamus, for example) areas of rat brain The radioenzymatic assays for “trace” ammes are based on the N-methylation of the B-hydroxylated amines by PNMT, preceded when necessary by B-hydroxylation of non-B-hydroxylated compounds with dopamme-B-hydroxylase (DBH) (Fig 12) 8 I I Purification of Phenylethanolamlne N-Methyltransferase (PlYMT, EC. 2.1.1.28). PNMT is obtained from bovine adrenal medullas Fresh tissues are homogenized with 4 vol of 0 25M sucrose, and the homogenate centrifuged at low speed The supernatant is filtered through gauze to remove cell debris and lipids and 1s then centrifuged at high speed Ammonmm sulfate is added to the clear supernatant, and the 30-60% precipitate is extensrvely dialyzed against 0.05M potassnun phosphate buffer, pH 7 4 (Axelrod,

RadIoenzymatIc

MIcromethods

113

for Bra/n Amines

l

0

-CH,-CH,-NH,

-$

@-!:-C”,.,

$

Q-i::

-Cy-:

0

/WHENYLETHYLAMINE

PHENYLETHANOlAMlNE

N-METHYL PHENYLEWIANOLAMINE

PNMT hII-SAME

DSH

.

0

“O 0

-CH,-CH,-NH,

,.($:,,,

TYRAMINE

F%

,.@-!:-CH2-?

OCTOPAMINE

12

phenylethanolamme,

Radloenzymatlc tyramme,

assay for and octopamme

N-METHYL

OCTOPAMINE

B-phenylethylamme,

1962b, 1972) The dialyzed preparation is adjusted to pH 5.5 by a dropwrse addmon of 1N acetic acid, and centrifuged at high speed The supernatant IS adjusted to pH 7.4 with 1N NaOH, and extensively dialyzed against 0.05M potassmm phosphate buffer, pH 7.4 The final protem concentration IS about 8-10 mg/mL, wrth an enzymatic activity of about 500-800 UimL with octopamme as substrate (Axelrod, 1962; 1972, Saavedra et al., 1974e). 8.1.2

Purification

of Dopamme-P-Hydroxylase

(DBH,

E.C.

1.14 17.1). Chromaffm granules are Isolated from fresh bovine adrenals (Smith and Winkler, 1967) and lysed by freeze-thawing followed by hand homogemzatlon m a Teflon glass homogenizer in 4 m&l Trrs-HCl buffer, pH 7.5. The homogenates are centrifuged at high speed, and DBH in the supernatant is separated from contaminating proteins by means of affmrty chromatography on a concanavalm A-sepharose column (Pharmacla Fme Chemicals, Piscataway, NJ) (Tallman et al., 1976).

8.2. Assay of Phenyfethanolamine EndoFenous PEOH can be quantitated by incubation with PNMT and [ HImethyl-SAME (Saavedra and Axelrod, 1973). The routme appllcatron of a thm layer chromatographrc step to further purify the radroactlve product formed is necessary for adequate specrfrcrty. The method could be used for the simultaneous determmatron of PEOH and OA (Saavedra, 197413) (Fig. 13, Table 7). To measure PEOH, rats are killed by decaprtatlon, the organs

40 1 54

840 408 1463

blank

26 472 28 8

%k

Apparent

and

80 70 70

5% of apparent

20 8 331 20 2

“&

Authentic

Phenvlethanolamme

Octopamme

TABLE 7 of Octopamme

692 362 1205

cpm over blank

Phenylethanolamme

20 489 18

“&

‘Tissues were processed after homogemzatlon m 1 mL of ice-cold 0 02M Tns-HCI buffer, pH 8 6, contammg lpromazld, 1 x 10-'M Blank values were 180 cpm for OA and 92 cpm for PEOH Authentic ammes added to tissue supernatants gave 400&4700 cpm (OA) and 3000-3800 cpm (PEOH)ing

gestation)

Rat hypothalamus” Rat pmeal gland Fetal rat brain (16 d

mg

cpm over

Determmatlon

Weight,

Simultaneous

115 rapidly removed, frozen on dry ice, weighed, and homogenized in 5-10 vol. of ice-cold 20 mM Tris-HCl buffer, pH 8 6, contammg the monoamme oxidase inhibitor iproniazid (50 bg/mL). The homogenates are heated at 90°C for 3 mm and centrifuged at high speed m a refrigerated centrifuge. A 200 WL aliquot of the supernatant fluid is transferred to an assay tube and incubated for 20 min at 37°C after addition of a mixture containing 10 PL of partially purified PNMT, 5 PL of [3H]methyl-SAME and 35 FL of 20 mM Tris-HCl buffer, pH 8.6. A 1 ng portion of PEOH is added to another aliquot as an internal standard. The mcubation IS stopped by addition of 0.5 mL of 0.5M borate buffer, pH 10, and the radioactive product IS extracted with 6 mL of a mixture containing 95% heptane and 5% isoamyl alcohol, v/v, by shaking for 5 mm in a mechanical shaker. After centrifugation, 5 mL of the organic phase is transferred to counting vials and evaporated to dryness under reduced pressure at 40°C. A 1 mL portion of ethanol and 10 mL of counting solution are added, and the radioactivity is measured by liquid scmtillation spectrometry (Fig 13) Normally occurrmg B-hydroxylated compounds, such as metanephrine, OA, catecholamines, and other phenylethylamines (PE and TA) give negligible interference (less than 3%) when present at 100 times the concentration of PEOH. Identification of [3H]-methylphenylethanolamine in tissues is made by means of TLC on precoated Eastman chromagram sheets (Saavedra and Axelrod, 1973).

8.3. Assay of ,B-Phenylethylamine B-Phenylethylamine is quantitated by a procedure similar to the one used for PEOH, after conversion to PEOH by B-hydroxylation with DBH (Saavedra, 1974a) (Fig. 14). To measure PE, rats are killed by decapitation, and their organs are rapidly removed, frozen on dry ice, weighed while still frozen and homogenized in 4 vol of ice-cold O.lN HCl containing pargylme (50 kg/mL). After high speed centrifugation in a refrigerated centrifuge, the supernatant IS divided mto 2 mL aliquots and transferred to assay tubes. To one of the aliquots, 10 ng of authentic PE are added as internal standard. The pH is adjusted to 11-11.5 with 1N NaOH, and the aqueous phase is extracted with 8 mL of toluene by shaking for 10 min in a mechanical shaker After centrifugation, 6 mL of the organic phase are transferred to a centrifuge tube containing 300 PL of O.lN HCl. The tubes are shaken for 10 min, and after centrifugation, the organic phase is carefully aspirated and discarded. The tubes are

116

Saavedra HOMOGENIZE t HEAT

IN BUFFER

AT 95OC

t CENTRIFUGE t INCUBATE t STOP

SUPERNATANT

WITH

t EXTRACT

BORATE

WITH

PNMT

AND

3H-SAME

BUFFER

INTO ISOAMYLALCOHOL-TOLUENE

MIX

t DRY t SEPARATE

BY THIN

LAYER

CHROMATOGRAPHY

(optlord)

t COUNT

Fig 13 Flow sheet for the radloenzymatlc ethanolamme and octopamme

assay

of phenyl-

placed m a vacuum desiccator at room temperature for 20 mm m order to remove traces of toluene, and 200 FL of the HCl are transferred to new assay tubes After addrtron of 20 PL of 1N NaOH, the centrifuge tubes are mcubated at 37°C after the addition of (a) 50 PL of a mixture A, freshly prepared, contammg 5 FL of 0.24214 ascorbic acid, pH 5.5, 25 PL of 0.5M sodmm fumarate, pH 5.5, 10 PL of 0 012M pargylme, 2 PL (or 1500 U) of catalase, and 8 FL of l.OM Tns-acetate buffer, pH 5.5, (b) 50 PL of partially purrfred DBH. After 60 min of mcubatron, 100 FL of a mixture B, contammg 70 FL of 1M Trrs-HCl, pH 8 6, 5 FL of [3H]-methylSAME, 5 PL of drstrlled water, and 20 PL of partially HOMOGENIZE IN HCI t CENTRIFUGE + EXTRACT SUPERNATANT IN ORGANIC SOLVENT t BACK EXTRACT INTO HCI t EVAPORATE TRACES OF ORGANIC SOLVENT t INCUBATE WITH OSH t INCUBATE WITH PNMT AND 3H-SAME t STOP WITH BORATE BUFFER t EXTRACT INTO TOLUENE-ISOAMLYLALCOHOL MIXTURE t DRY ORGANIC SOLVENT I SEPARATE BY THIN LAYER CHROMATOGRAPHY loptmall

i

COUNT

Fig 14. Flow sheet for the radloenzymatlc ethylamme and tyramme

assay of P-phenyl-

RadloenzymatlcM1cromethod.s

for Brain Amlnes

117

purified PNMT are added, and the mcubatron IS continued for 30 min Blanks consist of the same incubatron mixture, replacing the DBH by 50 FL of drstrlled water. The reaction 1sstopped with 0.5 mL of 0 5M borate buffer, pH 10, and the product extracted with 6 mL of a mixture containing 97% toluene and 3% isoamyl alcohol 4 mL of the organic phase are (v/v). After centrrfugation, transferred to counting vials and dried under vacuum for 1 h at 40°C After drying, the actrvrty is measured by liquid scintrllatron spectroscopy. (Fig 14). The identification of the [3H]-methylated product formed m the reaction 1smade by using TLC, m a manner similar to the one used for the assay of PEOH (Saavedra, 1974a) The assay for PE 1s reasonably easy to perform, fast, mexpensive and very sensitive. The double enzymatrc procedure allows trssue blanks to be used and endows the assay with marked specificity. The presence of endogenous PEOH IS corrected for by measurement of “blank’ values when DBH IS omitted from the incubation medium. The use of the present assay for the simultaneous determmatron of PE and PEOH, however, is not recommended due to the low extraction of the latter amme from strongly alkaline solutions.

8.4. Assay of Octopamine The enzymatic method for OA 1s based upon the N-methylation of octopamine by the use of a partially purified preparation of PNMT in the presence of [3H]-methyl-SAME (Molmoff et al , 1969; Saavedra, 1974b) (Fig. 12). The radioactive products formed, N-methyland NJ-drmethyloctopamme (synephrine and N-methylsynephrme) are isolated and identified by solvent extraction, evaporation procedures to remove volatrle contaminants, and TLC The routine separation of the radioactive synephrines formed m the reaction by a TLC step provides a necessary measure of specificity (Saavedra 1974b) (Fig 13). This assay can also be adapted for the simultaneous measurement of both OA and PEOH m the same sample (Saavedra, 1974b) (Table 7). Rat organs are dissected m the cold, immediately frozen on dry ice, weighed while still frozen and homogenized m 25 vol. of ice-cold 0.02M Trrs-HCl buffer, 8.6, contammg the monoamme oxidase inhibitor iproniazid, 10e3M. The homogenates are heated in a water bath at 90°C for 3 min The proteins are removed by centrifugatron and 200 ~.LLaliquots of the clear supernatant are mcubated m assay tubes at 37°C for 20 min, after addition of a mixture contammg 10 PL of partrally purrfred PNMT, 5 PL of [3H]-methyl-SAME, and 35 PL of 0.02M Trrs-HCl buffer, pH 8 6

118

Saavedra

Blanks are prepared by replacing the tissue with 200 FL of buffer. A 1 ng portion of octopamme is added to another aliquot as an internal standard. The reaction is stopped by the addition of 0 5 mL of 0 5M borate buffer, pH 10 A 10 pg portion of authentic synephrine (N-methyloctopamine, 1 mg/mL m distilled water) is added as a carrier, and the products are extracted mto 6 mL of a mixture contammg 3 parts toluene to 2 parts isoamyl alcohol, v/v, by shaking m a mechanical shaker for 10 mm. After centrifugation, the organic phase is quantitatively transferred to tubes containing 1 mL of 0.5M borate buffer, pH 10, and the tubes are shaken and centrifuged. A 4 mL alrquot of the organic phase is transferred to counting vials contammg 2 mL of tolueneisoamyl alcohol mixture, and the contents are dried overnight m a chromatography oven at 80°C The radioactivity is determined by liquid scintillation spectroscopy Identification of radioactive synephrme and N-methylsynephrme is made by TLC on precoated Eastman chromatogram sheets. (Saavedra, 1974b). The percentage of authentic OA in the tissue is determined as the fraction of radioactivity isographic with synephrine and N-methylsynephrine (Saavedra, 1974b, Molmoff et al , 1969). The simultaneous determination of PEOH and OA m the same sample is performed as follows (Table 7 and Fig. 13) the tissue supernatants are divided and processed as described above, with the exception of another allquot that is carried through the procedure with the addition of 1 ng I’EOH as internal standard. After stopping the reaction as described above, the [3H]-methylphenylethanolamine is separated from the [3H]-methyl-octopamine by extraction with 6 mL of heptane containing 5% isoamyl alcohol, v/v. A 4 mL aliquot of the organic mixture IS transferred to counting vials and dried at 40°C under vacuum for 1 h The remainmg heptane*isoamylalcohol mrxture is carefully aspirated, the [3H]-methyl-octopamme IS extracted with 6 mL of toluene:isoamyl alcohol (3 : 2, v/v) and this solution is processed as in the OA assay.

8.5. Assay of Tyramine Tyramme is quantitated by a procedure similar to the one used for OA, after being converted to OA by DBH (Figs 12 and 14) This method depends on the B-hydroxylation of TA by DBH followed by the transfer of a [3H]-methyl group to the N-terminal of OA by PNMT; t3H]-synephrine is extracted mto an organic solvent in a manner similar to that employed for the OA assay, and the radioactivity is determmed (Saavedra, 197413, Tallman et al,, 1976). The

Radioenzymatic

MIcromethods

for Brain Amlnes

119

apphcation of a TLC step to all samples IS necessary for specrfrcrty (Tallman et al., 1976) (Fig. 14, Table 8). To measure TA, rats are decapitated and their organs rapidly removed and Immediately frozen on dry ice Tissues are werghed while still frozen and homogenized in 5-10 vol. of Ice-cold O.liV HCl. After centrrfugation, the supernatant IS dlvlded mto 1 mL aliquots and placed m assay tubes. To one of the samples, 10 ng of authentic TA are added as an internal standard. The mrxture IS neutralized with 100 FL of 1.ON NaOH and 0.5 mL of 1M Tris-HCl buffer, pH 10.0 The aqueous phase 1s extracted with 7mL of methyl acetate. After centnfugatlon, 6.0 mL of the aqueous phase are transferred to a tube containing 0.5 mL of 0 1N HCl. The tubes are shaken, centrrfuged, and the orgamc phase IS asprrated. The water phase is washed with 4 mL of toluene to remove traces of methyl acetate and the tubes containing HCl are placed m a vacuum desiccator at room temperature for 1 h. Tubes could be frozen overnight. A 300 PL alrquot of this solutron IS transferred to a new tube and neutralrzed with 30 PL of l.ON NaOH The tubes are incubated for 1 h at 37°C after the addrtion of a solution containing 6 FL of 0.3M ascorbrc acid, 25 ~J,Lof 0.5M sodium fumarate, pH 5.5, 3 PL (200 U) of catalase, 16 PL of 1M Trrs-actetate buffer, pH 5.5, and 50 PL of DBH Blanks either contam water mstead of DBH or are not incubated for the l-h period After this incubation, 100 PL of a mixture contammg 85 ~J,Lof 1M Trrs-HCl buffer,

pH 8 6,lO

p,L of PNMT

and 5 p,L of [3H]-methyl-SAME

are

TABLE 8 Speclflcity Amme Tyramine” p-Phenylethylamme Phenylethanolamme Octopamme Noradrenalme Dopamme Serotonin Tryptamme Normetaneuhrme

of Tyramme

DBH omltted kpm)

620 660 2320 4250 660 570 480 560 680

Assay

Complete system (cpm)

24710 630 2410 4160 630 590 460 610 620

Difference (cum)

24090 0 90 0 0 20 0 50 0

“TA (10 ng), OA (10 ng), PEOH (10 ng), and other ammes (100 ng) were added to 1 0 mL of 0 1N HCl and carrled through the entlre procedure.

120

Saavedra

added and the mcubatron 1s contmued for another 25 mm The reaction IS stopped with 0.5 mL of 0.5M borate buffer, pH 10.0, contaming 10 t.~g of nonradloactrve synephrme as carrrer, and the product IS extracted mto 6 mL of toluene * rsoamyl alcohol (3 2, v/v). The rest of the extractlon IS identical to the one used for the assay of OA Several precautions should be taken when performing the TA assay The tissue supernatant must be extracted mto methyl acetate at pH 10.0, the use of either a higher or lower pH markedly reduces the recovery of TA The use of toluene to remove traces of methyl acetate from the HCl phase 1s essential Toluene IS easrly removed by vacuum, methyl acetate IS not, and traces of this organic solvent, rf present, will drastically reduce the enzymatic actlvlty. The punfled DBH and PNMT are stored at -15°C m small alrquots and remam stable for several months, repeated freezing and thawing of either enzyme should be avoided 13H]-methylSAME, once thawed, should be kept at 5°C and not refrozen TISsue blanks are necessary since B-hydroxylated amines such as OA and PEOH are extracted to a minor extent and would otherwise lead to high values for TA. A 50-55% recovery 1s found for bran-r TA, losses should be corrected by the use of mternal standards. The additional borate buffer wash and final drying procedure at 80°C are necessary to remove various volatrle and soluble contammants that would increase the blank values. 13H]-Synephrme IS identified by TLC (Tallman et al., 1976). The enzymatrc-radloisotoprc assay has been modrfred by a dansylatlon procedure and three sucesslve TLC separations to isolate the n~eta- and para-OA isomers (Danielson et al., 1977). The orfho isomer, however, can not be separated by this technique, which does not consider the formatron of N-methylsynephrme derrvatlves (Molmoff et al , 1969), and might therefore be not specific for determmatlon of posmon isomers

9. Assays for Enzymes of Synthesis and Degradation of Biogenic Amines The general principle outlmed here applies to the quantrtatron of the actrvlty of enzymes related to blogenlc ammes. The sensltlvrtres of the methods are very high, at the femtomole level, allowmg the quantltatlon of enzyme actrvrty n-r mdrvrdual rat bram nuclei Tissues are homogenized m appropriate buffers, essentral cofactors are added as required, and the enzymes are quantitated after mcubatron with exogenous substrates. Extraction proce-

Rad~oenzymatlcMlcromethods

for Bra/n Amrnes

121

dures for the product formed m the reactions are similar to those outlined for the assays of biogemc ammes. Quantitation of HIOMT, NAT, tryptophan hydroxylase, histidme decarboxylase, HMT, COMT, PNMT, tyrosme hydroxylase, L-ammo acid decarboxylase, and monoamine oxidase are performed with sensitiveties at the femtomole level (Deguchi and Axelrod, 1972; Brownstem et al., 1975, Saavedra et al., 1974a, 197413, 1975, Saavedra and Zivm, 1976; Taylor and Snyder, 1972).

10. Conclusions Radioenzymatic micromethods for biogenic amines have been developed over a period of 25 yr, starting with the discovery of the methylating enzymes (Axelrod and WeIssbach, 1961, Axelrod, 1962a; 1962b; Brown et al., 1959a, 1959b, Weissbach et al , 1961) and the first attempt of their use to measure biogenic amines by the principle of radioactive methyl group incorporation (Snyder et al., 1966). In the process, sensitivity was increased by three orders of magnitude, specificity was greatly improved, and cost was substantially reduced. The radioenzymatic techniques are now standard procedures m many laboratories around the world. Several important studies have been possible with the use of these techniques The steady-state concentrations of biogenlc amines and the activity of their related enzymes have been mapped in specific brain nuclei throughout the rat brain and in individual rat pituitary lobes (Brownstein et al., 1975, Palkovits et al., 1974a; 197413; Saavedra, 1976; 1977; Saavedra et al , 1976a, 1974a; 197613, 197413, 1976~; 1974d; 1974e, 1975; Saavedra and Zivm, 1976). Physiological and pharmacological experiments demonstrated great anatomical selectivity in changes m biogenic amine levels and turnover, as well as in enzyme activities, for each biogemc amme system. Brain areas previously considered to be homogeneous in their amine content have now been showed to be highly heterogenous (Saavedra et al., 1976~). Reciprocal influences between aminergic systems occur in selected brain areas (Saavedra et al., 1976~). The metabolism of biogemc ammes is altered m selected brain areas m animal models of human disease, indicating a participation of specific biogenic amme systems. These findings are relevant for the study of cardiovascular diseases (Correa and Saavedra, 1981; Saavedra et al., 1978, Saavedra and Alexander, 1983), alterations m fluid and electrolyte regulation (Correa and Saavedra, 1983), regulation of the stress

response (Kvetnansky et al., 1978, Saavedra et al., 1979;Saavedra

122

Saavedra

and Torda, 1980), endocrine abnormalities (Chevlllard et al., 1981a; 1981b), and depression and other psychotic disorders. The “trace” ammes (PE, PEOH, OA and TA) have been unequivocally demonstrated m the mammalian brain (Boulton, 1979; Molmoff and Axelrod, 1972; Saavedra, 1974a, Saavedra and Axelrod, 1973; Saavedra et al., 1974c, Tallman et al , 1976). Their levels and turnover (Molmoff and Axelrod, 1972) can now be easily studied under a number of physiological condltlons and pharmacological treatments. Future developments should be expected m radloenzymatlc procedures The methods could be highly automatized, with reduced cost and easy performance. Their sensltivlty could be further increased with the use of highly purified, high speclflc actlvity [3H]-methyl-SAME and additional separation techniques. Of these, high performance liquid chromatography could provide the most reliable, easy and sensitive method for separation of multiple radloactive substances after methylatlon. The combmatlon of radloenzymatic methods and HPLC has not been exploited in the past, but it IS hoped that interest in such a combination approach will increase in the future

References Arglolas, A. and Gessa, G L (1981) A simple radloenzymatlc method to measure picogram amounts of DOPA m brain and blologlcal fluids 1 Neurochem 36, 290-292 Axelrod J (1962a) The enzymatic N-methylatlon of serotonm and other ammes ] Pharmacol Exp Ther 138, 28-33 Axelrod J. (1962b) Purlflcatlon and properties of phenylethanolamme-Nmethyl transferase ] Brol Cheln 237, 1657-1660 Axelrod J (1972) Phenylethanolamme N-methyl transferase, in The Thyvoid and Btogemc Amlnes (Rall and Kopm, I. J., eds), pp 536-540. North-Holland Publlshmg Co , Amsterdam Axelrod J and Tomchlck R (1958) Enzymatic 0-methylatlon of epmephrme and other catechols ] Blol Chem , 233, 702-705 Axelrod J and Weissbach H. (1961) Purlflcatlon and properties of hydroxymdole O-methyl transferase 1 BloI Chem 236, 211-213 Boulton A A. (1979) Trace Ammes m the Central Nervous System, m In terna tzonal Review of Brochermstry, Physrologrcal aud Pharmacological Blochemzstry (Tlpton, K F ed), pp 179-206 Umverslty Park Press, Baltimore, Maryland. Brown D D , Axelrod J , and Tomchlck R (1959a) Enzymatic N-Methylatlon of histamine Nature, (Lond.) 183, 680 Brown D D , Tomchlck R , and Axelrod, J (195910)Dlstrlbutlon and

Radloenzymatlc

Micromethods

for Bra/n Amlnes

123

properties of a hrstamme-methylatmg enzyme ] &ol Che~n 234, 2948-2950. Brownstem M , Saavedra J M , and Axelrod J (1973) Control of N-acetylserotonm by a B-adrenergrc receptor. Mel P~U~JJI~CO~ 9, 605611 Brownstem M , Saavedra J M., Palkovrts M., and Axelrod J. (1974) Hrstamme content of hypothalamic nuclei of the rat Bruin Res 77, 151-156 Brownstem M , Palkovrts M , Saavedra J M , and Krzer J. S (1975) Tryptophan hydroxylase m the rat brain Brawn Res 94, 163-166 Chevrllard C , Barden N , and Saavedra J M (1981a) Twenty-four hour rhythm rn monoamme oxrdase actrvrty m specrfrc areas of the rat brain stem Buorn Rcs 223, 205-209 Chevrllard C , Barden N , and Saavedra J. M. (1981b) Estradrol treatment decreases type A and increases type B monoamme oxrdase m specific brain stem areas and cerebellum of ovarrectomrzed rats. Brawn Res 222, 177-181 Correa F M A and Saavedra J M (1981) Increase m hrstamme concentrations m discrete hypothalamrc nuclei of spontaneously hypertensive rats Bran Res 205, 445-451 Correa F M A and Saavedra J M. (1983) High hrstamme levels m specific hypothalamic nuclei of Brattleboro rats lacking vasopressm Brm Res 276, 247-252 Coyle J T and Henry D (1973) Catecholammes m fetal and newborn rat brain. ] Neirrochem 21, 61-67. Dahlstrom A and Fuxe K (1964) Evidence for the existence of monoamme-contammg neurons m the central nervous system I Demonstratron of monoammes m the cell bodies of brain stem neurons Acta PIryszol Scaild 62, Suppl 232, l-55 Danielson T. J., Boulton A A., and Robertson, H. A. (1977) JW Octopamme, p-octopamme and phenylethanolamme m rat brain A sensitive, specific assay and the effects of some drugs, 1 Neurochem 29, 1131-1135. Da Prada M and Zurcker G (1976) Simultaneous radioenzymatrc determmatlon of plasma and tissue adrenaline, noradrenalme, and dopamine within the femtomole range, Lzfe Scz 19, 1161-1174 Deguchr T and Axelrod J (1972) Sensrtrve assay for serotonm N-acetyl transferase activity m rat pmeal Anal Bzochem 50, 174-179. Dennis T and Scatton B (1982) A radroenzymatrc technrque for the measurement of free and conlugated 3,4-drhydroxyphenylethyleneglycol m brain tissue and brologrcal fluids J Neuroscl Methods, 6, 369-382 Durden D. A., Phrlrps S. R , and Boulton A A (1973) Identrfrcatron and drstrrbutron of B-phenylethylamme m the rat Can ] Brochem 51, 9951002 Elk-Nes K. B , and Brrzzee K R (1965) Concentratron of trmum u-r brain tissue of dogs given (1,2-3H2)-cortrsol mtravenously. Bmchem. Brophys Acta, 37, 320-333.

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Fekete M. I K , Kanyicska B., and Herman J I’ (1978) Simultaneous radioenzymatic assay of catecholammes and dihydroxy-phenylacetic acid (dopac), comparison of the effects of drugs on the tuberomfundibular and striatal dopamme metabolism and on plasma prolactm level Lzfe Scz 23, 1549-1556 Fuxe, K (1965) Evidence for the existence of monoamine neurons in the central nervous system. IV. Distribution of monoamme nerve terminals m the central nervous system Acta Physrol Stand 64, Suppl 247, 39-85 Helfti F. and Lichtensteiger W (1976) An enzymatic-isotopic method for DOPA and its use for the measurement of dopamme synthesis m rat substantia mgra J Netrrochem 27, 647-649 Kebabian J W., Saavedra J M., and Axelrod J. (1977) A sensitive enzymatic-radioisotopic assay for 3,4-dihydroxyphenylacetic acid 1 Neurochem

28, 795-801

Kobayashi, K , DeQuattro, V , Kolloch, R and Miano, L (1980). A radioenzymatic assay for plasma normetanephrme m man and patients with pheochromocytoma. Life Scl., 26, 567-573 Kvetnansky R , Kopm I J , and Saavedra, J. M (1978) Changes m epinephrine m mdividual hypothalamic nuclei after immobilization stress Brain Res. 155, 387-390 Lowry 0 H., Rosebrough N J , Farr A L , and Randall R. J (1951) Protem measurement with the Folm phenol reagent ] Blol Chem 193, 265-275. Molmoff, I’. B. and Axelrod, J (1972) Distribution and turnover of octopamme m tissues. J Neurochem 19, 157-163. Molmoff I’. B., Landsberg L , and Axelrod J. (1969) An enzymatic assay for octopamme and other B-hydroxylated phenylethylammes. ] Pharmacol. Exp Ther 170, 253-261 Molmoff P. B , Wemshllboum R., and Axelrod J (1971) A sensitive enzymatic assay for dopamme-p-hydroxylase I Pharmacol Exp Ther 178, 425431 Palkovlts M (1973) Isolated removal of hypothalamic or other brain nuclei of the rat, Brazn Res 59, 449450 Palkovits M., Brownstem M , and Saavedra J M. (1974a) Serotonm content of the brain stem nuclei m the rat Brazn Res 80, 237-249 Palkovits M , Brownstem M , Saavedra J M , and Axelrod, J (197413) Norepmephrme and dopamme content of the hypothalamic nuclei of the rat Brazn Res 77, 137-149 Philips S. R., Davis B A , Durden D A, and Boulton A A. (1975) Identification and distribution of m-tyramme m the rat Can ] Blochem

53,

65-69

Saavedra, J. M (1974a) B-phenylethylamme Saavedra, J M (1974b) the picogram level

Enzymatic lsotoplc assay for and presence of in brain ] Neurochem 22, 211-216 Enzymatic-isotopic method for octopamme at Anal

Bzochem 59, 62%633

RadloenzymatrcMlcromethods

for Brain Amines

125

Saavedra J, M., (1976) 5-Hydroxy-L-tryptophan decarboxylase actlvlty microassay and dlstrlbutlon m discrete rat bram nuclei J Neurochem

26, 585-589.

Saavedra J. M. (1977) Dlstrlbution of serotonm and synthesizing enzymes m discrete areas of the brain. Fed Pvoc 36, 2134-2141 Saavedra J. M. (1979) Microquantitatlon of neurotransmltters in specific areas of the central nervous system lnf. Rev Neuroblol 21,259-274 Saavedra J M (1982) Changes m dopamme, noradrenalme, and adrenalme m speclflc septal and preoptlc nuclei after acute lmmoblllzatlon stress. Neuroendocrmology 35, 396-401, Saavedra J M (1983) Radioenzymatic assay of blogenlc ammes, m Mcfhods in BlogenIc Anrtm Research (Parvez S , Nagatsu T , Nagatsu I and Parvez H., eds ), pp 257-283, Elsevler Science Pubhshers B V , Amsterdam Saavedra J M (1984) /3-Phenylethylamme, phenylethanolamme, tyramine and octopamme, m Hnlzdbook of Phnrrnacology Cafecllolalnlnes (Weiner N. and Trendelenburg U., eds), SprmgerVerlag, New York (m press) Saavedra J M and Alexander N (1983) Catecholammes and phenylethanolamme N-methyltransferase m selected brain nuclei and m the pmeal gland of neurogenlcally hypertensive rats Bvnln Res 274, 388-392 Saavedra J M and Axelrod J (1973) Demonstration and distribution of phenylethanolamme m brain and other tissues Proc Nat1 Acad Scl USA 70, 769-772 Saavedra, J M and Torda, T (1980) Increased brain stem and decreased hypothalamic adrenaline-forming enzyme after acute and repeated lmmoblllzatlon stress in the rat Neuroendocm 31, 140-146 Saavedra, J M and Zlvm, J (1976) Tyrosme hydroxylase and dopamme-p-hydroxylase: dlstrlbutlon in discrete areas of the rat llmblc system Brallr Res 105, 517-524. Saavedra J M , Brownstem M,, and Axelrod J (1973) A specific and sensitive enzymatic-isotopic microassay for serotonin in tissues. 1 Phavmacol Exp Ther. 186, 508-515. Saavedra J M , Brownstem M , and Palkovlts M (1974a) Serotonm dlstrlbutlon m the hmblc system of the rat. Brarn Res 79, 437-441 Saavedra J M , Brownstem M , Palkovlts M , Klzer S., and Axelrod J (1974b) Tyrosme hydroxylase and dopamme-P-hydroxylase dlstnbutlon m the mdlvldual rat hypothalamic nuclei. ] Neurochem 23, 869-871 Saavedra J M , Coyle J T , and Axelrod J (1974~) Developmental characterlstlcs of phenylethanolamme and octopamme m the rat brain / Newochein 23, 511-515 Saavedra J M , Palkovlts M , Brownstem M J., and Axelrod J. (1974d) Serotonm dlstrlbutlon m the nuclei of the rat hypothalamus and preoptlc region Brarn Res 77, 157-165 Saavedra J M , Palkovlts M , Brownstem M J., and Axelrod J (1974e)

126

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Localizatron of phenylethanolamme N-methyltransferase m the rat brain nuclei Nuttlre (Lond ), 248, 695-696 Saavedra, J. M , Palkovlts, M., Sizer, J S,, Brownstem, M and Zrvm, J A (1975) Dlstrrbutlon of brogemc ammes and related enzymes m the rat pmutary gland 1 Neurochem 25, 257-260 Saavedra J. M , Brownstein M. J , Klzer J S , Palkovrts, M (1976a) Brogemc ammes and related enzymes in the cncumventrrcular organs of the rat Brain Res. 107, 412-417 Saavedra J. M , Brownstem M. J , and Palkovrts M (1976b) Drstrrbutlon of catechol-0methyltransferase, hrstamme N-methyltransferase and monoamme oxrdase m specific areas of the rat brain Brnm Res 118, 152-156 Saavedra J M , Grobecker H , and Zrvm J. (1976~) Catecholammes m the raphe nuclei of the rat. Brain Res 114, 339-345. Saavedra J M , Grobecker H., and Axelrod J. (1978) Changes m central catecholammerglc neurons m the spontaneously (genetic) hypertensive rat. Clrc Res 42, 529-534 Saavedra J, M , Kvetnansky R , and Kopm I J (1979) Adrenaline, noradrenalme and dopamme levels m specific brain areas of acutely rmmobrllzed rats Brufn Res 160, 271-280 Saller Ch F. and Zrgmond M J (1978) A radroenzymatic assay for catecholammes and dlhydroxyphenylacetrc acrd Lzfe Scz 23, 1117-1130 Schlumpf M (1973) Analytlsche Mrkromethode zur Fluorrmetrlschen Bestlmmung von Monoammen unter Vervendug enter TopoExrzrsrons techmk fur Ratten-und graphrsch Standardrsrerten Mausegehrrn. Doctoral Thesis, N” 5060, Erdgenoessischen Techmschen Hochschule, Zuerrch, Switzerland Schlumpf M., Waser P. G , Llchtenstelger W , Langemann H and Schlup I’. (1974) Standardized excrslon of small areas of rat and mouse brain with topographrcal control. Bloc/tern PhnrmncoI 23, 2447-2449 Smith, A D and Wmkler, H (1967) A simple method for the lsolatron of adrenal chromaffm granules on a large scale. Blochem 1. 103, 480-482. Snyder S H., Baldessarim R , and Axelrod J (1966) A sensmve and specrfrc enzymatic lsotoplc assay for tissue hlstamme ] Phavmacol Exp. Ther 153, 544549 Tallman, J F , Saavedra, J M and Axelrod, J. (1976) A sensrtrve enzymatrc-lsotoprc method for the analysis of tyramme m brain and other tissues ] Neurochem 27, 465469 Taylor, K. M and Snyder, S. H (1971) Hlstamme m rat brain* sensitive assay of endogenous levels, formatron m VIVO and lowering by mhlbltors of hlstrdme decarboxylase. J. Pharmacol. Exp. They. 173, 619-633 Taylor, K M and Snyder, S H (1972) Isotopic mlcroassay of histamine, hrstldme, hlstidine decarboxylase and hrstamme methyltransferase m brain tissue 1 Neurochem 19, 1343-1358

Radioenzymatic

Micromethods

for Brain Amines

127

Vlachakis, N D and DeQuattro, V. (1978). A sample and specrfrc for measurement of radroenzymatrc assay urinary normetanephrme Bzochem Med. 20, 107-114. Vlachakrs N. D , Alexander N , Velasquez M T., and Maronde R F. (1979) A radroenzymatlc microassay for simultaneous measurement of catecholammes and their deaminated metabohtes B&em Med. 22, 323-331 Weissbach H., Redfreld B G and Axelrod J (1961) The enzymlc acetylatron of serotonm and other naturally occurrmg ammes Bzochm. Bzophys. Acta 54, 190-192 Zurcker, G. and Da Prada, M (1979) Radioenzymatrc assay of femtomole concentratrons of dopa m trssues and body fluids 1 Neurochem. 33, 631-639.

Chapter 4

Liquid Chromatographic Analysis of Monoamines and Their Metabolites GEORGE

M. ANDERSON

1. Introduction It 1s evident that an explosive growth has occurred over the past ten years m the high performance liquid chromatographic (HPLC) analysis of catecholammes, indoleammes, and the trace amines In part, this expansion can be attributed to a growmg interest m the monoamines; however, the increased sensitivity and specificrty afforded by the HPLC methods and their low cost must be vlewed as the malor reason for their wide acceptance. It is also clear that the vast preponderance of HPLC methods m neurochemistry utilize either amperometric or fluorometric detection systems. The bases of these methods of detection are well established and the benefits resulting from their couplmg to HPLC systems and their pervasive use in neurochemistry have been previously reviewed (Anderson and Young, 1981, 1982; Davis and Kissinger, 1982, Kissinger et al., 1977, 1981; Krstulovic, 1982; Mefford, 1981, Warsh et al., 1982). This chapter will be concerned with making an up-to-date review and critical analysis of the HPLC-amperometric (LC-EC) and HPLC-fluorometnc (LC-F) methods that have been employed for the determmation of noradrenalme (NA), adrenaline (A), dopamine (DA), 5-hydroxytryptamme (serotonm;5-HT), a number of trace ammes, and the compounds’ metabolites, m brain, CSF, blood, and urine The primary divrsion will be along sampletype lmes: methods will be discussed under major headings of brain, CSF and blood, and urine. This division 1snot capricious as the sample matrices presented by the different samples, and the 129

130

Anderson

typical levels of many of the compounds of interest, are characteristic and determine to a large degree the routes taken for analyses. The catecholammes (NA, A, DA) themselves ~111 be discussed m a section separate from their metabolites. The acidic catecholamme metabolites homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC), and vamllylmandelic acid (VMA) will be grouped together, along with the neutral metabolites 3-methoxy-4-hydroxyphenylglycol (MOPEG) and 3,4-dihydroxyphenylglycol (DOPEG), the basic 3-O-methylated amines metanephrme (MN), normetaphine (NMN), and 3-methoxytryamine (3-MTA), and the precursor 3,4-dihydroxyphenylalanine (DOPA). The 5-HT precursors tryptophan (TP) and 5-hydroxytryptophan (5-HTP) will be discussed together with 5-HT itself and its acid metabolite, 5-hydroxymdoleacetic acid (5-HIAA) and the alcohol, 5-hydroxytryptophol (5-HTOL). A separate section will cover the determination of the pmeal mdoles melatonm (5-methoxy-N-acetyltryptamme, MEL) and N-acetylserotonm, along with other indoles determined u-r the pmeal Finally, the determination of the trace amme tryptamme (T) and its metabolites, mdoleacetic acid (IAA) and tryptophol (TOL), will be reviewed along with the LC-EC and LC-F methods available for the measurement of other trace amines There is an arbitrary aspect to a subdivision of the methods according to the chemical groups outlined. In many cases, and especially so m brain tissue analysis, a number of precursors, monoamines, and metabolites-catechols and mdoles alikehave been simultaneously determined. In general, any method which includes one or more of the catecholammes (NA, A, DA) will be discussed initially, and usually most extensively, in the section covermg catecholammes A brief general discussion of three of the compound classes outlined-the catecholamines, the catechol metabolites, and the 5-hydroxyindoles-is included m the initial section covermg their analysis in brain. General information on the pineal indoles and on the trace amines and their metabolites is given in the separate sections dealing with the determination of those compounds in physiological samples.

2. Brain 2.1, Catecholamines in Brain The catecholammes, NA, physiochemical properties, basis of all HPLC methods

A, and DA have four distmctive several of which are, as a rule, at the developed for their determination.

Liquid

Chroma tographic

Analysis ofMonoamlnes

131

These are their catechol moiety (aryl 1,2-dihydroxy), a basicity that results from their amine function, their easily oxidized nature, and their native fluorescence. The catechol and/or amme functions determine which methods of sample purification and analytical separation can be employed, although all four aspects of the catecholamines have been used to advantage in their detection. The methods that have been developed m brain, CSF, blood, and urine differ due to the varying levels of the compounds present and because of the different nature of the sample matrices present in the various tissues and fluids. Methods for the HPLC determination of NA, A, and DA m brain can be described m terms of the extent and type of sample purification employed, the chromatographic mode used for their analytical separation, and the final means of detection An addltional important aspect of the methods to be discussed concerns the number of additional related species, catechols and indoles, which are simultaneously determined. The mmal application (Refshauge et al., 1974), and other early applications (Fuller and Perry, 1977; Hashimito and Maruyama, 1978, Keller et al., 1976), of LC-EC to the determination of brain catecholammes utilized alumina extraction, followed by analytical separation on a cationWhile the separations were relatively exchange column inefficient, sensitivity (detection limits of approximately 10 pg) and selectivity were such that brain areas could be analyzed, A chromatogram demonstrating the high absolute sensitivity obtained is presented m Frg. 1 (Keller et al., 1976). Several related methods also used analytical cation-exchange chromatography; however, brain homogenates were injected on an LC-EC system after a butanol extraction (Sasa and Blank, 1977, 1979) or without any prelimanary purification (Anderson et al., 1980). Most LC-EC brain catecholamine methods have employed ion-pair chromatography after a prehmmary alumma extraction (Felice et al., 1978; Hegstrand and Eichelman, 1981; Mefford et al., 1980, Maruyama et al., 1980, Remhard and Roth, 1982; Taylor et al., 1983; Wagner et al., 1979, 1982). Typical of the efficient separations obtained is the chromatogram shown m Fig. 2 (Felice et al., 1978) The trace is free of significant extraneous peaks and NA and DA, along with the internal standard 3,4-dihydroxybenzylamine (DHBA), are easily determined. Ion-pair chromatography of the catecholamines usually has been accomplished using heptanesulfonic or octanesulfonic acid. These agents result in a greater retention of the extremely hydrophilic catecholammes and also appear to mcrease efficiency by masking the interaction of residual silanol groups contained on reverse-phase packings with the amine func-

132

Anderson

Cm.

1

’ 0

I 4

I 8

T ime”(

I

I

I

20

mini6

Fig. 1. Brain NA (NE) and DA determined, along with the internal standard DHBA, in an alumina extract of rat brain by LC-EC. Separation was on a pellicular cation exchange resin (Keller et al., 1976, by permission).

Several ion-pairing methods have been reported using small preparative columns of Sephadex G-10 (Westerink and Mulder, 1981) or cation exchange resin (Kempf and Mandel, 1981; Warsh et al., 1982) to purify brain homogenates before injecting a catecholamine-containing fraction on an LC-EC system. Many of the methods mentioned permit the simultaneous determination of related catechols and indoles. Most often this is accomplished by injecting appropriate fractions produced in a purification scheme. However, a few ion-pairing LC-EC methods have permitted the direct injection of centrifuged brain homogenate. Figure 3 presents the determination of NA and DA, along with 5-HIAA and 5-HT, in unpurified rat midbrain (Zaczek and Coyle, 1982). Although a lengthy chromatographic run is required, and information is lacking on the retention time of HVA, the straightforward nature of the method is commendable. More recently, a direct injection method for mouse brain has been reported using ion-pair chromatography on a 3 km average particle size column as seen in Fig. 4 (Lin and Blank, 1983). The great efficiency of the 3 Frn column employed allows the catecholamines to be determined along with a number of related species. In general, the LC-EC methods are not sensitivity-limited; with everyday detection limits of 10-25 pg for the catecholamines, the compounds are tion.

Liquid

i5 DA”5

Chroma tographic

Analysis of Monoamines

II DAY5

I I 1

Fig. 2. Brain NA (NE), DA, and the internal standard DHBA, measured in rat hypothalamus (age in days given). The compounds were separated by ion-pair reverse-phase HPLC (10 km C18 packing, 30 x 0.39 cm column) and were detected by amperometry (Felice et al., 1978, by permission).

easily detected at the 100 r-q/g and above levels usually seen in brain. However, when determining NA and DA in many small areas, or adrenaline (A) in any brain sample, the increased concentration detection limits afforded by the alumina clean-up and concentration step are beneficial. In addition, the area-to-area variations in the extent of significant interferences with the catecholamines, especially with early eluting NA, are reduced using an alumina extraction. The fluorometric methods for catecholamines in brain are most easily classified according to whether or not a derivative is formed. Better detection limits are achieved after derivatization; in fact limits of less than 10 pg have been reported after derivatization with o-phthalaldehyde (OPT) (Todoriki et al., 1983), whereas after reaction to trihydroxyindole derivatives a 1 pg detection limit was obtained for NA and DA (Yui and Kawai, 1981). The methods employing fluorescamine derivatives (Imai et al., 1977) or native fluorescence (Jackman et al., 1980; Krstulovic and Powell, 1979; Peat and Gibb, 1983) report detection limits of - 100-300 pg for the catecholamines. The two procedures using

Anderson

Fig. 3. Rat midbrain NA (NE), DA, 5-HIAA, and 5-HT determined by direct injection of centrifuged homogenate on an ion-pairing reverse-phase (10 pm) LC-EC system (Zaczek and Coyle, 1982, by permission).

native fluorescence without alumina extraction are difficult to judge in terms of specificity and possible interferences with NA and DA. However, the procedure using an alumina extraction (Jackman et al., 1980) would appear to offer excellent specificity, with sensitivity sufficient to measure the compounds in all but the smallest or least concentrated areas. An example of the determination of NA and DA in an alumina extract from hypothalamus is shown in Fig. 5 (Jackman et al., 1980). Whereas the derivative

Liquid Chromatographic

Analysis

of Monoamines

135

Fig. 4. Mouse whole brain NA (NE), DOPAC, DA, 5-HIAA, HVA, and 5-HT measured, along with the internal standards DHBA and N-methylserotonin, after the direct injection of centrifuged homogenate. The species were separated by ion-pair reverse chromatography on a 3 km (7.5 x 0.46 cm) column and detected amperometrically (Lin and Blank, 1983, by permission).

methods offer advantages in sensitivity compared to native fluorescence, they have sensitivities similar to, or only slightly better than, the LC-EC methods. The extra trouble involved with derivatization does not encourage their use. On the other hand, methods measuring native fluorescence after an alumina extrac-

136

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tion would appear to be underutilized given the greater ease of everyday operation of a fluorometer compared to hydrodynamic amperometry at thin-layer carbon electrodes (LC-EC).

2.2. Catecholamine

Metabolites

and Precursors

in Brain

The major catecholamine metabolites can be categorized as having acidic (HVA, DOPAC, VMA), neutral (MOPEG, DOPEG), or basic (the metanephrines MN, NMN, and 3-MTA) side chains. The compounds are also distinguishable with respect to the presence (HVA, VMA, MOPEG, metanephrines) or absence (DOPAC, DOPEG) of the 3-O-methyl group. The majority of the HPLC

W 0 z W v

(Cl)

(b)

-5 20 0I

; E 3 LL

1

I

024

I

1

6

TIME (mln)

I

1

I

1

0246 TIME (mln)

Fig. 5. Noradrenaline (A), epinephrine (B), and dopamine (C) standards (left) and NA and DA determined after alumina extraction in rat midbrain (right) by LC-F with reverse-phase separation (Jackman et al., 1980, by permission).

Liquid

Chroma

tographic

Analysis

of Monoamines

137

methods reported in brain and other sample types use amperometric detection (LC-EC), thereby taking advantage of the easily oxidizable nature of the dihydroxy (catechol) and hydroxymethoxy (vanillyl) species. Although fluorometric methods have been reported, they are fewer in number and (as with the catecholamines, themselves) offer detection limits approximately one order of magnitude higher. The preliminary separations employed for the metabolites either depend on (and in some S

r

6

20

10

10

0

TIME

0

(minutes)

Fig. 6. On the right the catechols determined after alumina extraction of rat hypothalamus: DOPAC (l), NA (2), A (3), DHBA (4), and DA (5) were determined at 0.5, 100, 2, 20, and 10 nA/V sensitivities, respectively. On the left compounds determined in unextracted brain homogenate include 5-HT (3), TP (5), 5-HIAA (6), MOPET (7 - the internal standard), and HVA (8) (Mefford et al., 1980, by permission).

138

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cases are frustrated by) the chemical nature of the side chain moiety or the presence of the catechol group. A number of the assays for brain catecholammes referred to in the previous section also measure the acid metabolites HVA and DOPAC. These methods detect one or more of the metabolutes by isolating an appropriate fraction from a small column purification scheme or by direct mlection of an unextracted and/or alumma-extracted brain sample. Examples of the latter strategy are the methods shown m Figs 6 and 7. Demonstrated m Fig. 6 (Mefford et al., 1980) is the determination of DOPAC along with the catecholamines NA and DA The compounds had similar recoveries through the alumma isolation procedure and DHBA was used as an internal standard. Homovanillic acid was determined m the supernate usmg 3-methoxy-4-hydroxyphenylethanol (MOPET) as an mternal standard In Fig. 7 (Remhard and Roth, 1982), DOPAC is similarly determined in an alumina extract whereas HVA (and several 5-hydroxyindoles) is also measured m the supernate. As discussed m Mefford’s (1981) review, this efficient combining of a direct mlection method and an alumina extraction permits a range of catechols and indoles to be determined from small brain areas. This IS not to fault the utile separations based on small column purification procedures; shown m Fig. 8 (Westerink and Mulder, 1981) is a chromatogram of a fraction obtained from one such scheme. Certainly this and similar methods demonstrate a flexible approach to analysis of the monoamines and metabolites, however, the simplicity of alumina extract/supernate methods makes them more readily accepted Another group of methods for determining DOPAC and/or HVA (along with other compounds) consists of analyses that do not report, or appear not to have as their mam oblective, the simultaneous determination of the catecholamines. In most of these methods it would not be difficult to add provisions for determining NA and/or DA in a suitable fraction or ahquot after alumma extraction. A number of these determinations depend upon the solvent extraction of DOPAC and HVA from bran-t homogenate (Cross and Joseph, 1981; Van Valkenberg et al., 1982, Saraswat et al , 1981, Heft1 1979). In Fig. 9 (Cross and Joseph, 1981) the two acids are determined, after ethyl acetate extraction, m human brain using both electrochemical and fluorometric detection. Extraction efficiency and consistency eliminated the requirement for an Internal standard. Several other groups have reported analyses of DOPAC and/or HVA after the direct mlection

Liquid Chroma tographic Analysis of Monoamines

139 C.

B.

1LA.JL, T a :

I

It, ”

0

5

IO

15

20

25

0

5

IO

I5

20

0

5

IO

15

20

25

30

MINUTES

D

I=.

I

! L.-L 0

5

IO

I5

20

25

0

5

IO

15

20

I 25 0

I 5

I IO

1 15

1 20

I 25

MINUTES

Fig. 7. Upper traces are from left to right; 10 pmol (-2ng) of standards (NMS, N-methylserotonin), control rat brain cortex, and treated rat cortex. Separation was by reverse-phase chromatography (5pm, 4.6 X 0.25 cm column), detection was by amperometry. Lower traces were from left to right; 2.5 pmol catechol standards, treated rat cortex, control rat cortex separated by ion-pair LC-EC. Tissue samples shown in lower traces were alumina-extracted (Reinhard and Roth, 1982, by permission).

140

Anderson DOPAC

T

0.5 nA 1

I 0

I

I 4

I

I 8

I

I 12

I

I 16

I

I

I

20 min *

Fig. 8. The labeled peaks are determined by reverse-phase LC-EC in a small column-purified fraction of brain (cortex) homogenate using a pH 3.5 8% methanol mobile phase (Westerink and Mulder, 1981, by permission).

of deproteinized and centrifuged brain homogenate (Magnussen et al., 1980; Anderson et al., 1981a; Kilts et al., 1981; Sperk, 1982). Shown in Fig. 10 (Magnussen et al., 1980) is the determination of DOPAC and HVA, along with other catechols and indoles, in unpurified brain (striatum) homogenate using epinine as an internal standard. A similar determination of HVA, DOPAC, and other compounds was accomplished by Kilts et al. (1981). They used N-methylserotonin as an internal standard and heptanesulfonic acid was employed as the ion-pairing agent (for the amines being determined simultaneously) instead of the hexylsulfonic acid used in the method of Fig. 10. Reports of the determination of the neutral diol metabolites

Liquid

Chromatographic

Analysis ofMonoamines

t

Y

’

1 7 Fig. 9. LC-F (top) and DOPAC (2), 5-HIAA (3), and tracts of human hypothalamus HVA (4) on the reverse-phase Joseph, 1981, by permission).

LC-EC (bottom) traces of MOPEG (l), HVA (4) determined in ethyl acetate ex(left) and cortex (right). Retention time of LC-EC system was 14.1 min (Cross and

MOPEG and DOPEG in brain are relatively rare. Although abstracts have appeared concerning MOPEG’s measurement in rat brain, the author is unaware of a published HPLC method suitable for MOPEG in rat brain. Reports have detailed its determination in mouse (Towel1 and Erwin, 1981; Ishikawa and McGaugh, 1982) and primate (Cross and Joseph, 1981) brain, either after organic solvent extraction or by direct injection. The usual levels of free MOPEG in rat brain (10-20 “g/g) are lower than those seen in mouse or human brain due to a greater degree of sulfate conjugation in the rat. However, the measurement is not frustrated by sensitivity aspects, but rather by a lack of specificity resulting from the hydrophilic nature of MHPG that makes a selective extraction difficult and causes the compound to have a short retention time on reverse-phase columns.

142

Anderson

2nA I

3

6

I 4

il

7’

-7-----T-

O

4

8

12

16

20 min

Fig. 10. Reverse-phase ion-pair separation of DOPAC (l), DA (2), epinine (3)(the internal standard), 5-HIAA (4), HVA (5), 3-methyoxytyramine (6), and 5-HT (7) in unpurified rat striatum. A pH 4.25 citrate buffer containing 8% methanol and 1.7 mM hexyl sulfate eluted a 5 km Cl8 column (Magnussen et al., 1980, by permission).

As a class, the basic metabolites, the metanephrines and 3-methoxytyramine (3-MTA), have also been only rarely measured in rat brain using HPLC. The DA metabolite, 3-MTA, has been determined using LC-EC following a small column isolation (Westerink and Spaan, 1982) or after an extraction (Ponzio et al., 1981). This paucity is probably due to the low levels of metanephrines found in brain (rig/g) and a general dismissal of

Liquid

Chromatographic

Analysis

ofMonoamines

143

their importance. The final catechol whose measurement in brain will be discussed is the precursor DOPA. As with MOPEG, its determination is hindered by its relatively low levels and its hydrophilic nature. Several methods have as their main thrust the measurement of DOPA (Westerink et al., 1982; Freed and Asmus, 1979), whereas others determine DOPA along with a variety of other catechols and/or indoles (Reinhard and Roth, 1982; Wagner et al., 1979, 1982; Westerink and Mulder, 1981). In a previously presented chromatogram, DOPA was determined (in an aromatic

lric acid

I

DA /

T 1 nA 1_

OOPA-

-

1 I

I

I

I

0

4

6

6 10

2

I

min *

Fig. 11. Endogenous DOPA determined in normal rat cortex after reverse-phase separation on a 5 pm CIR column. A small-column purified fraction was also analyzed for uric acid and DA (Westerink and Mulder, 1981, by permission).

144

Anderson

ammo acid decarboxylase Inhibitor-treated animal) along with other catechols m an alumma extract (Fig 7) Endogenous levels of DOPA m untreated ammals have been determined with greatest specificity using the method of Westermk and Mulder (1981), as shown m Fig. 11. The DOPA-containing fraction obtamed from a small column (Sephadex G-10) isolation procedure was separated on a reverse-phase column usmg a mobile phase of pH 5.5 citrate-phosphate buffer contammg no organic solvent

2.3. Indoles in Brain Most of the mdoles related to serotonm (5-HT) exhibit strong native fluorescence and are also amenable to detection by amperometry. Detection limits of 5-25 pg can be obtained for most mdoles using fluorometry-values similar to those observed for hydroxymdoles with electrochemistry. In some cases, fluorometric detection will afford greater selectivity due to the ability to choose excitation and emission wavelengths. The compounds of interest m brain, namely TP, 5-HTP, 5-HT, 5-HIAA, and 5-HTOL are of varied chemical type, bemg ammo acids (TP, 5-HTP), an amme, acid, and alcohol. Generally, techniques for purifymg the compounds m bram homogenates can isolate only one or two of the mdoles m a particular fraction Unlike the catechols, no class-specific extraction technique exists for the mdoles. This lack of selectivity is often mitigated by the specificity engendered by fluorometric detection using an excitation wavelength of 280-290 nm and an emission wavelength of 345-360 nm. Some of the earliest HPLC methods for the determmation of hydroxymdoles (Meek, 1976, Meek and Neckers, 1975, Neckers and Meek, 1976) m brain depended upon the even more specific detection of acid-shifted fluorescent emissron (emlsslon wavelength 550 nm m strong acid) In Fig. 12 (Neckers and Meek, 1976), 5-HIAA and 5-HT are determined in unpurified brain homogenate with absolute detection limits of approximately 100 pg after separation on a cation-exchange analytical column In addition to the large number of electrochemical methods mentioned previously for the determmation of one or more of the indoles along with catecholammes and/or catechol metabohtes, several LC-EC methods have been developed specifically for the detection of mdoles. These methods have employed prepurihcation with a small preparatory column (Koch and Kissinger, 1979, 1980; Lyness, 1980, Warsh et al., 1979) or solvent extraction (Ponzlo and Jonsson, 1979), or have directly mlected brain homogenates (Remhard et al., 1980; Mefford and Barchas, 1980,

Liquid

Chromatographic

145

Analysis ofMonoamines

Lackovic et al., 1981). A number of investigators have reported LC-F methods for the indoles in unpurified brain homogenates (Anderson et al., 1981a; Flatmark et al., 1980; Hori et al., 1982; Wolf and Kuhn, 1983; Yamada et al., 1983a, 1983b). Most use an excitation wavelength of 280-290 nm and emission wavelengths of 345-360 nm, although 254 nm radiation from a Pen-Ray low pressure mercury lamp was used for the analysis of 5HT, TP, and 5HIAA in whole rat brain as shown in Fig. 13 (Anderson et al., 1981a). The compounds were separated on a reverse-phase analytical column and 5HIAA (and HVA) was also determined using amperometric detection. A similar determination of indoles and catechols using amperometric and fluorometric detection was shown in Fig. 9 (Cross and Joseph, 1981). A related method, using N-acetylserotonin as an internal standard and employing

STANDARDS

DORSAL RAPHE NUCLEUS

PARGVLlNf

MINUTES

Fig. 12. Serotonin and 5-HIAA determined in standards (1 ng) and rat hind-brain samples following separation on a cation-exchange column. Acid-shifted fluorescence is detected at 550 nm after excitation at 300 nm (Neckers and Meek, 1976, by permission).

Anderson

146 0 I

2 1

4 1

6 1

8 1

IO mln I

v II HVA

,

“lAA

AMPEROMETRIC

I

INJ

i

0 5 nA (0 2 nA after SHIAA)

5t -RP

WHOLE

BRAIN

SAMPLE

FLUOROMETRIC I 0 sens TRP 20 mV (IO mV after TRP) I

INJ

L 5H

i!i SHIAA

1

I

1

I

0

2

4

’

’

1

6

8

IO mln

LC-EC (upper) and LC-F (lower) traces of mdoles and Fig 13 HVA in directly qected rat whole brain homogenate. Separation was effected on a 10 pm Cl8 reverse-phase column (30 X 0 39 cm) (Anderson

et al , 1981a, by permlsslon)

Liquid

Chromatographic

Analysis offlonoamlnes

147

fluorometric detection solely, is shown in Fig. 14 (Wolf and Kuhn, 1983). Here a Perkin-Elmer spectrophotofluorometer (650-10 LC) was used with an excitation wavelength of 285 nm.

2.4. Pineal Indoles Among the indoles of interest determined in the pineal gland are TP, 5HTP, 5HT, 5HIAA, 5HTOL, A!-acetylserotonm (NAS), and MEL. Measurements of two additional 5-O-methyl compounds, 5-methoxytryptophol (5-MTOL) and 5-methoxyindoleacetic (5-MIAA), as well as tryptamine (T) and indoleacetic acid (IAA), have also been of Interest Melatonin, with known hormonal effects, is thought to be the most important pmeal indole. Levels of MEL m the rat vary from a high of -1-3 ng/pineal at night, down to a low of several hundred picograms during the day. Diurnal variations are also present m the levels seen for all of the mdoles studied. The precursors to MEL (TP, 5HT, and NAS) along with MEL and the 5HT metabohtes 5HIAA and 5HTOL have been determined m rat and human pineal using an LC-F system (Anderson et al., 1981a, Anderson et al., 1982) as shown in Fig. 15. There the more hydrophilic species are measured after using reverse-phase separation with pH 4.5 buffer containing 15% methanol. The predominant peak, particularly m these daytime samples, is 5HT. Melatonin was determined, as seen m Fig. 16 (Anderson et al , 1982), using a mobile phase containing 35% methanol. The compounds were also determined in human pineal, but not without increasing the retention time so as to minimize the possible interferences from the greater number of fluorescent species present m the human sample. A similar scheme was employed when amperometric detection was used to determine pineal indoles m the rat. Chromatograms from the LC-EC determination of indoles in directly mlected pineal homogenates are shown in Fig. 17 (Mefford and Barchas, 1980). Here 5HTP was determined rather than 5HTOL and this necessitated a longer chromatographic run, otherwise the methods are quite comparable-both having detection limits in the lo-100 pgipmeal range. Levels of the mdoles observed using these two HPLC methods were m good agreement (Anderson et al., 1982; Young and Anderson, 1982; Mefford and Barchas, 1980) However, upper limits established for 5MTOL, 5MIAA, and TAM using the LC-F method were lower than the

148

Anderson

18

16

1’4 li

MINUTES

lb

8

6

AFTER

i

b

INJECTION

Fig. 14. Indoles determined in untreated rat midbrain tegmenturn by direct injection of homogenate on a reverse-phase LC-F system. The compounds: 5-HTP (l), 5-HIAA (2), TP (3), 5-HT (4), and N-acetylserotonin (NAS, the internal standard) (Wolf and Kuhn, 1983, by permission). RAT

STANDARDS

PINEAL

SAMPLES

I

2mV (20mV for SHT) 10 18flS

RP 5HTOL

il 1

2

I

/

6

1

/

10

1.)

1

2

6

10

II

2

6

2

6

IO

MINUTES

Fig. 15. Determination of pineal indoles in homogenized rat pineals by LC-F. Note sensitivity change (increase) after 5-HT peak (Anderson et al., 1982).

Liquid

Chromatographlc

Analysis ofMonoamines

149

previously reported concentrations using gas-chromatographic analyses Although MEL can be fairly easily measured in the pmeal using LC-F or LC-EC systems, no report of a HPLC MEL assay m plasma or CSF has been forthcommg. This is undoubtedly due to the lower concentrations of MEL present (and, in the case of blood, the more complex sample matrix). Given the good recovery of MEL through a chloroform extraction it is not inconceivable that an HPLC method will be developed. The LC-F approach would probably offer greater selectivity advantages, but these ideas await practical testing An easier, and perhaps more meaningful measure, would be the analysis of the major melatonin metabohte, 6hydroxymelatonin (6HMEL), m urine. Occuring at the -10-100 ng/mL level, its measurement should not be frustrated by sensitivity aspects. However, again, no HPLC method is currently available as an alternative to the laborious gas chromatographrc-mass spectrometric GC-MS technique (Tetsuo et al., 1981).

3. Blood 3.1.

Catecholamines

in BIood

In the development of assay methods for plasma catecholammes one must wrestle with the fact that extremely low levels (tens to hundreds of pg/mL) of the compounds are present. The various approaches taken m plasma analysis have been admirably reviewed by Holly and Makm (1983). The HPLC methods for catecholammes m blood are easily categorized according to whether detection is accomplished using amperometry (LC-EC) or fluorometry (LC-F) after a fluorogemc reaction. The first reported LC-EC determmations of plasma catecholamines used alumma extraction followed by chromatography on cation-exchange columns (Hallman et al., 1978; Fenn et al., 1978). In the chromatograms of Fig 18 the catecholammes are determined m an alumina extract of 0 75 mL of plasma after separation on a 50 X 0 2 cm column of Vydac (-35km particle size) cation exchange packing (Hallman et al., 1978). The catecholammes were determined with concentration detection limits of 5-10 pg/mL and average levels (N = 6) of -500 and 40 pg/mL were reported for NA and A respectively Dopamine values were typically less than 10 pg/mL. Subsequently, several LC-EC methods were reported using higher efficiency 10 km cation-exchange packings (Hlemdahl et al , 1979; Watson, 1981, Eriksson and Persson,

Anderson

RAT PI NEAL SAMPLES

STANDARD Sng MEL

a3

*1

IOmV 10 I se”, INJ

INJ

/

INJ ?

I

2mV 10 98”).

MEL

I

Fig. 16. Melatonin determined in three different directly injected homogenized pineal samples. Concentrations in the sample shown ranged from 0.93 ng to 0.17 ng/pineal (Anderson et al., 1982). 1982). The selectivity that resulted from using an alumina extraction and high-performance ion-exchange chromatography appeared sufficient to determine NA, A, and DA. Unfortunately little normative data was published in these latter reports. Since 1981 a plethora of LC-EC methods using ion-pair reverse-phase separation after initial alumina extraction have been published 1981; Davies and (Causon et al., 1983; Davis and Kissinger, Molyneux, 1982; Goldstein and Feurstein, 1981; Goldstein et al., 1981a, 1981b; Goto et al. 1981, 1983; Jenner et al., 1981; Mefford et al., 1981; Westerink, 1981). In general the methods require great care and, even under optimum conditions, inferences are often a problem with NA and ultimate sensitivity a factor with A and DA. The determination of NA, A, and DA (and DOPAC) in an alumina extract of human plasma is depicted in Fig. 19 (Mefford et al., 1981). The amines are separated with ion-pair chromatography on a 5 km 25 X 0.46 cm Cl8 column; mean levels of 292, 81, and 29 pg/mL were reported for NA, A, and DA, respectively. Reviews of the previous chromatographic and radioenzymatic methods (Davis et al., 1981; Holly and Makin, 1983; Mefford et al., 1981) indicate usual normal levels are in that range, or slightly lower. As evident in Fig. 19, sensitivity is a problem with A and DA and efforts have been made to improve the aspect with re-

Liquid Chromatographic

151

Analysis ofMonoamines

spect to A (Goldstein and Feurstein, 1981) by injecting the total alumina extract obtained from 1 mL of plasma. A number of groups have reported on correlation and precision studies comparing the LC-EC and radioenzymatic methods and agreement and precision for NA appears good as long as NA is clearly separated from other early eluting species. The validity of the A measurement remains to be established. The two-step alumina/cation exchange purification recommended by Frayn and Maycock (1983) seems warranted. In Fig. 20 chromatograms of alumina and alumina/cation-exchange extracts of human plasma are contrasted; the benefit of the additional sample pre-

3 7

3

k x10

c1

4

8

J

I““1 108

6

4

/

L4 2

0

I 15

-1 I 10

I 5

I 0

MINUTES Fig. 17. The reverse-phase LC-EC determination indoles in rat pineal homogenates. On the right, 5-HTP (l), 5-HT (3), TP (5), and 5-HIAA are determined using a mobile phase containing 10% methanol. On the left, MEL (3) is determined with a mobile phase containing 25% methanol (Mefford and Barchas, 1980, by permission).

152

Anderson

d

0-m

18

o

18 min

Fig. 18. The LC-EC detection of approximately 150 pg of catecholamine standards: NA (a), A (b), DA (c), and wmethyldopamine (d) after separation by cation-exchange chromatography (left trace). On the right the compounds are determined in an alumina extract of human plasma (Hallman et al., 1978, by permission). is evident when the NA and A peaks are compared (Frayn and Maycock, 1983). The higher detection limit obtained for DA and the apparent presence of interferences in some of the reverse-phase LC-EC methods, coupled with the extremely low levels of free DA in human plasma, have made it the most difficult of the catecholamines. Parenthetically, this might not be of great importance given the uncertain meaning of peripheral DA, particularly of the small free fraction (l-5%) of plasma DA. An attempt to validate an LC-EC procedure for DA by comparison to a radioenzymatic method actually throws each of the methods employed in doubt as levels of over 200 pg/mL were seen by both (Goldstein et al., 1981b). The catecholamines have been determined in plasma extracts using LC-fluorometric systems, but only after precolumn (Davis et al., 1978) or postcolumn (Hamaji and Seki, 1979; Yui et al., 1979, 1980) derivatization. The precolumn o-phthalaldehyde method of Davis et al. (1978) does not appear to have sufficient sensitivity to determine the compounds in normal human plasma. However, in elegant work, several groups have demonstrated that a post-column trihydroxyindole reaction sys-

purification

Liquid

Chromatographic

Analysis ofMonoamines

153

minutes

Fig. 19. LC-EC determination of DOPAC (l), NA (2), A (3), DHBA (4 - the internal standard), and DA (5) in an alumina extract of human plasma after separation by ion-pair reverse-phase chromatography on a 5 km (25 x 0.46 cm) column (Mefford et al., 1981, by permission). tern coupled to HPLC separation permits the sensitive and selective measurement of NA and A in human plasma. The methods of Yamatodani and Wada (1981) and of Hamaji and Seki (1979) both utilize a cation-exchange purification step followed by analytical separation on a cation exchange column. In Fig. 21 (Yamatodani and Wada, 1981), NA and A are determined fluorometrically at the 250 and 35 pg/mL level in human plasma after an automated two-dimensional cation exchange separation. Both of the methods suffer only from a relatively lengthy separation and the complexity of the postcolumn reaction systems. The former drawback has been reduced in the method of Yui et al. (1980), where, after alumina extraction, separation was effected on Zipax SCX cation exchange column in 15 min. The chromatogram obtained is pre-

154

Anderson

sented in Fig. 22 (Yui et al., 1980). Average values of 185 and 32 pg/mL were reported for NA and A, respectively, and, even though no internal standard was employed, excellent coeffecients of variation were reported. Although Holly and Makin (1983) have stated that “there is no reason why fluorometric detection (of LC effluents), which is sufficiently sensitive, should not be more widely adopted,” it is apparently the difficulties attendant to setting up a multi-component postcolumn reaction system that have limited the use of LC-F systems in the determination of plasma catecholamines.

3.2. Catecholamine Metabolites in Blood Determinations of the catecholamine metabolites in blood by HPLC are few in number. This scarcity is due to the low levels of the compounds of interest, the relatively complex nature of the

DHBA

DHBA

I

5

I

I

I

10 15 20 Time, min

I

25

I

d

5

I

I

I

10 15 20 Time, min

J

25

Fig. 20. Noradrenaline (NE) and adrenaline (E) measured in human plasma by LC-EC following ion-pair separation on a 5 pm (25 x 0.46 cm) column. The chromatogram on the left is an alumina extract while that on the right is of plasma purified by cation-exchange and alumina extraction (Frayn and Maycock, 1983, by permission).

Liquid

Chroma tographic

Analysis of Monoamines

155

I, G r EN

I 0

I 10

I 20

I

30 min

Fig. 21. Plasma catecholamines NA (NE) and A (EN) determined in human plasma after automated cation-exchange chromatography and fluorometric (trihydroxyindole) detection (Yamatodani and Wada, 1981, by permission). sample matrix and the absence of selective purification techniques. The compounds to be discussed, MOPEG, HVA, and VMA, normally occur at levels of less than 15 ng/mL in human plasma. Chromatograms from the two reported methods for MOPEG in plasma are presented in Fig. 23 (Ong et al., 1982) and Fig. 24 (Scheinin et al., 1983). In Fig. 23 both VMA and MOPEG are determined in an extract isolated from a 1 mL plasma sample. A small reverse-phase preparatory column was used, followed by an ethyl acetate extraction of a selected fraction. Recoveries of VMA, MOPEG, and the internal standard hydroquinone average approximately 60%. As can be seen, VMA and MOPEG are easily detected (using amperometry) at their normal levels of 4-5 ng/mL. The alternative method of Scheinin et al. (1983) uses 3-ethoxy-4-hydroxyphenylglycol (EHPG) as an internal standard and determines EHPG and MOPEG after an ethyl acetate extraction. The MOPEG peak (Fig. 24) is apparently free of interfer-

156

Anderson

NE

-19

‘Q

min

min

Fig. 22. Determination (LC-F) of NA (NE) and A (E) standards (left, 200 pg each) and measurement of the compounds in an alumina extract of 0.5 mL human plasma (Yui et al., 1979, by permission).

05nA

I

OSnA

I

-tT-Gloljm

IO IS

TIME

20

25

! MINI

Fig. 23. From left to right: LC-EC chromatograms of standards (in order: 5 ng hydroquinone, VMA, and MHPG) A, plasma blank B, plasma blank with added standards C, and plasma sample from normal subject D. Plasma samples were prepared by reverse-phase small column-extraction, then separated as shown on a 5 pm (25 x 0.3 cm) C,s column (Ong et al., 1982, by permission).

Liquid Chromatographic

MINUTES

Analysis

I

L

1

15

10

5

of Monoamines

157

.L

AFTER INJECTION

Fig. 24. LC-EC determination of MOPEG (1) and EHPG (2 - the internal standard) in standards (A, 20 pmol each) and an ethyl acetate extract of human plasma (B) (Scheinin et al., 1983, by permission). ences, although judging from the standard peak and the injection volume (100 PL of 300 FL redissolved extract from 1 mL of plasma), an elevated level of -10 ng/mL is present in the sample shown. Comparison of the method to a GC-MS assay gave a correlation of 0.96 (N = 12) and C.V.s of 7-9% were reported at the 5 and 16 ng/mL level. A direct injection LC-EC method has been reported for HVA in human plasma (Javaid et al., 1983), using vaIVY 1

I 0

!! 5 8

I I5

?O

Fig. 25. Chromatogram of tyrosine (Tyr), TP (Try), and the internal standard (B-2-thienyl-D,L-alanine) in 50 FL of deproteinized human plasma. Compounds were separated on a 25 x 0.4 cm reverse-phase column and detected fluorometrically (280 nm excitation, >330 nm emission wavelengths) (Neckers et al., 1980, by permission).

158

Anderson

nillic acid as an internal standard. Although the method is not particularly well characterized, HVA is apparently determined accurately at normal 10 ng/mL levels. As shown (Fig. 19), the other major DA metabolite, DOPAC, has been determined m human plasma after an alumina extraction (Mefford et al., 1981)

3.3. Indoles in Blood Serotonm (5HT) and its precursors TP and 5HTP, and the malor metabolite 5HIAA, have been widely determined m serum, plasma, and whole blood. Tryptophan, typically occurrmg at 10 kg/mL levels, is easily determined in blood using ultraviolet absorbance (Krstulovic et al., 1977), amperometric (Koch and Kissinger, 1979; Krstulovic et al., 1981) or fluorometric (Beck and Hesselgren, 1980, Krstulovic and Matzura, 1979, Neckers et al., 1980; Anderson et al , 1981; Morita et al., 1981, Inoue et al., 1983) detection after HPLC separation. Fluorometry appears most well suited to determining TP m plasma. when using emission and excitation wavelengths optimized for the mdoles, TP is the one malor peak observed m an unpurified sample (Beck and Hesselgren, 1980, Krstulovic and Matzura, 1979). If emission and excitation wavelengths of 280 and 330nm are used, TYR can be determined in human plasma, along with TP and an internal standard (B-2-thienyl-D,L-alanme), as shown in Fig. 25 (Neckers et al., 1980). This chromatogram can be compared to Fig. 34 where TYR and TP are determined in human CSF using fluorometrlc detection with excitation and emission wavelengths of 254 and 360 nm, respectively. Tryptophan often has been measured along with a host of other amino acids using fluorometric (Lmdroth and Mopper, 1979; Turnell and Cooper, 1982) or amperometric (Joseph and Davies, 1982) detection of OPT derivatives. A chromatogram of the reverse-phase gradient separation of TP and a number of other important amino acids m human serum is presented m Fig. 26 (Turnell and Cooper, 1982) The LC-F OPT methods for ammo acid analysis appear to offer advantages m terms of sensitivity, selectivrty, and ease and rapidity of measurement over the ninhydrin and dansyl derivative procedures In contrast to TP, 5HTP, the immediate precursor to 5HT, occurs at extremely low (approximately 1 ng/mL) levels m human plasma. At this concentration difficulties are encountered m obtaming sufficient selectivity and sensitivity. Two LC-F methods (Anderson and Purdy, 1979; Engback and Magnussen, 1978) and an LC-EC method (Tyce and Cragan, 1981) have been reported for determining 5HTP in human plasma. Only one of the meth-

Liquid Chroma tographic

Analysis of Monoamines

159

ods is capable of determining 5-HTP in at least some normal human plasma samples (Anderson and Purdy, 1979) as the other LC-F procedure and the LC-EC method both have detection limits of -10 ng/mL. In the sample shown in Fig. 27 (Anderson and Purdy, 1979) 5HTP is determined at the 3.3 ng/mL level in a patient treated with a decarboxylase inhibitor. Even with the 0.5 ng/mL detection limit obtained using reverse-phase LC-F, 5HTP levels are often undetectable in normal subjects (Young et al., 1982). The measurement of 5HT in blood presents some special problems. Nearly all of the 5HT in blood is contained in the platelet and 5HT may be fairly easily determined in platelet-richplasma (PRP-prepared by low-speed centrifugation of whole blood) by LC-EC (Koch and Kissinger, 1979; Sasa et al., 1978; Petrucelli et al., 1982) or LC-F (Morita et al., 1981). Measurements made in PRP are complicated by difficulties in obtaining plasma with high and consistent (inter and intrasubject) yields of platelets. Several LC-EC methods also have been developed in order to (a)

. .. ..

,.,’ ,: . ,/

..

..___ . . . ..*

,:.

Fig. 26. Reverse-phase gradient (methanol) separation of OPT derivatives of serum amino acids with fluorometric detection. Peaks labeled 20, 25, and 26 are TYR, phenylalanine, and TP, respectively (Turnell and Cooper, 1982, by permission).

160

Anderson

measure 5HT in platelet-poor-plasma (PPP), where low levels (-10 ng/mL) are found (Koch and Kissinger, 1980; Tagari et al., 1984). These measurements are difficult to interpret as it is not clear what amount of the 5HT in PPP simply arises from the blood drawing and PPP preparation procedure. Serotonin is usually most usefully measured in whole blood, and this may be accomplished with either LC-F (Anderson et al., 1981b) or LC-EC (Korpi, 1984). The LC-F assay measures 5HT after deproteinization in the presence of the antioxidant ascorbic acid. Direct in-

-HTP tanda

10 mV

Aasma ;ample

rd

I 5-HTP

L

4-J

inj.

t

I

I

L

0

4

8

12

I I

I

16

20

*

min

of 5-HTP in 40 PL of Fig. 27. Reverse-phase LC-F determination deproteinized plasma (Anderson and Purdy, 1979).

Liquid

Chroma tographic

Analysis of Monoamines

161

jection of the supernate results in the chromatogram shown in Fig. 28 where 5HT is determined along with the internal standard (5HTP) and TP. The LC-EC method (Korpi, 1984) requires a prior deoxygenation of the whole blood using carbon monoxide due to a limitation on the amount of the electroactive ascorbic acid that may be added. The major metabolite of 5HT, 5HIAA, has been widely measHPLC-FLUOROMETRIC DETERMINATION OF SEROTONIN IN HUMAN WHOLE BLOOD (204)

I!

5HTP

5H 5f7+J, 3.0secm.

TRP (50mV)

INJ

I

I

1

0

2

JL

1 4

1 6

I 8 mm

Fig. 28. Determination of 5-HT, TP, and the internal standard (5-HTP) in deproteinized human whole blood after reverse-phase separation (10 pm, 30 X 0.39 cm Cl8 column) and fluorometric detection. Previous studies had established that endogenous 5-HTP levels were insignificant compared to the 400 ng/mL added as an internal standard (Anderson et al., 1981).

162

Anderson

ured in urine; however, only a handful of HPLC methods have been reported for its determination rt plasma Of these, two use fluorometric detection with (Morita et al , 1981) or without (Krstulovrc et al., 1981) prehmmary sample purification, and two utilize amperometric detection, again with (Koch and Kissinger, 1979) or without (Martmez et al., 1983) a prepurification. Although the LC-F methods present chromatograms showing well defined 5HIAA peaks neither gives data on normal levels observed in plasma. The two LC-EC methods report normal levels of 10.2 ng/mL (Koch and Kissinger, 1979) and 9 9 ng/mL (Martinez et al., 1983) m human plasma, demonstrating excellent interassay agreement

4. Cerebral Spinal Fluid (CSF) 4.1. Catechofamine Metabolites in CSF The inrtral method for catecholamme metabolites m CSF determined HVA and DOPAC (with 5HIAA) m 5 FL of rabbit ventrrcular CSF after direct mlection on an anion-exchange column (Wightman et al., 1977) The compounds were separated in approximately 12 min and could also be determined in rat CSF samples although no chromatograms or data on rat samples were presented. A number of methods for one or more of the catecholamine metabolites have followed employmg reverse-phase separation and electrochemical (Anderson et al., 1981~; Elghozl et al., 1983; Frattmi et al., 1982; Krstulovic et al , 1982; Mignot et al., 1982; Le Quan-Bui et al., 1982; Schemin et al., 1983; VanBockstaele et al., 1983; Wagner et al., 1982) or electrochemical/fluorometric (Anderson et al., 1979) detection. Several reports have u-tvolved the measurement of MOPEG alone (Anderson et al , 1981~; Frattini et al., 1982; Mignot et al., 1982), whereas others have measured MOPEG along with HVA (Krstulovic et al., 1982), or with HVA and 5HIAA (Langlais et al., 1980, VanBockstaele et al., 1983; Scheinin et al., 1983). In Fig. 29 (Anderson et al., 1981~) MOPEG is determined amperometrically after direct inlection of 40 PL of human lumbar CSF onto a reverse-phase column; in Fig. 30 MOPEG and HVA are determined in an ethyl acetate extract of human CSF using a gradient separation (Krstulovic et al., 1982). A direct inlection method for MOPEG, 5HIAA, and HVA in human CSF is presented in Fig. 31 (Scheinm et al. 1983). An internal standard of 5-fluorohomovanillic acid was used, separation was on a 5pm reverse-phase column, and electrochemical detection

Liquid Chromatographic Analysis ofMonoamines

CSF SAMPLE (40@)

163

CSF SAMPLE

*I

X2

(4Opl)

T 0.2

MHPG

nA

1

: I

I

0

4

8

12 TIME

16 (mm)

20

24

0

I

I

4

I

I

(

8 TIME

11

II

12

16

fi

20

11

24

(min)

Fig. 29. The determination of MOPEG (MHPG) in two different CSF samples using different C 1s columns. The MOPEG levels for the samples shown are 6.7 ng/mL (sample 1) and 10.5 ng/mL (sample 2). Electrode sensitivities established at the time each sample was run were 0.336 and 0.125 nA/ng MOPEG injected for samples 1 and 2, respectively (Anderson et al., 1981~).

was employed. In rat CSF, DOPAC (and 5HIAA) has been determined using reverse-phase LC-EC as shown in Fig. 32 (Le QuanBui et al., 1982). A 5 PL ventricular CSF sample was injected and then eluted using a pH 3.3 buffer containing 15% methanol. Depending on the species and compounds of interest, little difficulty should be presented in measuring MOPEG, DOPAC, HVA (and various indoles as will be discussed) in small volumes of CSF. In terms of the catechols, the method of Scheinin et al. (1983) would appear to offer the most flexibility, assuming that DOPAC can also be measured when appropriate (e.g., in rat), and assuming that the use of an internal standard is not an absolute requirement.

164

Anderson

4.2. Indoles in CSF Several of the CSF methods discussed in the previous section also measured 5HIAA All but one (Anderson et al , 1979) of the methods for catechols and 5HIAA used amperometric detection solely. Here the LC-F methods (and LC-F/EC method) for TP and/or 5HIAA m CSF will be reviewed Of the two mitral LC-F methods for determining 5HIAA m CSF, one determined 5HIAA after an ethyl acetate extraction (Beck et al , 1977) whereas the other measured 5HIAA and TP after the direct mlection of CSF (Anderson and Purdy, 1977). In Fig 33 (Beck et al., 1977) 5HIAA is determined along with the internal standard, 5-hydroxymdolepropiomc acid, after mlectmg 10 FL of a redissolved extract (equivalent to 0.4 mL of CSF) The method was well correlated (r = 0 97) with GC-MS In most of the direct inlection LC-F reports both TP and 5HIAA are determined (Anderson et al , 1978, Anderson and Purdy, 1977, 1979), m one method TP alone was determined 1980). By adding an electrochemical de(Beck and Hesselgren, tector and slightly changing the mobile phase composition, TYR and HVA were determined along with the mdoles shown in Fig. 34 (Anderson et al., 1979). The 5HIAA level is determined both amperometrically and fluorometrically, whereas TYR and TP were determined with fluorometry and HVA was measured using amperometric detection. The levels determined for 5HIAA were highly correlated (v = 0.99 and 0.95) in the two studies comparmg the LC-EC and LC-F values (Anderson et al., 1979, Anderson et al., 1983) These and other (Westermk, 1982, Beck et al , 1977) comparisons of LC-F and LC-EC methods with each other and with GC-MS methods have validated the HPLC assays and have suggested that they are at least as accurate as the GC-MS assays

5. Trace Amines and Metabolites 5.1. Tryptamine and Metabolites The behaviorally active trace amme tryptamme (T) is formed from TP after simple decarboxylation. The low levels (cl rig/g) of T present m brain and CSF have frustrated its determination in those samples by HPLC. However, higher levels of its malor metabolite, indoleacetic acid (IAA), are present, and the strong fluorescence of IAA has enabled it to be determined m brain and CSF using LC-F It should be mentioned that, as with the monoamine neurotransmitters, the measurement of malor metab-

Llquld

0

Chromatographrc Analysis ofMonoamlnes

5

IO

I5

20 TIME

25

165

30

35

(mm 1

Fig 30 LC-EC determmatlon of MOPEG, HVA, and VMA m an ethyl acetate extract of human CSF after reverse-phase gradlent separation The equivalent of 180 FL of CSF was injected on a 5 km (25 x 0 46 cm) C18 column (Krstulovlc et al , 1982, by permlsslon)

olites will often provide more mformatlon concernmg turnover and functional status than the determination of the amine itself Brain levels of IAA have been determined in the rat (Anderson et al , 1979, Anderson and Purdy, 1979) and mouse (Yamada et al,, 1983a, 1983b). As shown m Fig 35 (Anderson and Purdy, 1979),

166

Anderson -

-1

15

II

10

5 MINUTES

0 AFTER

_L

I

15

10

5

J

0

INJECTION

Fig. 31 LC-EC determmatlon of MOPEG (l), 5-HIAA (2), HVA (3), and 5-fluorohomovamlllc acid (5, the internal standard) after lsocratlc reverse-phase separation on a 5 pm (250 x 0 46 cm) Cl8 column (Schemm et al , 1983, by permlsslon)

IAA can be determined m rat brain after the direct mlectron of 50 FL of centrifuged brain homogenate An absolute detection limit of 5 pg was observed for IAA using excitation and emission wavelengths of 254 and 360 nm, respectively. Average levels of 8 3 rig/g were observed m rat brain, similar to the 9 0 rig/g levels seen m the mouse brain (Yamada et al , 1983b), and in good agreement with GC-MS determinations (Warsh et al., 1977). Indoleacetic acid has also been determined m rat and human CSF (Beck et al., 1977, Anderson and Purdy, 1979, Anderson et al , 1979) In the method of Beck et al (1977), previously presented m Fig 33, IAA 1s determined along with 5HIAA m an ethyl acetate extract of 2 mL of human CSF The direct mlection method of Anderson and Purdy (1979) illustrated m Fig 36 is suitable for both human and rat CSF The volume hmitations (25-100 PL total sample) imposed on determmations m the rat necessitate the use of an LC-F system with absolute detection limits m the low observed picogram range due to the -5 ng/mL concentrations Although similar concentrations are observed m human lumbar CSF, samples of several mL are often available and extracts contammg ng quantities can be prepared, enabling less sensitrve mstruments to be employed

Llquld

Cbromatographlc

Analysis

167

ofMonoamines

1;

I

c

/

I 15

\o

I 10

I 5

I RflENllON

7r II

TIME

(min)

Analym of standards (right trace) and a 5 ~J,L dlrectlyFig 32 mlected rat crsternal CSF sample DOPAC and 5-HIAA are determmed by LC-EC, separation was achieved using a pH 3 5 15%’ methanol mobile phase to elute a 10 km (30 X 0 39 cm) reverse-phase column (LeQuan-l3ul et al , 1982, by permrsslon)

Indoleacetrc acid has also been determined in blood (Anderson and Purdy, 1977; Martinez et al., 1983) and urine (Anderson and Purdy, 1979; Beck et al., 1977). Both of the methods m blood Involve direct mlection of deprotemized plasma on LC-F reverse-phase systems. The determination in urine is performed either after an extraction (Beck et al., 1977) or by direct mlectron

of 1 PL of urine

(Anderson

and Purdy,

1979). The extrac-

tion method also permits analysis of 5HIAA, whereas the du-ectmlection techniques allows for the simultaneous determination of mdoxyl sulfate and tryptophan. Most of the trace amines are not sensitively detected by native fluorescence and, although a fluorogenic reaction (e g., OPT fluorescamine, dansyl) with the amme

group

could

theoretrcally

produce

the sensrtrvrty

required

168

Anderson

to determine the compounds, the derivative been widely used (Davis et al., 1978)

methods have not

5.2. Phenolic Trace Amines The phenolic trace ammes, such as tyramme (TA) and octopamme (OA), can be determined with high sensitivity using LC-EC systems. In Fig. 37 (Shoup and Kissinger, 1977), TA 1s determined m human urine, along with other related ammes, using 3-methoxy4-hydroxybenzylamme as an internal standard A two-step purlflcatlon scheme was employed to isolate an amine-contammg fraction that was separated using ion-pair reverse-phase HPLC More recently, Bailey et al (1984) and Martin et al (1984) have 2

1

‘0

I

I 4

I 8

I

MIN Fig 33 LC-F determmatlon of 5-HIAA (I), 5-hydroxymdoleproplonlc acid (2, the internal standard), and IAA (3) m an ethyl acetate extract of human CSF The equivalent of 0 4 mL of CSF was mjetted, the compounds were separated on a reverse-phase Cs column (10 pm, 30 x 0 4 cm), and fluorescence excited at 272 nm (Beck et al , 1977, by permlsslon)

Liquid

Chromatographic

Analysis

169

ofMonoamines

AMPEROMETRIC

FLUOROME T RIG IO Lens

I

5 mV TRP

(after peak)

Fluorometric (lower trace) and amperometric (upper Fig. 34 trace) detection of TYR, TP, 5-HIAA and HVA in 20 I.LL of human CSF. Separation was by reverse-phase chromatography on a 10 km (30 x 0.39 cm) Cl8 column (Anderson et al., 1979) employed HPLC with a dual coulometric detection system for analysis of TA and OA m rodent brain and insect nervous tissue

6. Urine 6.1. Urine Catecholamines Quite unlike the situation in brain, in urine the catecholamines occur at substantially lower levels than their major metabolites And, although the absolute amounts (tens to hundreds of nanograms) of the catecholammes contained m a typical urine sample are similar to the levels seen m brain samples, a greater

170

Anderson

spiked

brain

sample

10 mV

IAA

IPA

. b

LL

4 .

*8

12

16

q

mln

Fig 35 LC-F measurement of IAA m rat bram homogenate Separation was on a 30 X 0.39 cm 10 km CIR column eluted with pH 4.25 buffer contammg 30% acetonltrlle Endogenous concentration of IAA was 9 rig/g (Anderson and Purdy, 1979, by permlsslon).

Liquid

Chromatographlc

Analysis

of Monoamines

171

degree of sample purification is usually required. As with brain determmations, LC-EC and LC-F (with or without derivatization) techniques have been widely employed and provide a convenient division for discussion of the methods Many of the LC-EC methods have employed a two-step purification procedure usmg a small cation-exchange column followed by alumma extraction of an appropriate fraction (Kissinger et al., 1977; Riggm and Ktssmger, 1977, Hansson et al , 1979, Hoeltke and Stetson, 1980). In Fig 38 (Riggm and Kissmger, 1977), a typical LC-EC chromatogram obtained after such a twostep procedure IS presented The chromatogram is notable for the absence of any sigmficant noncatecholamme peaks. Various other preliminary purification procedures have been employed before reverse-phase LC-EC Chromatograms obtained after boric acid gelialumma (Moyer et al., 1979), reverse-phase/silica gelialumma (Goldstein, 1983), and ion-pair (Smedes et al , 1982) extraction appear quite similar All are free of interferences and appear able to determine A m the low-normal range (1-5 ng/mL) On the basis of recovery and simplicity, the ion-pair extraction procedure of Smedes et al (1982) seems to offer advantages over the two-step procedures However, this is only to suggest that it might be the best of several good alternatives. Several groups have commented on the difficulties encountered when attempting to determine the catecholammes by ionpair reverse-phase LC-EC after only alumma extraction However, the compounds can be determined m an alumma extract if cation-exchange HPLC 1s used for the analytical separation (Eriksson et al., 1983) As with plasma, the combmation of selectIves obtained when using alumma extraction and cation-exchange HPLC appears superior to that observed with alumina and ionpair HPLC. However, when alumina m a packed precolumn is used followed by ion-pair LC-EC, the catecholammes apparently can be measured without interference (Goto et al , 1981) Unfortunately, the procedure is lengthy and the apparatus complicated Initially, the urme catecholammes were detected using LC-F systems only after pre- or postcolumn fluorogemc reactions Numerous LC-F procedures have been reported using trihydroxymdole (Yoshida et al , 1982), dansyl, and fluorescamme (Imai and Tamura, 1978) derivatives None of the various derivative-based methods (of which the above are listed by way of example only) appear to offer any advantages m selectivity over the native fluorescence LC-F procedures now available (Anderson et al , 1981d, Jackman, 1981) Shown m Fig. 39 (Anderson et al., 1981d)

172

10 mV

Anderson

I IAA

I

in]

I

IPA .

0

2

*

L IPA

I

0

IAA

inj

.

4

.

6

.

8 ,u

8

min

Fig 36 The determmatlon of IAA and mdoleproplomc acid (IPA) m two different rat cisternal CSF samples Fifty PL of CSF was mlected on a reverse-phase LC-F system. levels of IAA and IPA were -5 and -2 ng/mL, respectively (Anderson and Purdy, 1979, by permission).

Llquld

Chromatographic

Analysis ofMonoamlnes

173

3MT

I 5HT

I

I

I

I

I

0

4

8

12

16

I

20

minutes

Fig 37 The LC-EC determmatmn of normetanephrme (NM), metanephrme (M), tyramme (T), the internal standard 3-methoxy-4hydroxybenzylamme (IS), 3-methoxytyramme (3-MT), and 5-HT m a cation-exchange/solvent extraction-purified urine sample (Shoup and Klssmger, 1977, by permlsslon).

174

Anderson

is the chromatogram obtained after mlection of a cation exchange/ alumina purified urine sample on an ion-pair LC-F system Given the ability of the native LC-F procedure to measure A (at subnanogram/mL levels) along with NA and DA, the potential sensitivity advantage of some of the derivatives is not compellmg In many cases a one-step prelimmary purification can be used with the derivative methods, but this advantage is usually mmgated by the difficulties involved m the derivatization process

6.2. Catecholamine Metabolites in Urine The acidic catecholamme metabolltes VMA, HVA, and DOPAC occur at -1-10 kg/mL levels in normal human urine At those levels sensitivity should not be a malor problem when using LC-EC or, at times LC-F and HPLC-UV absorbance systems. However the specificity problems that might be encountered when determining the compounds m urine can be appreciated by referring to the chromatograms of Molnar and Horvath (1977) where a tremendous number of UV-absorbing and fluorometric peaks were observed in an ethyl acetate extract of acidified urine. Several of the HPLC methods for VMA employ an ethyl acetate extraction followed by reverse phase LC-EC analysis (Morrisey and Shihabi, 1979; Joseph et al., 1981; Moleman and Borstrok, 1983). The methods appear able to determine VMA at normal levels, however, the presence of late eluting peaks and/or the absence of internal standards compromises the techniques. Other LC-EC approaches have been to oxidize extracted VMA to van&n before HPLC separation (Felice and Kissinger, 1977), to use a small-column anion exchange preparatory step before ionpair (tetrabutylammoma) HPLC (Soldm and Hill, 1980) or to directly mlect urine on an LC-EC system with a low (0 6 V vs Ag/ AgCl) electrode potential (Fulita et al , 1983) UV absorbance detection has also been employed after ethyl acetate (Yoshida et al , 1982) or carbon black (Lagana and Rotatori, 1983) extraction. An LC-F method using precolumn dansylation of VMA and the internal standard, p-hydroxybenzoic acid, m an ethyl acetate extract, has also been reported (Yamada et al , 1981) Although all the methods discussed are workable, none is especially compelling. Urinary HVA has also often been determined using LC-EC systems Several of the methods were previously mentioned regarding the simultaneous determination of VMA (Joseph et al , 1981, Yoshida et al., 1982; Fulita et al., 1983; Soldm and Hill, 1980) Other LC-EC methods have determined HVA after a com-

Liquid

Chromatographlc

175

Analysis ofMonoamlnes

NE

I L

DA

EPI

x10

x2

dI I I Xl -J-.1-

1

0

2

0

6

8

MIN Fig 38 The LC-EC analysis of NA (NE), A (El?), and DA m a cation exchange/alumma extract of human urine DA 1s determined at a reduced sensltlvlty relative to the other catecholammes (Rlggm and Klssmger, 1977, by permlsslon).

176

Anderson

plicated extraction and TLC isolation procedure (Felice and Krssmger, 1976), following a small-column alumma fractronation (Mitchell and Coscia, 1978), or by direct mlection of urine (Seegal et al., 1983). As with VMA, none of the procedures and/or chromatograms are especially attractive, and nearly all of the methods appear borderline in terms of sensitivity and specificity when measuring HVA at normal levels of -1-5 kg/mL (Seegal et al , 1983; Mitchell and Coscia, 1978; Fugita et al , 1983, Soldm and Hill, 1980; Yoshida et al., 1982). A well-defined robust HVA peak (albeit, partially spiked) is observed m the method of Joseph et al. (1981), but unfortunately a chromatographic run of over 25 min was required and recoveries were rather variable (74.6 + 11.5%, C.V. 15.4%). The less commonly determined DA metabohte DOPAC was first assayed, after ethyl acetate and alumina extraction, using anion exchange chromatography (Felice et al., 1977). In addition, it has been measured along with HVA and VMA by the method of Joseph et al. (1981). A relatively consistent recovery of DOPAC (80.1%; C.V., 10%) through an ethyl acetate extraction was observed; however, it was not clear how well the identity of the DOPAC peak was established. DA

URINE SAMPLE

STANDARDS NE

DHEA NE

IOnl” (losens) (loransl , I I I I I I ! I,+\ , II 50mV

EPI

I

0

2

4

6

E

IO ml”

02468

IO min

Frg 39. Chromatograms of catecholamme standards (25 ng) and a cation-exchangeialumma extract of human urine The equivalent of 0 4 mL of urine was injected on a reverse-phase ion-pair LC-F system and the compounds detected fluorometrlcally with excltatlon and emlsslon wavelengths of 285 and 305 nm, respectively (Anderson et al , 1981d, by permission)

Llquld Chromatographic Analysis ofMonoamines

177

The LC-EC determination of MOPEG (1) and ISOFig 40 MOPEG (2) m an extracted standard (left) and human urme (right) Approxrmately 1 @mL of the internal standard was added (Shipe et al , 1984, by permlssron)

1

I

36

-Y 5nC. I i IWJ I Iru I 1 4

32

26

24

20

16

12

6

4

0

MINUTES

Fig. 41 Chromatograms of MOPEG (MHPG) standards (right) and MOPEG determined m a reverse-phase purified fraction of unextracted hydrolyzed human urine The standard was collected m 1.58 mL, collectron volumes for the samples are given below the trace One hundred FL of the collected purified fraction was iqected on the reverse-phase analytical system and detected by amperometry as shown (Anderson et al , 1983, by permission)

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Anderson

One of the first LC-EC methods for the analysis of the hydrophilic alcohol MOPEG in urine employed an ethyl acetate extraction followed by periodate oxidation to vamllin and reduction to vamllyl alcohol (Buchman et al., 1979). This approach was taken after unsuccessful LC-EC attempts were made to determine MOPEG directly in an ethyl acetate extract. Subsequently, a number of LC-EC methods have been reported usmg an ethyl acetate extraction as the first step of more extensive isolation procedures. Several of the more well characterized procedures use back extractions into borate and acetate buffers (Santagostmo et al., 1982, Moleman and Borstrok, 1982; Shipe et al., 1984) The LC-EC chromatogram resulting from such a multistep extraction procedure is shown m Fig. 40 (Shipe et al., 1984) Iso-MOPEG was used as an internal standard and a day-to day coefficient of variation of 3.8% was observed Similar chromatograms were obtained usmg the other two LC-EC back-extraction methods, however several assays using one-step ethyl acetate extraction required more lengthy chromatography, used no internal standards, and were not fully described (Joseph et al., 1981, Krstulovic et al., 1980). The lack of an internal standard also compromises to some extent the LC-F method of Taylor et al. (1981), although specific detection of MOPEG’s native fluorescence (265 nm excitation, 310 nm emission) does permit MOPEG to be easily determined, with a retention time of 5 mm, in a urine extract. Two additional LC-EC techniques have measured urme MOPEG after two-dimensional chromatography. One used thm layer chromatography (TLC) of an ethyl acetate extract, followed by analytical separation by reverse phase HPLC (Alonso et al., 1981). The resulting LC-EC chromatogram (not shown) is probably the most free of extraneous peaks of any of the LC methods, but the laborious multiple-step procedure and lack of internal standard causes a rather high C.V. (12.5% day-to-day). An alternate two-dimensional separation uses reverse-phase columns for both the purification and analytical separation. The chromatogram shown m Fig. 41 (Anderson et al., 1983) illustrates the analysis of MOPEG m a fraction that was purified by a previous HPLC collection step. Diluted, hydrolyzed urme was directly inJected for the collection step that took approximately 8 mm Recovery of MOPEG was quantitative (-loo%), the total separation time required (collection and analysis) was less than 15 mm per sample, and no extraction was necessary. The basic catecholamine metabolites metanephrme (MN) and normetanephrine (NMN) occur at lower levels than most of the

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179

HT

l-

i 1 0

1

1

4

1

1 8

1

1

12

minutes

Fig 42 Determination of urinary 5-HT by LC-EC. A 5-HTcontammg fraction was isolated from a cation-exchange purification scheme and mlected on a reverse-phase 10 km Cl8 column. Concentration in the sample shown was 103 ng/mL (Koch and Kissinger, 1979, by permission) other commonly measured urine metabohtes. Typical concentrations are 100-200 ng/mL and the compounds are usually found in the coqugated form The method of Shoup and Kissinger (1977) involves a small column cation-exchange separation followed by a complex solvent extraction with final LC-EC detection after reverse phase chromatography. In the chromatogram shown m Fig. 37, the metanephrmes are determined along with the internal standard, 3-methoxy-4-hydroxybenzylamine. The preliminary purification procedure was shortened to single-step small column

180

Anderson

cation exchange chromatography m the method of BertoniDziedzic et al. (1981) Two well resolved peaks were apparent for NMN and MN after gradient reverse phase HPLC, however, determmations in normal sublects were made with a precision of only one or two sigruficant figures. No internal standard was used, and no mformation was given on recoveries observed. Postcolumn oxidation of the metanephrmes to vanillin was employed by Flood and McComb (1981). The compounds were separated by reverse phase HPLC after cation-exchange purification and were detected by UV absorbance Here, too, no internal standard was used and the utility of the method m measuring low-normal levels of the metanephrmes is difficult to ascertain The more complicated cation exchange/solvent extraction procedure of Shoup and Kissinger (1977) was used by Jackman (1982) preparatory to determmmg the compounds by HPLCfluorometry after ion-pair reverse phase separation The chromatogram (not shown) appears remarkably similar to that of Shoup and Kissinger (1977) (Fig 37), although the compounds are determined in about half the time. Jackman (1982) used tritiated NMN as the internal standard and excited the native fluorescence of the compounds with a deuterium lamp (200 nm). The most extensively characterized of the LC methods is that of Orsulak et al, (1983). Also using the purification procedure of Shoup and Kissinger (1977), these workers separated the compounds using a slightly different reverse phase column The separation was evidently improved and speeded-up (-10 mm/sample) and CVs of 1 4% and 2.9% (within-day) were obtamed for NMN and MN. A comparison of previous GC-MS, fluorometric, and LC-EC determmations was given and mdrcated good agreement across methods. Although urine metanephrines usually occur at higher levels than the catecholamines, no really simple selective extraction procedure exists for their isolatron and this contmues to make their analysis difficult if, m some cases, accurate and precise

6.3. Indoles in Urine Tryptophan occurs m urine at levels similar to those seen in plasma (-10 kg/mL). Levels of urinary 5HIAA are also in the pg/mL range (l-5 kg/mL), whereas 5HT concentrations are substantially lower (-100 ng/mL) Although TP was not detected m normal urine using HPLC with IJV absorbance detection (Grushka et al., 1977), the relatively high levels could be detected by directly mjectmg diluted urine on reverse phase LC-F systems

Liquid Chromatographic Analysis ofMonoam/nes

181

(Anderson and Purdy, 1979; Graffeo and Karger, 1976). Neither of the LC-F methods appears completely optimized for determining TP in urine as in both the TP is not baselme-separated. An LC-EC method (Koch and Kissmger, 1979) is better characterized; however, a lengthy multistep small column isolation procedure is required before reverse phase LC-EC separation and measurement. Urmary TP can also be determined along with many other ammo acids using OPT LC-F methods (e.g , see Turnell and Cooper, 1982).

It is unclear what meaning should be given to urine 5HT levels, and the neurotransmitter has been only rarely determined in urine. One of the HPLC procedures measures 5HT after a onestep small column cation exchange isolation (Koch and Kissinger, 1979); the other follows the small column-step with a solvent extraction (Shoup and Kissinger, 1977) This latter procedure was also used to determine the metanephrmes, and a chromatogram was previously presented m Fig 37. The simpler, but apparently more specific, one step procedure results m the chromatogram shown m Fig 42. The numerous HPLC methods for 5HIAA in urine are fairly evenly divided into those using amperometric (LC-EC) (Koch and Kissinger, 1979; Fugita et al., 1983; Joseph et al., 1981), fluorometric (LC-F) (Beck et al., 1977; Rosano et al., 1982; Tracy et al., 1981), and UV absorbance detection (Draganac et al., 1980, Fornstedt et al., 1978; Yamaguchr et al , 1982). The LC-EC methods using a solvent extraction (Joseph et al., 1981) or small column preparation (Koch and Kissinger, 1979) determine 5HIAA specifically; however, the direct inlection method of Fugita (1983) determine 5HIAA in the midst of other peaks and, because a concentration was not given for the chromatogram presented, it is difficult to Judge the potential for interferences A high correlation (Y = 0.98) was observed between direct inlection and solvent extraction methods and the low electrode potential (+0 45 mV vs Ag/AgCl) probably contributes to the method’s apparent specificity (Fugita et al., 1983) None of the LC-EC procedures uses an internal standard. The three LC-F procedures all utilize a solvent extraction followed by reverse-phase separation and detection of 5HIAA’s native fluorescence. Internal standards of 5-hydroxymdolepropiomc acid (5HIPA) (Beck et al., 1977; Rosano et al , 1982) or 5-hydroxyindole-2-carboxylic acid (5HICA) were used The method of Rosano et al. (1982) (Fig. 43) appears to be a slight improvement over that of Beck et al. (1977) in that fewer extraneous peaks were observed. However, it IS unclear whether this im-

Anderson

182

Standard

4

ua f ;o

: i i?,

00 c’ -I--di4

1,

t0 Z

00 ‘Z

bLi

b h 4 Minutes

After

Li

In jectlon

Fig. 43. Analysis of 5-HIAA and the internal standard 5-HIPA m an ether extract of normal human urine by reverse-phase LC-F The equivalent of -5 FL of urine was mlected, the compounds were detected with excitation and emlsslon wavelengths of 300 nm and 350 nm, respectively (Rosano et al., 1982, by permlsslon)

1s due to the use of different excltatlon and emlsslon wavelengths (300 and 360 nm instead of 272 and >370 nm) or results from using ether instead of ethyl acetate in the extraction procedure. It 1s also not clear why an IAA peak, present m the method of Beck et al (1977), IS not seen

provement

7. Conclusions The foregoing overvlew of the methods that have been developed for the monoammes and then metabolltes m physlologlcal samples demonstrates

the great flexlblhty

and umversahty

of the

HPLC approach. The ease, specificity, and sensitivity of the methods have been most welcome in neurochemical research lab-

Liquid

Chromatographic Analysis ofMonoamines

183

oratories and the clmlcal chemistry milieu. Especially attractive is the relatively low cost involved m setting up state-of-the-art LC-EC and LC-F systems and the fact that such systems (especially in combmatlon) allow a tremendously wide range of compounds and sample types to be analyzed. Even without further improvement in the instrumental aspects of the techniques they should continue to be extensively employed in the coming decade

Acknowledgments I would like to thank Karm Schlicht, Frederick Felbel, Lisa Wetlaufer, and, especially, Marjorie Buccmo for their help m preparing this review.

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and 5-hydroxymdoleacetlc acid m human cerebrospmal fluid. ] Ckromatogr 277, 282-286 Anderson G M and Purdy W. C (1977) A liquid chromatographlcfluorometrlc method for the analysis of picogram amounts of tryptophan metabolltes m cerebrospinal fluid. Anal. Lett 10, 493499 Anderson G M and Purdy W C. (1979) Liquid chromatographlcfluorometrlc system for the determination of mdoles in physlologlcal samples. Anal Ckem. 51, 283-287 Anderson G M , Purdy W. C , and Young S N (1978) The determmatlon of neurologlcally important tryptophan metabolltes m brain and cerebrospmal fluid, m Trace Organic Analysis. A New Frontier tn Analytxal

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Anderson G. M and Young J G. (1981) Appllcatlons of liquid chromatographlc fluorometrrc systems m neurochemistry L$e Scl 28, 507-517 Anderson G M and Young J G (1982) Determmatlon of neurochemrcally important compounds m physlologlcal samples using HPLC. Schzzophrenta Bull 8, 33%348. Anderson G. M , Young J. G , Batter D K , Young S N , Cohen D. J,, and Shaywrtz B A (1981a) Determmatlon of mdoles and catechols m rat brain and pmeal using lrquld chromatography with fluorometrrc and amperometrrc detection 1 Chvomatogv 223, 315-320 Anderson G M , Young J G , and Cohen D J (1979) Rapid llquld chromatographrc determmatlon of tryptophan, tyrosme, 5-hydroxymdoleacetlc acid and homovamllrc acrd m cerebrospmal fluid. 1. Chromatogr 164, 501-505 Anderson G M , Young J G , Cohen D J , Schlrcht K R , and Pate1 N (1981b) Liquid-chromatographrc determmatlon of serotonm and tryptophan in whole blood plasma Clan Chem 27, 775-776 Anderson G M , Young J G , Cohen D J., Shaywltz B A , and Batter D K (1981~) Amperometrlc determmatron of 3-methoxy-4-hydroxyphenylethylenglycol m human cerebrospmal fluid ] Chromatogv 222, 112-115 Anderson G M., Young J G , Cohen D J , and Young S N. (1982) Determmatron of mdoles m human and rat pmeal ] Chromatogr 228, 155163 Anderson G. M , Young J G., Jatlow I’ , and Cohen D J (1981d) Urrnary free catecholammes determined using a llqurd chromatographic-fluorometrrc procedure Clan Chem 27, 2060-2063 Barley B A., Martin R J , and Downer R. G. H. (1984) A rapid and specific technique for the extraction of tyramme and octopamme from brologlcal tissues for HPLC analysis, m Neurobiology of Trace Amznes (Boulton A A , Baker G B , Dewhurst W.G and Sandler M , eds.), pp 85-90, Humana Press, Clifton, NJ Beck 0 and Hesselgren T (1980) Method for the determmatlon of tryptophan m serum and cerebrospmal fluid 1 Chromatogr 181, 10@102. Beck O., Palmskog G., and Hultman E. (1977) Quantltatrve determmanon of 5-hydroxymdole-3-acetic acid m body fluids by hrghperformance llquld chromatography Clan Chrm Ada 79, 149-154 Bertam-Dzredzrc L M , Krstulovlc A M , Dzledzlc S W , Gltlow S E , and Cerqueua S (1981) Analysis of urmary metanephrmes by reverse-phase high performance lrquld chromatography and electrochemrcal detection Clan Chrm Acta 110, l-8 Buchanan D N , Fucek F R , and Dommo E F (1979) Analysis of urrnary 4-hydroxy-3-methoxyphenylethylene glycol as vamllyl alcohol

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by high-performance liquid chromatography with amperometric detection ] Chromatogr. 162, 394400 Causon R. C. and Brown M J. (1983) Analytical differences m measurement of plasma catecholamines. C~UZ. Chem 29, 735737. Cross A. J, and Joseph M. H. (1981) The concurrent estimation of the malor monoamme metabohtes m human and non-human primate brain by HPLC with fluorescence and electrochemical detection LlfE SCl 28, 499-505. Davies C L and Molyneux G (1982) Routine determmation of plasma catecholammes using reversed-phase, ion pair high-performance liquid chromatography with electrochemical detection 1 Chromatogr. 231, 41-51 Davis G. C. and lssmger I’ T (1981) Strategies for determmation of serum or plasma norepmephrme by reverse-phase liquid chromatography. Anal Chem 53, 15f%159 Davis T. I’., Gerhke C W , Gerhke C W Jr , Cunnmgham T D., Kuo K C , Gerhardt K. O., Johnson H D., and Williams C H (1978) High-performance liquid-chromatographic separation and fluorescence measurement of biogemc ammes in plasma, urine and tissue Clan Chem 24, 1317-1324 Draganac P S , Stemdel S J , and Trawick W G (1980) Liquidchromatographic separation of urinary 5-hydroxy-3-mdoleacetic acid with measurement at 254 nm Clan Chem 26, 910-912 Elghozi J L , Mignot E , and Lequan-Bui K H (1983) Probemcidsensitive pathway of elimmation of dopamme and serotonm metabelites m CSF of the rat. ] Neural Trunsm. 57, 85-94. Engback F and Magnussen I (1978) Determination of 5-hydroxytryptophan m plasma by HPLC and fluorometric detection after phthaldialdehyde reaction. Clan. Chem. 24, 376-378. Eriksson B. M , Gustafsson S , and Persson B A (1983) Determmanon of catecholammes m urine by ion-exchange liquid chromatography with electrochemical detection ] Chromatogr 278, 255-263 Eriksson B M and Persson B A (1982) Determmation of catecholammes m rat heart tissue and plasma samples by liquid chromatography with electrochemical detection 1 Chromatogr 228, 143154. Felice L. J., Bruntlett C S., and Kissinger P T. (1977) A liquid chromatographic assay for 3,4-dihydoxyphenylacetic acid (DOPAC) in urine J Chromatogr 143, 407-410 Felice L J. and Kissmger I’ T. (1976) Determmation of homovamllic acid m urine by liquid chromatography with electrochemical detection Anal Chem 48, 794796 Felice L J and Kissinger P T (1977) A modification of the Pisano method for vamlmandelic acid using high pressure liquid chromatography. Clzll Chum Acta 76, 317-321

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Felice L. J.. Felice J D , and Kissinger I’ T (1978) Determmation of catecholammes m rat bram parts by reverse phase ion-pair liquid chromatography / Neurochem. 31, 1461-1465 Fenn R J , Srggra S , and Curran D. J. (1978) Liquid chromatography detector based on single and twm electrode thin-layer electrochemrstry Application to the determmatron of catecholammes m blood plasma Anal Chem. 50, 1067-1073 Flatmark T , Jacobsen S W , and Haavik J (1980) Fluorometrrc detection of tryptophan, 5-hydroxytryptophan, and 5-hydroxytryptamme (serotonm) m high performance lrqurd chromatography Anal Blochem 107, 71-74. Flood J G and McComb R B. (1981) Urinary metanephrmes as measured by liquid chromatography with an on-line post-column reaction detector Clan CIzem 27, 12681271. Fornstedt N (1978) Determmation of 5-hydroxymdole-3-acetic acid m urine by high performance liquid chromatography Anal Chem 50, 1342-1346 Frattml P , Santagostmo G , Cucchi M L , Corona G L , and Schmelli S (1982) 3-Methoxy-4-hydroxyphenylglycol m human cerebrospmal fluid Clln CIzlm Acfa 125, 97-105 Frayn K N and Maycock I’ F (1983) Sample preparation with ionexchange resin before liquid chromatographic determmation of plasma catecholammes Clan Chem 29, 1426-1428 Freed C. R. and Asmus I’. A (1979) Brain tissue and plasma assay of L-DOPA and a-methyldopa metabolites by high performance liquid chromatography with electrochemical detection ] Neurochem 32, 163-168 Fulita K , Maruta K , Ito S , and Nagatsu T (1983) Urmary 4-hydroxy-3methoxymandelic (vamllylmandelic) acid, 4-hydroxy-3-methoxyphenylacetic (homovamlhc) acid, and 5-hydroxy-3-mdoleacetic acid determined by liquid chromatography with electrochemical detection Clan Chem. 29, 87fG-878 Fuller R W and Perry K W (1977) Lowermg of epmephrme concentration m rat bram by 2,3-dichloro-a-methylbenzylamme, an mhibitor of norepmephrme N-methyltransferase Blochem Pharmacol 26, 2087-2090 Goldstem D S (1983) Modified sample preparation for high performance liquid chromatographic-electrochemrcal assay of urmary catecholammes. ] Chromafogr 275, 174-177 Goldstem D S and Feuerstem G. (1981) Improved reliabrlrty of the liquid chromatography-electrochemical detection assay technique for measuring plasma epmephrme Clan Chem 27, 508. Goldstem D. S , Feuerstem G , Izzo J L , Jr , Kopm I J , and Keiser H R (1981a) Validity and reliability of liquid chromatography with electrochemical detection for measuring plasma levels of norepmephrme and epmephrme n-t man L$e Scl 28, 467-475 Goldstein

D S , Feuerstem

G Z , Kopm I J , and Keiser H R (1981b)

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Validity of llquld chromatography with electrochemical detection for measuring dopamme m human plasma. Clm Ckzm Acta 117, 113-120. Goto M , Nakamura T , and Ishll D. (1981) Micro high performance llquld chromatographlc system with micro precolumn and dual electrochemlcal detector for direct mJectlon analysis of catecholammes m body fluids J Ckromatogr 226, 3342. Goto M., Zou G , and Ishll D. (1983) Determination of catecholammes m human serum by micro high-performance liquid chromatography with micro precolumn and dual electrochemical detection 1 Chromafogr. 275, 271-281 Graffeo A. P and Karger B. L (1976) Analysis for mdole compounds m urme by high-performance llquld chromatography with fluorometrlc detection Clm Chem 22, 184-187. Grushka E., Klkta E. J Jr., and Naylor E. W. (1977) Tryptophan and kynurenme determination m untreated urine by reverse-phase high pressure liquid chromatography J. Ckromatogr 143, 51-56. Hallman H , Farnebo L 0 , Hamberger B., and Jonsson G. (1978) A sensltlve method for determination of plasma catecholammes using hquld chromatography with electrochemical detectlon. Lffe Scr 23, 1049-1052 Hamall M. and Sekl T (1979) Estimation of catecholammes m human plasma by ion-exchange chromatography coupled with fluorlmetry J Chromatogr

163, 329-336

Hansson C., Agrup G , Rorsman H , Rosengren A M.. and Rosengren E (1979) Analysis of cystemyldopa, dopa, dopamme, noradrenalme and adrenaline in serum and urine using high-performance liquid chromatography and electrochemical detection. ] Chromatogr 162, 7-22 Hashimoto H. and Maruyama Y (1978) Development of an electrochemical detector for high-performance liquid chromatographlc assay of brain catecholammes ] Chromatogr 152, 387-393. Heft1 F (1979) A simple, sensitive method for measuring 3,4-dlhydroxyphenylacetlc acid and homovamlllc acid m rat brain tissue using high performance llquld chromatography with electrochemical detection. Lzfe Scz 25, 775-782 Hegstrand L R, and Elchelman B (1981) Determmatlon of rat brain tlssue catecholammes using liquid chromatography with electrochemical detection J Chromafog 222, 107-111. Hlemdahl I’., Daleskog M., and Kahan T. (1979) Determination of plasma catecholammes by high performance liquid chromatograwith electrochemical detection Comparison with PhY radloenzymatlc method I-+ Scz 25, 131-138 Hoeldtke R. D and Stetson I’. L (1980) Separation of urmary catecholammes and catecholamme metabohtes by high-pressure hquld chromatography Anal Btochem 105, 207-217. Holly J M. P and Makm H L J (1983) The estlmatlon of catecholammes m human plasma Anal Bzochem 128, 257-274

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Horl S., Ohtaru D., Ohtam S., Kayanuma K., and Ito T (1982) Srmultaneous determmatlon of tryptophan, serotonm, and 5-hydroxymdoleacetlc acid m rat brain by high-performance liquid chromatography using weak acidic cation-exchange resin ] Chromatogr 231, 161-165 Imar K and Tamura Z (1978) Liquid chromatographic determmahon of urinary dopamme and norepmephrme as fluorescamme derivatives Clm Chrm Acta 85, l-6 Imai K , Tsukamoto M , and Tamura Z (1977) High performance liquid chromatographic assay of rat brain dopamme and norepmephrme J Chromatogr 137, 357-362. Inoue S., Tokuyama T , and Taki K. (1983) Picomole analysis of tryptophan by denvatization to 9-hydroxymethyl-B-carbolme Anal Blochem 132, 468480. Ishikawa K and McGaugh J L (1982) Simultaneous determmation of precursors and metabohtes m a single mouse brain. J Chromafogr 229, 3546 Jackman G P. (1981) Differential assay for urinary catecholammes by use of liquid chromatography with fluorescence detection. Clan. Chem 27, 1202-1204 Jackman G. I-’ (1982) A simple method for the assay of urinary metanephrmes using high performance liquid chromatography with fluorescence detectron. C/m Chum Acta 120, 137-142 Jackman G I’ , Carson V J., Bobik A , and Skews H (1980) Simple and sensitive procedure for the assay of serotonm and catecholammes m brain by high-performance liquid chromatography using fluorescence detection 1, Chromafogr 182, 277-284 Javaid J I , Lm T S , Maas J W , and Davis J. M (1983) Measurement of 3-methoxy-4-hydroxyphenylacetic acid (HVA) m plasma by high performance liquid chromatography with electrochemical detection (HPLC-EC) Anal Biochem 135, 326-331 Jenner D. A , Brown M. J., and Lhoste F J. M (1981) Determmation of a-methyldopa, a-methylnoradrenalme, noradrenalme, and adrenalme m plasma using high-performance liquid chromatography with electrochemrcal detection. ] Chromafogr 224, 507-512 Joseph M H. and Davies P (1982) Electrochemical detection of ammo acids m plasma and brain using OPT derivation Bloanalytical Systems product literature, West Lafayette, IN. Joseph M. H , Kadam B. V , and Risby D. (1981) Simple high performance liquid chromatographic method for the concurrent determmation of the amme metabolites vamllylmandelic acid, 3-methoxy-4hydroxyphenylglycol, 5-hydroxymdoleacetlc acid, dihydroxyphenylacetic acid, and homovanillic acid in urine using electrochemical detection J Chromafogr 226, 361-368 Keller R , Oke A., Mefford I , and Adams R N (1976) Liquid chromatographlc analysis of catecholammes, Routme assay for regional brain mapping. Lzfe Scz. 19, 995-1004 Kempf E. and Mandel I’. (1981) Reverse-phase high-performance liquid chromatographic separation and electrochemical detection of nor-

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Analysis ofMonoamlnes

dopamme,

serotonm,

189

and related malor metabolrtes.

Blochem 112, 223-231

Kilts

C D , Breese G R , and Mailman R B (1981) Simultaneous quantification of dopamme, 5hydoxytryptamme, and four metabolrcally related compounds by means of reverse-phase hrghperformance lrqurd chromatography with electrochemical detection J Chromafogr 225, 347-357 Krssmger I’. T , Bruntlett C S , Davis G. C., Felice L J , Rlggm R M., and Shoup R E (1977) Recent developments m the clmrcal assessment of the metabolrsm of aromatics by high-performance, reversephase chromatography with amperometrlc detection Clan Chem 23, 1449-1455 Klssmger I’ T , Bruntlett C S , and Shoup R. E (1981) Neurochemrcal apphcatlons of lrqurd chromatography with electrochemical detection L$e Scr 28, 455465. Koch D D and Kissinger I’ T (1979) Determmatron of tryptophan and several of Its metabolrtes u-r physlologlcal samples by reverse-phase lrqurd chromatography with electrochemical detection I Chromatop 164, 441455. Koch D D and Kissinger P T (1980) Liquid chromatography with precolumn sample enrichment and electrochemical detection Regional determmatlon of serotonm and 5-hydoxymdoleacetrc acid m brain tissue Life Scl 26, 1099-1107 Koch D. D and Krssmger P T (1980) Determmatron of serotonm m serum and plasma by hqurd chromatography with precolumn sample enrichment and electrochemrcal detection Anal Chem 52, 27-32 Korpl E R (1984) Serotonm determined m whole blood by lrquld chromatography with electrochemical detection C/m Chem 30, 487488

Krstulovlc A. M (1982) Investrgatlons of catecholamme metabolrsm using high-performance lrqurd chromatography Analytical methodology and clmlcal applrcatrons. ] Chromatogr 229, 1-34 Krstulovlc A. M , Bertam-Dzledzlc L., Bautrsta-Cerquelra S , and Grtlow E (1982) Simultaneous determination of 4-hydroxy-3-methoxyphenylacetrc (homovamlllc) acid and other monoamine metabolites in human lumbar cerebrospmal fluid ] Chromatogr. 227, 379-389 Krstulovlc A. M , Brown P R , Rosre D M , and Champlm I’. B. (1977) High performance llquld-chromatographrc analysis for tryptophan m serum Clm Chem 23, 1984-1988 Krstulovrc A M , Friedman M J , Smclau P R , and Felice J (1981) Complementary use of amperometrlc and spectrophotometnc detection for concurrent momtormg of serum tryptophan metabohtes by reverse-phase llquld chromatography. Clan Chem 27,1291-1295. Krstulovrc A M and Matzura C (1979) Rapid analysis of tryptophan metabohtes using reverse-phase high performance liquid chromatography with fluorometrrc detectron. J Chromafogr 163, 72-76 Krstulovlc A. M , Matzura C. T., Bertam-Dzredzlc L., Cerquelra S., and Grtlow S. E (1980) Endogenous levels of free and conlugated urrnary 3-methoxy-4-hydroxyphenylethyleneglycol m control sublects

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and patients with pheochromocytoma determined by reverse-phase liquid chromatography with electrochemical detection Clan Chum Acta 103, 109-116 Krstulovlc A M. and Powell A M (1979) Use of native fluorescence measurements and stopped-flow scanning technique m the high performance liquid chromatographic analysis of catecholammes and related compounds. 1 Ckromatogr 171, 345-356 Lackovic Z , Parent1 M , and Neff M. H (1981) Simultaneous determmation of femtomole quantities of 5-hydroxytryptophan, serotonm, and 5-hydroxymdoleacetic acid m brain using HPLC with electrochemical detection Eur 1 Pkarmacol 69, 347-352 Lagana A and Rotator1 M (1983) High performance liquid chromatographic procedure for the analysis of urmary 3-methoxy-4hydroxymandelic acid 1 Ckromatogr 275, 168-173 Langlais I?. J , McEntee W J., and Bird E D (1980) Rapid liquidchromatographlc measurement of 3-methoxy-4-hydroxyphenylglyco1 and other monoamme metabolites m human cerebrospmal fluid Clan. Ckem 26, 786-788. LeQuan-Bul K H., Elghozi J L , Devynck M A , and Meyer I’. (1982) Rapid liquid chromatographic determmation of 5-hydroxy mdoles and dihydroxyphenylacetic acid m cerebrospmal fluid of the rat Eur J Pkarmacol 81, 315-320 Lm P Y T and Blank C L. (1983) Utilizmg 3 pm reverse-phase columns for LC-EC determmation of biogenic ammes, m Current Separatzons Bzoanalytrcal SystemsProduct Lderature, Vol 5(l), West Lafayette, IN, PP. 3-6 Lmdroth P and Mopper K (1979) High performance liquid chromatographic determmation of sub-plcomole amounts of ammo acids by precolumn fluorescence derivatlzation with o-phthaldialdehyde Anal Ckem 51, 1667-1670 Lyness W H., Friedle N M., and Moore K E (1980) Measurement of 5-hydroxymdoleacetic acid m discrete brain nuclei using reverse phase liquid chromatography with electrochemical detection Lzfe Scz 26, 1109-1114 Magnusson 0 , Nilsson L B , and Westerlund D (1980) Simultaneous determination of dopamme, dopac, and homovamllic acid: Direct mlection of supernatants from brain tissue homogenates m a liquid chromatography-electrochemical detection system J Ckromatogr 221, 237-247 Martm R J , Barley B A., and Downer R G H (1984) Analysis of octopamme, dopamme, 5-hydroxytryptamme and tryptophan m the brain and nerve cord of the American cockroach, m Neurohology of the Trace Amrnes (Boulton A A , Baker G B., Dewhurst W.G , and Sandler M , eds.), pp. 91-96, Humana Press, Clifton, NJ Martinez E , Artigas F , Sunol C , Tusell J M , and Gelpi E (1983) Liquid chromatographic determmation of mdole-3-acetic acid and 5-hydroxymdole-3-acetic acid m human plasma Cllir Ckem 29, 1354-1357

Maruyama Y , Oshima T., and Nakalima E (1980) Simultaneous deter-

Liquid

Chromatographic

Analysis

ofMonoamlnes

191

mmation of catecholammes m rat bram by reverse-phase liquid chromatography with electrochemical detection. Life Scl 26, 1115-1120 Meek J. L. (1976) Application of inexpensive equipment for high pressure liquid chromatography to assays for taurine, ammobutyric acid, and 5-hydroxytryptophan Anal Chem 48, 37.5379. Meek J. L and Neckers L. M (1975) Measurement of tryptophan hydroxylase m a single brain nuclei by high pressure liquid chromatography. Brain Res 91, 336-340. Mefford I N. (1981) Application of high performance liquid chromatography with electrochemical detection to neurochemical analysis Measurement of catecholammes, serotonm and metabolites m rat brain J Neuroscl Methods 3, 207-224 Mefford I. N and Barchas J D. (1980) Determination of tryptophan and metabolites in rat brain and pmeal tissue by reverse-phase high performance liquid chromatography with electrochemical detection ] Chromatogr 181, 189-193 Mefford I. N., Gilberg M., and Barchas J. D. (1980) Simultaneous determmation of catecholammes and unconlugated 3,4-dihydroxyphenylacetic acid m brain tissue by ion-pamng reverse-phase high performance liquid chromatography with electrochemical detection. Anal Blochem 104, 469472 Mefford I N., Ward M M., Miles L , Taylor B., Chesney M. A., Keegan D L., and Barchas J D (1981) Determination of plasma catecholammes and free 3,4-dihydroxyphenylacetic acid m contmuously collected human plasma by high performance liquid chromatography with electrochemical detection L$e Scz 28, 477483. Mignot E , Laude D , Elghozi J. L , LeQuan-Bui K. H., and Meyer I’. (1982) Central admmistration of yohimbme increases free 3methoxy-4-hydroxyphenylglycol m the cerebrospmal fluid of the rat. Eur. 1. I’harmacol 83, 135-138. Mitchell J and Coscia C. J, (1978) Application of paired-ion highpressure liquid column chromatography to the analysis of 3,4-dihydroxyphenylalanine metabolites. J. Chromatogr 145, 295301. Moleman I’ and Borstrok J. J. M. (1982) Analysis of urinary 3-methoxy4-hydroxyphenylglycol by high performance liquid chromatography and electrochemical detection. J Chromatogr 227, 391405. Moleman I? and Borstrok J J M. (1983) Determmation of urinary vamllylmandelic acid by liquid chromatography with electrochemical detection Urn. Chem 29, 878881 Molnar I., and Horvath C (1977) Rapid separation of urinary acids by high performance liquid chromatography. 1 Chromatogr 143, 391400 Morita I. Masulima T , Yoshida H., and Imai H (1981) Enrichment and high performance liquid chromatography analysis of tryptophan metabohtes in plasma Anal Bzochem 118, 142-146. Morrisey J L and Shihabi Z K (1979) Analysis of homovanlllic acid by liquid chromatography with electrochemical detection Clan Chern 25, 2045-2047

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Moyer T P., Jiang N , Tyce G M , and Sheps S G (1979) Analysis for urinary catecholammes by liquid chromatography with amperometric detection Methodology and clmlcal mterpretatlon of results. Clan Chem. 25, 256263. Neckers L M , DeLisi L E., and Wyatt R J (1980) Lrquidchromatographlc quantification of plasma phenylalanme, tyrosme, and tryptophan Clan Chem 27, 146148 Neckers L M. and Meek J L (1976) Measurement of 5HT turnover rate m discrete nuclei or rat brain. Lzfe SU 19, 1579-1584 Ong H., Capet-Antonuu F , Yamaguchi N , and Lamontagne D (1982) Simultaneous determmation of free 3-methoxy-4-hydroxymandelrc acid and free 3-methoxy-4-hydroxyphenylethyleneglycol m plasma by liquid chromatography with electrochemical detection. ] Chromatogr 233, 97-105. Orsulak I’. J., lzuka P , Grab E , and Schildkraut J J (1983) Determmanon of urinary normetanephrme and metanephrme by radialcompression liquid chromatography and electrochemical detection Clm Chem 29, 30.5-309. Peat M A and Gibb J. W (1983) High performance liquid chromatographic determination of mdoleammes, dopamme, and norepmephrme m rat brain with fluorometnc detection Anal Btochem 128, 275-280 Petrucelli B , Bakrls G , Miller T , Korpl E R , and Lmnoila M (1982) A liquid chromatographic assay for 5-hydroxytryptophan, serotonm, and 5-hydroxymdoleacetic acid m human body fluids Acta Pharmacol Toxzcol 51, 421427. Ponzio F , Achill G., and Algeri S (1981) A rapid and simple method for the determmation of picogram amounts of 3-methoxytyramme m brain tissue using lrquid chromatography with electrochemrcal detection ] Neurochem 36, 1361-1367 Ponzio F. and Jonsson G (1979) A rapid and simple method for the determmation of picogram levels of serotonm m brain tissue using liquld chromatography with electrochemical detection J Neurochem 32, 129-132 Refshauge C , Kissmger P T , Drellmg R , Blank L , Freeman R , and Adams R N. (1974) New high performance liquid chromatographic analysis of brain catecholammes Lzfe Scz 14, 311-322 Remhard J F., Jr , Moskowitz M A , Sved A. F , and Fernstrom J,D (1980) A simple, sensitive and reliable assay for serotonm and 5-HIAA m brain tissue using liquid chromatography with electrochemical detection L$e SCZ 27, 905-911 Remhard J F , Jr and Roth R H (1982) Noradrenerglc modulation of serotonm synthesis and metabolism I Inhibition by clorudme m vlvo ] Pharmncol Exp Therap 221, 541-546 Rlggm R M and Kissinger P T (1977) Determmation of catecholammes u-r urine by ion-pair reverse-phase liquid chromatography with electrochemical detection Anal Chem 49, 2109-2111 Rosano T. G , Meola J M , and Swift T A (1982) Liquid chromatographic determmation of urinary 5-hydroxy-3-mdole-

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193

acetic acid, with fluorescence detection. Clm. Chem. 28, 207208. Santagostmo G., Frattmi I’., Schmelli S., Cucchi L., and Corona G. L (1982) Urinary 3-methoxy-4-hydroxyphenylglycol determmation using reverse-phase chromatography with amperometric detection. I. Chromatogr. 233, 89-95 Saraswat L D., Holdmess M R., Justice J B , Salamone J D , and Neil1 D. B. (1981) Determmation of dopamme, homovamllic acid and 3,4-dihydroxyphenylacetic acid in rat brain strlatum by high performance liquid chromatography with electrochemical detection ] Chroma togr 222, 353-362. Sasa S and Blank C. L. (1977) Determmation of serotonm and dopamme m mouse brain tissue by high performance liquid chromatography with electrochemical detection Anal Chem 3, 354-359 Sasa S and Blank C L. (1979) Simultaneous determmation of norepmephrme, dopamme, and serotonm m brain tissue by highpressure liquid chromatography with electrochemical detection. Anal Chrm Acta 104, 29-45. Sasa S , Blank C L , Wenke D. C , and Sczupak C. A (1978) Liquid chromatographic determmation of serotonm m serum and plasma Clan. Chem 24, 1509-1514. Schemm M , Chang W -H., Jimerson D C , and Lmnoila M (1983) Measurement of 3-methoxy-4-hydroxyphenylglycol m human plasma with high performance liquid chromatography using electrochemical detection Anal. Blochem. 132, 165-170. Schemm M., Chang W -H , Kirk K L , and Linnoila M. (1983) Simultaneous determmation of 3-methoxy-4-hydroxyphenylglycol, 5-hydroxymdoleacetic acid, and homovamlhc acid m cerebrospmal fluid with high performance liquid chromatography using electrochemical detection Anal Bzochem 131, 245-253 Seegal R F., Brosch K 0 , and Bush B (1983) Direct determmation of 4-hydroxy-3-methoxyphenylacetic (homovamlhc) acid m urine by high performance liquid chromatography with amperometric detection. J Chromatogr 273, 253-261. Shipe J R , Savory J , and Willis M. R (1984) Improved liquid chromatographic determmation of 3-methoxy-4-hydroxyphenylethyleneglycol m urine with electrochemrcal detection Clzn Chem. 30, 140-243. Shoup R E and Kissmger I’. T. (1977) Determmation of urinary normetanephrme, metanephrme and 3-methoxytryamme utilizmg liquid chromatography with amperometric detection Clan Chem 23, 12681274 SoIdin S J and Hill J G (1980) Simultaneous liquid chromatographic analysis for 4-hydroxy-3-methoxymandehc acid and 4-hydroxy-3methoxyphenylacetic acid m urine Clin Chem 26, 291-294 Smedes F., Kraak J. C , and Poppe H. (1982) Simple and fast solvent extraction system for selective and quantitative isolation of adrenaline, noradrenalme, and dopamme from plasma and urine J Chromatogr 231, 25-39.

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Sperk G. (1982) Srmultaneous determmatron of serotonm, 5-hydroxymdoleacetrc acid, 3,4-dihydroxyphenylacetrc acrd, and homovamllrc acid by hrgh performance lrqurd chromatography with electrochemrcal detection J Netlrochem 38, 840-843 Tagarr P C , Boullm D J , and Davies C L (1984) Srmplrfred determmatron of serotonm m plasma by lrqurd chromatography with electrochemical detection Clrn Chem 30, 131-135 Taylor J, T , Freeman S , and Brewer I’ (1981) Liquid chromatography of 3-methoxy-4-hydroxyphenylethylene glycol m urine with fluorescence detectron. Clan. Ckem 27, 173-175 Taylor R B , Reid R , Kendle V. E , Geddes C , and Curie P. F (1983) Assay procedures for the determmatron of brogenrc ammes and then metabolrtes u-r rat hypothalamus using ion-pamng reverse phase high performance lrqurd chromatography J Chromatogr 277, 101-114 Tetsuo M., Markey S P , Colburn R W , and Kopm I J (1981) Quantltatrve analysis of 6-hydroxymelatonm m human urine by gas chromatography-negative chemical ronrzatron mass spectrometry Anal Blochem. 110, 208-215. Todorrkr H , Hayashr T , Naruse H., and Hrrakawa A. Y. (1983) Sensrtrve high performance lrqurd chromatographrc determmatron of catecholammes m rat brain using a laser fluorometnc detection system ] Chromatogr 276, 4.5-54 Towel1 J, F and Erwin V G (1981) Determmatron of the primary metabolrte of central nervous system norepmephrme, 3-methoxy-4hydroxyphenethyleneglycol, m mouse brain and brain perfusate by high performance liquid chromatography with electrochemrcal detection 1. Chromatogr 223, 295-303 Tracy R. I’ , Wold L. E , Jones J D., and Burrrtt M F. (1981) Colorrmetrrc vs liquid chromatographrc determination of urinary 5-hydroxymdole-3-acetic acid. Clan Ckem 27, 16&162 Turnell D C. and Cooper J. D H (1982) Rapid assay for ammo acids m serum or urine by precolumn derrvatrzatron and reverse phase lrqurd chromatography Cltn Ckem 28, 527-531 Tyce G. M and Creagan E. T (1981) Measurement of free and bound 5-hydroxytryptophan m plasma by lrqurd chromatography with electrochemrcal detection Anal Bzochem 112, 143150 VanBockstaele M , Drllen L , Claeys M , and DePotter W P (1983) Srmultaneous determmatron of the three malor monoamme metabolutes m cerebrospmal fluid by high performance lrqurd chromatography with electrochemrcal detection 1 Chromatogr 275, ll20 Van Valkenberg

C , Tladen

U , Van der Krogt

T , and Van der Leden

B

(1982) Determmatron of dopamme and rts acidic metabolrtes m brain tissue by HPLC wrth electrochemrcal detection m a single run after minimal sample pretreatment. 1 Neurockem 39, 990-997 Wagner J., Palfreyman M , and Zrarka M (1979) Determmatron of dopa, dopamme, dopac, epmephrme, norepmephrme, monofluoromethyldopa and drfluoromethyldopa m varrous tissues of mice and

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195

rats using reverse phase ion-pair lrqurd chromatography with electrochemrcal detection J Chronratogr 164, 41-54 Wagner J , Vital1 I’ V , Palfreyman M G , Zrarka M , and Huot S. (1982) Srmultaneous determmatron of 3,4-dihydroxyphenylalanme, 5-hydroxytryptophan, dopamme, 4-hydroxy-3-methoxyphenylalanme, norepinephrme 3,4-drhydroxyphenylacetrc acid, homovanrllrc acid, serotonm, and 5-hydroxymdoleacetrc acid m rat cerebrospmal fluid and brain by high-performance liquid chromatography with electrochemrcal detection ] Neurochenl 38, 1241-1254 Warsh J J,, Chan I’ W , Godse D D , Coscma D V., and Stancer H C (1977) Gas chromatographrc-mass fragmentographrc determmatron of mdole-3-acetic acid m rat brain 1 Neurockenl 29, 955-958 Warsh J J , Chm A S , and Godse D D (1982) Determmatron of brogenlc ammes and their metabohtes by high-performance liquid chromatography, in+ Technzquesaruz’ Imtrunrentcltm IPZ Amlytrcal Ckemstry, Vol 4 Eunluntm of Armlytuxl Metkods LIZBdogrcal Systems Part A Analysis of Brogemc Ammes (Baker G B and Coutts R T , eds ) pp 203-236, New York Elsevrer Warsh J J , Chm A , and Godse D D. (1982) Srmultaneous determmatron of norepmephrme, dopamme and serotonm m rat brain regions by ion-pair liquid chromatography on octyl srlane columns and amperometnc detection 1 Chromato,gr 228, 131-141 Warsh J J , Chm A , Godse D D , and Coscma D. V (1979) Determmatron of picogram levels of brain serotonm by a srmprfred liquid chromatographrc electrochemrcal detection assay Bmrn Res Bull 4, 567-570 Watson E (1981) Liquid chromatography with electrochemrcal detection for plasma norepmephrme and epmephrme L$e Scr 28, 493-497 Westermk B. H C and Mulder T B A. (1981) Determmatron of prcomole amounts of dopamme, noradrenalme, 3,4-drhydroxyphenylalanme, 3,4-dlhydroxyphenylacetrc acid, homovanrllrc acid, and 5-hydroxymdoleacetrc acid m nervous tissue after one-step purrfrcatron of Sephadex G-10, using high-performance lrqurd chromatography with novel type of electrochemrcal detection ] Neurochenr 36, 1449-1462 Westermk B H C and Spaan S. J (1982) Estrmatron of the turnover of 3-methoxytyramme m the rat strratum by HPLC with electrochemical detectron. Implrcatrons for the sequence m the cerebral metabolism of dopamme J Neuvochem 38, 342-347. Westermk B H C , van Es T I’., and Spaan S J (1982) Effects of drugs interfering with dopamme and noradrenalme brosynthesrs on the endogenous 3,4-hydroxyphenylalanme levels m rat brain ] Neurochem.

39, 4451

Wrghtman M. R , Plotsky P M , Strope E , Delcore R , Jr , and Adams R N (1977) Liquid chromatographrc momtormg of CSF metabolutes. Bram Res 131, 345-349. Wolf W A and Kuhn D M (1983) Simultaneous determmatron of 5-hydroxytryptamme, its ammo acid precursors and acid metabohte

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m discrete brain regions by high-performance liquid chromatography with fluorescence detection J Chromatogr 27.5, l-9 Yamada J , Kayama E , Alzawa Y , Oka K., and Hara S (1981) Determination of vanlllylmandelic acid in urme by precolumn dansylation using micro high-performance liquid chromatography with fluorescence detection. J Chromatogr 223, 176-178. Yamada J , Sugimoto Y., and Hosisaka K. (1983a) Determmation of endogeneous mdoleacetlc acid and tryptophol m mouse brain by high performance liquid chromatography with fluorometric detection Life Scz 33, 204s-2047 Yamada J., Sugimoto Y , and Horisaka K (1983b) Simultaneous determination of tryptophan and its metabohtes m mouse brain by highperformance liquid chromatography with fluorometric detection Anal

Blochem. 129, 460463

Yamaguchi T., Yokota K., and Uematsu F. (1982) Separation of mdole metabolltes from urine with an ODS type resin by high performance liquid chromatography. ] Chromatogr 231, 166-172. Yamatodam A. and Wada H (1981) Automated analysis for plasma epinephrme and norepmephnne by liquid chromatography, mcludmg sample cleanup procedure Clan Chem 27, 1983-1987. Yoshida H , Kite S , Akimoto M., and Nakalima T (1982) Multi-parallel detection m high performance liquid chromatography. 1 Chromatogr

240, 49-96

Yoshida A., Yoshioka M., Sakai T , and Tamura Z. (1982) Simple method for the determmation of homovanlllic acid and vamllylmandelic acid m urme by high performance liquid chromatography. ] Chromatogr 227, 162-167. Young S N. and Anderson G M (1982) Factors mfluencmg melatonm, 5-hydroxyindoleacetic acid, 5-hydroxytryptamme and tryptophan in rat pmeal glands. Neuroendocrrnology 35, 464-468. Young S N , Gautier S , Choumard G., Anderson G M., and Purdy W. C. (1982) The effect of carbidopa and benserazide on human plasma 5-hydroxytryptophan levels. 1 Neural Transm 53, 83-87 Yui Y., Fugita T., Yamamoto T., Itokawa Y., and Kawai C (1980) Liquid chromatographlc determmation of norepmephrme and epmephrme m human plasma Clan Chem 26, 194-196 Yui Y. and Kawai C (1981) Comparison of the sensitivity of various post-column methods for catecholamme analysis by highperformance liquid chromatography J Chromatogr. 206, 586-588 Yui Y , Kimura M , Itokawa Y., and Kawai C. (1979) Ultramicro method for the determmation of picogram amounts of norepmephrme and epmephrme by high performance liquid chromatography. J Chromatogr

177, 376-379

Zaczek R and Coyle J T. (1982) Rapid simple method for measuring biogenic ammes and metabolites in brain homogenates by HPLCelectrochemical detection ] Neural Transm 53, 1-5.

Chapter 5

In Vllo Voltammetry JOSEPH B. JUSTICE, JR., ADRIAN C. MICI-IAEL, AND DARRYL B. NEILL

1. Introduction One of the fundamental goals of neuroscience is an understandmg of the relationship between neurotransmissron and behavior. Although considerable informatron has come from methods such as push-pull perfusion, obtaining data about neurotransmitter release in behaving animals has been quite difficult. Recently voltammetry, a standard electroanalytical technique, has been shown to be applicable to monitormg the extracellular neurochemistry of monoammes and related compounds. Since the orrgma1 report from the laboratory of R.N. Adams (Kissinger et al., 1973), more than 100 papers have been published on in vlvo voltammetry and its apphcatron to the study of monoammes in the central nervous system. Adams and Marsden (1982) and Hutson and Curzon (1983) have recently revrewed the sublect. Because of the complexity of the extracellular environment of the brain, most of the work to date on in vivo voltammetry has addressed the issue of interpretatron of the data rather than the apphcatron of the technique. As the interpretation of m vivo voltammetrrc data has clarified, appllcatrons have started to appear that clearly demonstrate the utrhty of the method for obtainmg new mformatron about monoammes m vivo. One of the slgmfrcant advantages of the method IS the sampling rate. The extracellular dynamics of catecholammes and related species can be followed at a rate of a sample every 2 s m some cases (Ewing et al., 1983a). On the other hand, rt IS also pos197

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and Nell1

sible to record circadian changes (O’Nerll et al., 1982a) over a period of days, Multiple electrodes have been employed to monitor regional variations m extracellular chemistry (Justice et al , 1980, Salamone et al , 1982) Because the method detects only those compounds that oxidize easily, it IS limited relative to other more general methods However, for m vrvo monrtormg in the complex medium of the extracellular fluid of the brain, this can be an advantage, as the vast malorrty of compounds m the brain are not electroactlve under the conditions employed and thus do not interfere with the measurement. This chapter begins with an mtroductron to voltammetry that discusses the relationship of measured current to applied potential common to all voltammetrlc experiments This discussron is followed by a review of the electrochemistry of catecholammes The varrous voltammetric techniques are then covered, including normal pulse voltammetry, differential chronoamperometry, pulse voltammetry, differential double pulse voltammetry, and linear sweep voltammetry. The next section IS a discussron of the mstrumentatron that has been used to implement the above techniques for in vlvo monitormg. A section on the various electrodes and the modifications that have been made to improve selectivity is followed by a model of the m vlvo behavior of voltammetrrc electrodes. The section on electrodes ends with a drscusslon of calibration. The section on mterpretatlon examines the drffrcultres mvolved m analyzing the results of m VIVO voltammetrlc expenments and explains some of the strategies employed to overcome these difficulties. Most of the discussion concerns work done on the dopaminerglc nigrostriatal system One part of the section, however, deals specrfrcally with serotonm The section ends with a discussron of the detection of extracellular neurotransmitters. The chapter ends with a summary of some of the applications of in vrvo voltammetry to neurochemistry of catecholamines in the striatum, locus ceruleus, and median eminence, and its use m momtormg neurochemistry during behavior.

2. Introduction

to Voltammetry

Oxidation and reduction reactions mvolve either the loss (oxldatlon) or gam (reduction) of electrons by chemical species m solu-

Voltammetly

199

tion. The process may be described m a general form by the half reaction* Ox + ne = Red (1) that indicates that the oxidized form of the couple, Ox, reacts to produce the reduced form, Red As written, the half reaction is not complete. In solution rt will occur only in the presence of a second redox reaction to provide the electrons consumed in Reaction (1). In the complete redox reaction the molecules undergoing reduction consume electrons liberated by the molecules undergoing oxidation. Electrodes may mediate these reactions by actmg as sources or sinks of electrons. For example, a copper electrode m a solution of copper ions is described by the half reaction. Cu*+ + 2e Z Cu (2) A potential is established at the electrode solution interface according to the Nernst Equation: E = E” - g

log $$ i

1

(3)

where E is the observed potential , E” is the standard potential, R is the gas constant, T 1s the temperature m degrees Kelvin, F is Faraday’s constant, ~zis the number of electrons involved in the reaction, and a represents an activity. Potentials are relatrve, so the value of E may only be observed by measuring it against a second electrode of fixed potential. Hence, a reference electrode is required to provide both the balancing half reaction and a stable reference potential A high impedance voltmeter is used to measure the potential difference between the electrodes. If the high impedance device is replaced by a wire that short circuits the two electrodes, current will flow until the potential difference IS zero. At this point the two half reactions are at equihbrmm. If the wire is replaced with a power supply so that a potential difference is applied between the electrodes, the equilibrium is disturbed and current will flow such that the ratio of oxidized and reduced species at the electrode surface will be maintained m accordance with the Nernst Equation [Eq. (3)] f or a reversible reaction. The measurement of the current that flows m response to an applied potential is the basis of the various applied potential techniques collectively called voltammetry. The basic voltammetric experiment is depicted as follows In Fig 1 a three-electrode cell is shown rather than the simple

Justice,

Michael,

and Nell/

B

,/ j’ /

i

POTENTIAL

Fig 1 A Schematic of a three electrode cell A potential difference 1s maintained between the working (WE) and reference (RE) electrodes by the power supply and control electrode (CE) The applied potentlal is monitored by the voltmeter, V, and the current at the working electrode, I,,,,, is monitored by the ammeter, A B Data recorded from A and V yield the current-potential curve two-electrode cell described above. The three-electrode cell allows improved control of the potential between the reference and workmg electrode because the reference electrode is no longer m the current loop. Current flow m the reference electrode causes an ohmic drop, resultmg m a nonconstant potential between reference and working electrodes The control, or auxiliary electrode, as it 1s sometrmes called, IS used to maintain a potential drfference between the working and reference electrodes regardless of the amount of current flowing. This is not a serious concern m m vrvo voltammetry, where the currents are m the picoampere to nanoampere range The electrode and circuit arrangement 1s called a potentrostat and IS analogous to a voltage clamp circuit of electrophysiology m that a potential 1s held at some fixed value To understand the current-voltage or current-time curves of voltammetry, one must consider the effect of an applied potential on the concentration of electroactive components of a solution. It has been stated that the electrochemical reaction occurs at the electrode surface. In order for the reaction to take place, then, material must be at the electrode surface. A molecule m solution must first be transported to the surface before it can oxidize or reduce. The mode of mass transport of interest for m vrvo voltammetric work 1s diffusion along concentration gradients A concentratron gradient arises at the electrode surface and extends

201

Vo/tammetfy

mto solutron because the electrochemrcal reaction consumes material at the electrode surface, thus creating a difference in concentration between the electrode surface and the bulk solution concentration. This gradient is illustrated m Fig. 2 for several potentials less than the oxidation potential of a given compound and for a potential that drives the surface concentration to zero. As more material is consumed, the depletion extends farther from the electrode surface mto the solution. Diffusion along the concentratron gradient from higher concentratron to the region of lower concentratron supplies material to the electrode surface. The shape of the gradient is very dependent on the parameters of the experiment, in particular the characteristics of the applied potential waveform. Because the observed current is directly dependent on the rate at which material arrives at the electrode surface, the shape of the current-potential curve 1s also dependent on the nature of the applied potential waveform. An additional factor that complicates the relatlonshrp of current to potential is rate of electron transfer between the electrode and molecules at the electrode surface. In the simplest case the electron transfer is suffrcrently rapid that the observed current IS SURFACE REGION

I

BULK SOLUTION

C

DISTANCE

FROM

ELECTRODE

SURFACE Fig 2. The effect of applied potential on concentration of electroactlve compound The left llmlt of the horizontal axis represents the electrode surface. The concentration, C, is represented as a fraction of the bulk concentration, Cbulk.

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Justice, Michael, and Nerll

determined by the rate at which reactant reaches the electrode surface, as described above. For many organic electrochemical oxidations, however, the rate of electron transfer is relatively slow. This phenomenon broadens the current-potential curve and shifts the curve toward higher potentials. The electrochemical oxidation of ascorbic acid at carbon paste electrodes provides an example of this effect. Two types of information may be obtained from an electrochemrcal experiment. Quantitative mformatron on component concentratrons may be obtamed by comparison of observed oxidation currents with data from standard curves. Qualitative mformation is based on current-voltage curve shapes and their positron on the potential axis. The Nernst equation mdicates that the position of a current-potential curve is dependent on E; E IS a thermodynamic potential, however. As mentioned above, other factors such as the kinetics of the electrochemical reaction may affect the position and shape of the curve. Thus qualitative interpretation of a signal can be difficult. It is usually helpful to compare results to an authentic standard run under identical conditions. This is a particularly troublesome problem with m vrvo work, where simply placmg the electrode in brain tissue alters the shape of the current-potential curve, as recently pointed out by Freed and Echizen (1983) and O’Nelll et al. (1982b). A thorough discussion of voltammetry may be found m the text by Bard and Faulkner (1980). Adams has written a very useful text on electrochemistry at solid electrodes (1969).

3. Electrochemistry

of Catecholamines

In CNS tissue there are a number of molecules that will oxidize at low potentials at carbon electrodes, These include the neurotransmitters dopamme (DA), noradrenaline (NA), and 5-hydroxytryptamme (5-HT, serotonm), as well as the nonconlugated metabolites of these. Ascorbic acid, present in much higher concentration, is also oxidizable m the same potential range. Adams and coworkers have studied the electrochemistry of the catecholammes extensively (Hawley et al., 1967; Papouchado et al., 1972, Sternson et al., 1973, Tse et al , 1976) The pH dependence of the oxidation potential has been established for a number of the catecholammes and metabolites (Sternson et al., 1973). It was shown m this paper that although DA is slightly easier to oxidize than NA, the oxidation potentials are too similar

Vo/tammetfy

203

to distmguish these compounds. This is not necessarily a problem for in VIVO voltammetry, however, since the concentration of DA is often much higher than NA (and vice versa) m the bram regions where they are found. Much of m viva voltammetry to date has been done m the striatum, where the level of DA is much higher than that of NA It was also demonstrated that the methoxylated metabolites of DA and NA are considerably more difficult to oxidize than the neurotransmitters themselves 3,4-Dihydroxyphenylacetic acid (DOPAC) is the easiest of the acidic metabolites to oxidize and has an oxidation potential similar to DA An additional serious problem with m viva measurement of catecholammes is that ascorbic acid oxidizes at the same potential as DA at physiological pH The difficulty m resolving these compounds is illustrated m Figs 3a and 3b, which show the overlap of oxidation peaks from different electroactive compounds recorded using differential pulse voltammetry at carbon paste electrodes. These problems and the various solutions are discussed m the Interpretation section Cychc voltammetry has been used to identify the transient mtermediates, i.e , open chain o-qumones formed during the electrochemical oxidation of DA and related compounds, and to determme the rate of mtramolecular cyclrzation to the substituted mdole and its subsequent oxidation to the ammochrome (Sternson et al , 1973). This process is shown below m Fig. 4 It was shown that oxidized DA cyclized at about one-tenth the rate of oxidized NA at pH 7 0 At physiological pH the rate of cyclization is 2.63 x 10-i s-l for the oxidized DA The product of the cyclization oxidizes more readily than DA to form an ammochrome. Cyclization, however, is not the only reaction the o-quinone may undergo It will also react very rapidly (at 1800 times the rate of cyclization) with nucleophiles such as glutathione. The products of DA oxidation m viva have not been established, prmcipally due to the extremely small amounts of material formed. An additional aspect of the electrochemistry of DA relevant to measurements m viva is that DA mediates the oxidation of ascorbic acid. This means that after a DA molecule is oxidized at the electrode surface and the product diffuses into solution, it is reduced by the ascorbic acid to DA, which can again be oxidized at the electrode, as shown m Fig 5. The same molecule may cycle many times through this process Thus a given concentration of DA will generate a larger current m the presence of ascorbic acid than m its absence. The rate constant for the catalysis has been

204

Justice, Michael,

and Nell1

calculated to be 3.2 X lo+5 M-’ SC’ (Dayton et al., 1980b) which yields a half-life of 2.1 ms for the oxidized DA

4. Voltammetric Techniques Various applied potential waveforms are used to obtain information about electroactlve compounds m solution Each has its own advantages and IS discussed below.

APPLIED

PO I ENTIAL

(v)

Fig 3 In szttr voltammograms from the rat median eminence A the solid line (-) represents the endogenous electrochemical signal from the median eminence of a urethane-anesthetized rat The dashed lme (- -) was recorded after the qectlon of 5 PL of 1 x 10P4M DA adlacent to the electrode The dash-dot lme ( - ) was recorded followmg the mjectlon of ascorbic acid (10 pL, 1 x lOpaM) adlacent to the electrode The peak at +0 14 V 1sat least partially comprised of DA and ascorbic acid B An endogenous DPV 1sillustrated by the solld lme () After mlectlon of HVA (5 FL, 1 x lo-‘M) adjacent to the electrode (dashed line, - -), the oxldatlon wave at +0.43 V increased The other dashed lme (a-.) was recorded after the mlectlon of 10 FL DOPAC (1 x 10-‘M) adjacent to the electrode This caused an increase m the oxldatlon wave occurring at +0.14 V From these experiments it can be concluded that the oxldatlon wave appearing at +0 14 V represents the oxldatlon of DA, DOPAC, and ascorbate, whereas the wave at -to.43 V represents HVA The oxldatlon signal apparent beyond +0 60 V remains unidentified From I’ M Plotsky et al (1982), with permission.

Voltammetly

205

I

I

IOOmV

-0 2

‘0.2

0

APPLIED

‘06

POTENTIAL(v)

B

4.1. Chronoamperometty The simplest of the voltammetnc techniques is chronoamperometry, u-t which a short square wave pulse is applied and the resultmg current measured at some time after the initiation of the pulse (Fig. 6). Pulse lengths are typically 100 ms to 1 s and are applied at intervals as short as one pulse width or as long as several minutes. The current as a function of time measured at a planar electrode to which material diffuses normal to the surface is described by the Cottrell equation where n is the number of elec1=

nFAD112C #Zt1/2

trons transferred/molecule, F is Faraday’s constant, 96,458 coulombs/equivalent, A is the electrode area m cm, D is the diffusion coefficient of the reacting material m cm2/s, C is the bulk concentration of the compound being oxidized, and t is the time from the application of the potential m s. With these units the current is in A Because most of the above elements of the Cottrell equation are constants (n, F, A, D, IT) the equation states that the bulk concentration is proportional to the product of the current and the square root of the time. For a fixed time, then, the current is proportional to concentration. Note that this equation refers only to the faradaic, or reaction, current. It does not treat the

Justice,

206

Michael,

and Nell1

HO HO

+ 2Ht

+ 2e’

+ 2Ht

+ 2e’

HO HO

3.

Fig 4 The proposed mechanism for the electrochemical oxldatlon of dopamme Dopamme (1) IS oxldlzed to the dopamme o-qumone (2) Cycllzatlon to the mdole (3) 1s followed by further oxldatron to the ammochrome (4) nonfaradaic, or charging, current that results when a potential 1s applied to an electrode solution interface. This chargmg current IS substantral relative to the faradaic current at the instant the pulse is applied, but decays quite rapidly thereafter and is negligible by 1 s with the carbon paste or epoxy electrodes used in VIVO. The charging current is smaller with the carbon fiber electrodes due to their smaller surface area. The general form of the equation for the charging current due to the double layer capacitance IS I =

AAox

DOQ

/

-

DOQ + AA

/

Fig 5 The catalytic oxldatlon of ascorbic acid by dopamme The dopamme 1s recycled by oxldatlon at the electrode and reduction by ascorbic acid. DOQ 1s dopamme o-qumone

207

Vo/tammetry

ke-f/K, where l/K is the time constant of the electrode. The capacitance 1s directly proportional to the electrode area, so that the smaller the electrode, the faster the charging current decays to zero. As shown m Fig 6 below, the faradaic current decays with a t-“2 dependence The applied potential IS such that the surface concentration goes rapidly to zero for the reasons discussed above At short times following the onset of the applied potential, the concentration gradient from the electrode surface mto the bulk solution will be quite steep, leading to a high rate of mass transport to the electrode surface and therefore a large current. As material is depleted farther into the solution, the concentration gradient becomes smaller, resulting m a reduced rate of mass transport to the electrode surface and thus a smaller current. The advantage of chronoamperometry over other voltammetric methods, aside from its simplicity, is the frequency with which measurements may be made. This is not a strong advantage m pharmacological studies where effects may develop slowly

E

Ef aw

‘C

\

I

abs

‘\ , ‘a

! ’

Fig.

6

Chronoamperometry.

TIME

The applied potential IS stepped

from an mitral value, E,, to a final value, Ef, and returned to E, At E, no slgruflcant current 1sobserved The total current () 1sthe summation of the faradalc (- - -) and nonfaradalc ( a.) current The current measured at the time, f, 1s prlmarlly faradalc Conventionally, cathodic current, I,, 1sreported m the posltwe direction and anodlc current, I,, m the negative dlrectlon

208

Justice,

Michael,

and Neil1

and persist for several hours, but may be important m studies of neurotransmitter release and behavior. Apphcations include those of Conti et al (1978), Huff et al. (1979), Lmdsay et al (1980b,c), Salamone et al. (1982), Schenk et al., (1983) and Salamone et al. (1984).

4.2. Normal Pulse Voltammetry Chronoamperometry is a good choice for obtaining quantitative mformation about simple electrochemical systems or the sum total of current from multicomponent systems The diffusion limited current, however, provides no qualitative mformation regarding the identity of mdividual solution components. Such information can be obtamed by methods m which the current is observed as a functron of applied potential. Several methods are available for this purpose. The one most similar to chronoamperometry 1s normal pulse voltammetry. In this method, potential steps are applied from some resting potential through a range of applied values. The current is recorded as shown in Fig. 7 In normal pulse voltammetry, the potential is returned to baseline between each pulse, thus reducing the electrolysrs which E apt E

---d ---

‘d

I

obs

E app

Fig 7. Normal pulse voltammetry The potential waveform 1s a series of increasing pulses The current on each pulse 1s plotted against the pulse potential E 112IS the potential at which the current 1s one-half the diffusion limited value, Id

Voltammetly

209

occurs m methods where the potential remains constant or contmually mcreases In vivo, this 1s desn-able since the neurochemical environment is less affected. On the other hand, the nonfaradaic charging current is somewhat larger than m differential pulse voltammetry (described below). The current plateau may be used for quantitation whereas E i12, the potential at which the current is half the hmitmg current, is related to the standard or formal potential of the redox couple and can be used qualitatively. Normal pulse voltammetry with carbon fiber electrodes has been described by Ponchon et al (1979). It has also been used by Ewing et al (1982, 1983a)

4.3. Differential Pulse Voltammetly Differential pulse voltammetry (DPV) is a widely used quantitative electrochemical method for determining the concentration of oxidizable or reducible substances m solution. The method was developed as a way to eliminate or greatly reduce the effect of charging current relative to the faradaic current and to express the signal m a more easily quantifiable form. The waveform used to accomplish this is shown in Fig. 8A. The waveform is essentially a slowly mcreasmg ramp upon which is superimposed small, fixed-amplitude pulses of short duration (approximately 50 mV for 50 ms). The resulting current is sampled at two points relative to each small pulse, once lust before the pulse and again lust before the end of the pulse (Fig. 8B) The data are expressed as the differences between these two currents (Fig SC), hence the name. The procedure generates a peakshaped signal that is effectively the derivative of the sigmoidal normal pulse voltammogram This instrumental differentiation of the signal largely ehmmates the charging current since the only charging current present in the signal after the subtraction of the current obtamed lust prior to the pulse is that arising from the small pulse. The second advantage is seen m Fig. SC where a differential pulse voltammogram 1s illustrated. Linear sweep voltammograms do not provide as convenient a peak shape for quantitation. Generally the peak height is measured for quantitative purposes. In differential pulse voltammetry, compounds that oxidize at different potentials will show up as separate peaks if their oxidation potentials are sufficiently different. Otherwise one peak may appear as a shoulder on another or they may be completely mdistinguishable For such cases, considerable effort has gone mto

210

Justice,

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makmg electrodes that can better discriminate between compounds such as DA, its metabolrte DOPAC, and ascorbic acid (Lane et al., 1976; Lane and Hubbard, 1976, Gonon et al., 1981b; Blaha and Lane, 1983, Gerhardt et al., 1984) These compounds all oxidize at about the same potential when a carbon paste electrode is used. The development of such electrodes 1s drscussed m the interpretation section Differential pulse voltammetry has been A

Eapp

TIME

I

obs

TIME

E atv

Fig. 8. Dlfferentlal pulse voltammetry A The applied potential 1sa linearly mcreasmg ramp upon which small pulses of height AV are superimposed. B Two measurements are made fcr each pulse, one Just before the pulse 1sapplied and one lust before the end of the pulse, to yield the differential current value, AI C The differential current 1sreported against the applied potential to give the peaked voltammogram

211 used m viva by Lane et al. (1976, 1978), Gonon et al. (1980), Cespuglio et al. (1981a,b,c), and Buda et al. (1981).

4.4. Differential Double Pulse Voltammetry Differential double pulse voltammetry uses the method of normal pulse voltammetry described above, except that two pulses of unequal amplitude are alternated (Fig. 9) and the difference in current between the two measured. The technique is thus similar to differential pulse voltammetry except that the potential returns to baselme between each pulse. Its use at chemically modified platinum electrodes has been described (Lane and Hubbard, 1976). The method is an attempt to combme the attractive features of normal pulse and differential pulse voltammetry. The peaked signal of DPV IS achieved and between each differential measurement the potential is returned to its resting value where little or no oxidation occurs. It also has the advantage of minimizmg the perturbation of the extracellular neural environment during the scan since less material is consumed than in differential pulse voltammetry. It also tends to increase electrode lifetime since there is less reaction product to adsorb to the electrode surface.

4.5. Linear Sweep and Cyclic Voltammetly The preceding tial methods

(LSV and CV)

sections have described stepped and pulsed potenLinear sweep voltammetry mvolves applying a

--TIME

Fig 9 Differential double pulse voltammetry. The applred potential waveform 1s a double potential step As in differential pulse voltammetry, the differential current m response to the double step, AV, is reported against the applied potential but between each measurement the potential 1s returned to the mltlal value, E,, as in normal pulse voltammetry. The voltammogram IS similar to that m DPV

212

Justlce, Michael,

slowly

increasing

voltage

rent. Cyclic voltammetry lar waveform cified value,

and Nell1

ramp and observing the resulting curIS an extension of LSV m that a triangu-

is used such that when the potential reaches a spethe direction of the ramp is reversed and the

potential returns to its initial value, as illustrated in Fig 10. The current

is also observed

during

the reverse ramp or sweep to pro-

duce a cyclic voltammogram. During the forward scan there is a buildup of reaction product(s) near the electrode surface If the product(s) is electroactlve it will generate a current as it undergoes electrochemical reduction during the reverse scan Thus one observes the current resulting from the initial electrochemical reaction

on the forward

electrochemical

side of the triangular

waveform

and the

reaction of the products on the reverse side.

TIME B

I

Fig

10

obs

Cyclic voltammetry

A The applied potential waveform

1s triangular, starting at an mltlal value, E,, and ramping to a final potential, Ef At Ef the ramp direction 1s reversed and the potential returned to E, Single or multiple cycles may be used B The observed current durmg the cycle 1s reported against the applied potential Note that the right limit of the potential axis corresponds to E,, where the scan reverses dl-

rectlon

In linear sweep voltammetry,

only the forward ramp 1s used

213

Vokammetfy

Considerable mformation can be obtained about electrochemical reactions from the shape of cyclic voltammograms (Bard and Faulkner, 1980) Also, the triangular waveform may be repeatedly applied to detect additional electroactive compounds formed followmg the first electrochemical reaction. Thus, cyclic voltammetry is most frequently used as a diagnostic tool for understanding electrochemical reactions rather than as a quantitative method, although it has been used in viva by Curzon and Hutson (1981) and Kennett and Joseph (1982).

4.6. Linear Sweep Voltammetly

with Semidifferen tia tion

The in vivo oxidation current resultmg from linear sweep voltammetry may be further processed to yield sharper, more easily discriminated peaks with noticeably better baseline and more symmetrical peak shape (Lane et al., 1979). Semidifferential voltammetry is an extension of ordinary scan methods, except that instead of recording the usual current-voltage curve, the semidifferential function of current is calculated and recorded. The relationship of current to concentration in this method has been shown to involve an analytically unsolvable integral of current to the one-half power (seeNicolson and Shain, 1964, for a detailed derivation of this relationship). It was pointed out by Oldham (1969) that recording the semi-integral of current during linear sweep voltammetry scans gave signal-voltage curves of considerably improved symmetry and more sharply defined features than the unprocessed signal. Goto and Ishii (1975) used differentiation of this sigmoidal signal to get the peaked response that has considerable advantages over both the direct current and semi-integral modes. In particular, the semiderivative signal yields sharper peaks with an enhanced peak separation for the oxidation of multicomponent solutions. Also, the peak potential corresponds with the EiI2 of the redox couple, which enables qualitative identification Semidifferentiation 1sa mathematical procedure and does not affect the electrochemical technique or assumptions. Oldham (1973, 1981) has described both an analog semidifferentiation method and a straightforward numerical algorithm for digital evaluation that have been used by most investigators rmplementmg the method for m viva voltammetry (Lane et al., 1978; Morgan and Freed, 1981; O’Neill et al., 1982a) O’Neill has recently discussed the use of the method for chronic m vivo recordmg (O’Neill et al , 1983a).

214

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and IYell/

5. Instrumentation Although voltammetry can be performed m vivo with commercial mstrumentation such as a Model 174A or 374 pulse polarograph (EG and G Prmceton Applied Research, Prmceton, NJ), a Model PRG-5 pulse polarograph (Tacussel Electronique, Lyon, France) or a Model CV37 Voltammograph (Bioanalytical Systems, West Lafayette, IN), a number of laboratories have constructed their own equipment to perform the measurements (Cheng et al., 1980; Lindsay et al., 1980b,c, Ewing et al., 1981a; Blakely and DuVarney, 1983, Gerhardt and Adams, 1982; O’Neill et al., 1983b). The basic requirements of the instrumentation are that it serve as potentiostat to apply the various waveforms of interest for chronoamperometry, linear sweep voltammetry, or differential pulse voltammetry, and that it amplify the very small currents generated at the workmg electrode. Additional features include the provision for repetitively performing the experiment, computerized control of the applied waveform, storage of the data, and smoothing or other signal processmg such as that required m differential pulse voltammetry, semidifferential voltammetry, and background subtraction. The ability to process the currents from multiple electrodes is also of interest. Two different potentiostat-amplifiers are in use in our lab. The schematic of one of these is shown below m Fig. 11. In the design shown (Lindsay et al , 1980~) the current from up to 16 electrodes may be measured sequentially through an analog multiplexer to a single amplifier In the other design (Blakely and DuVarney, 1983), each electrode has its own amplifier, and up to 8 electrodes may be monitored simultaneously. Both systems require a microcomputer to apply the waveform and process and store the data. In chronoamperometry, for example, the microcomputer has control of the applied potential, the length of the pulse, the interval between pulses, the rate of data collection, the number of points to be collected, and the number of electrodes to be sampled. Ewing et al. (1981a,b) designed an instrument specifically for pulse voltammetry with carbon fiber electrodes. This instrument allows for correction of the signal by subtraction of the nonfaradaic current and for differential output and adlustable step and delay times. Experimental results with the equipment indicate that potential step times as short as 100 ms can be used with a faradaic accuracy of better than 5%. Gerhardt and Adams (1982) have constructed and evaluated a simple, inexpensive battery-powered chronoamperometry ap-

11 MultIelectrode electrochemical potentlostati’ampllfier IC-1 and IC-2 comprise the potentlostat E,, IS supplled from a DAC output from a mlcrocomputer IC-3 1s an analog multiplexer that selects one of 16 working electrodes The multlplexor 1s controlled via a parallel I/O port IC-4 and IC-5 are a high gam current-toIC-6 1s a filter and IC-7 1s an mverter V,,, IS momvoltage converter and variable gam amplifier, respectively tored by the computer via an analog-to-dlgltal converter From W S Lindsay, et al (198Oc), with permlsslon

I

MULTI -ELECTRODE ELECTROCHEMICAL POTENTIOSTAT/AM PLI FIER

1

Justlce,

216

Michael,

and Nell1

paratus which can be used for m VIVO measurements or as the potentiostat for an electrochemical detector for liquid chromatography The authors attribute the good signal-to-noise characteristics of the instrument to the absence of an AC power source. The equipment used by O’Neill et al. (1983b), like that of Ewing et al. (1981a), provides for the construction of difference voltammograms. Voltammograms obtained during a control period preceding the administration of a stimulus are averaged, this serves as a reference that may be subtracted from subsequent voltammograms It is also possible to scan contmuously to remove components from the fluid at the electrode and thus obtain a background scan minus the substances generating the peaks, as shown m Fig. 12. Changes m extracellular concentrations are more easily detected m the resulting difference voltammograms, as illustrated m Fig. 12. Another manipulation is to plot the current observed at a specified potential recorded during a series of scans. This is useful for demonstrating the time course of an effect (Ewing et al., 1982).

5.2. Electrodes In performing in vivo voltammetry, one uses a conventional 3-electrode cell, as depicted earlier m Fig. 1. The cell consists of a reference electrode, a control or auxiliary electrode, and a workmg electrode. Electrochemical potentials are always the difference of potential between two electrodes. The purpose of the reference electrode is to establish a potential relative to which the working electrode may be set. Since current flow would reduce the potential between the reference and working electrodes, a third electrode is added to the cell. This third electrode, the control electrode, maintains the potential difference between the reference and control electrodes when current flows m the cell. 5.2.1. Reference

Electrode

The reference electrode is a miniature Ag/AgCl electrode. Several designs have been used for work in vivo. Early electrodes used a silver wire coated with silver chloride that was located in a pulled glass capillary containing a solution of NaCl. Problems such as occlusion of the electrode tip led to elimmation of the NaCl and the glass capillary. Instead the Ag/AgCl wire is placed m direct contact with the extracellular fluid and tissue. The 0.15M Cl- of the extracellular fluid mamtams the potential of the electrode at a constant value. The location of the reference electrode m the brain is not critical. Multiple working electrodes may all use the same reference and control electrodes.

217

b

1

0.4

0.2

0.0

,188

280

mu

500

Fig. 12. The elimination of background current. Upper: Continuous scanning depletes the ECF of electroactive components at the electrode resulting in a background signal (circles). Scanning at 12 min intervals allows the components to diffuse back to the electrode and produces the control signal (*). Lower: The difference voltammogram obtained by subtracting the voltammograms shown in the upper graph. Peak 1 at 80 mV is caused by the oxidation of ascorbate and uric acid; peak 2, at 220 mV, to the 5-hydroxyindoles (principally 5-HIAA); and peak 3 (380 mV) to the methylated metabolites of the catechols (mainly HVA). From R. D. O’Neill et al. (1983a), with permission. An Ag/AgCl electrode is easily made by inserting a silver wire in O.lM HCl. The wire is connected to the positive terminal of a voltage source (a 1.5 V battery will do), while a stainless steel

218

Justice, Michael,

and Neil1

or platinum wire is attached to the negative terminal, forming the cathode. As current flows (a few milliamps is sufficient) through the cell, hydrogen evolves at the cathode while silver metal is oxidized to silver ion at the anode The silver ions immediately deposit on the silver wire as silver chloride. The evolution of hydrogen gas may be used as a gauge of the completeness of the coatmg on the silver wire. When the evolution has slowed considerably, the wire is sufficiently coated with silver chloride. Several electrodes may be prepared at one time and stored m physiological saline for later use. 5.2.2.

Control

Electrode

The control or auxiliary electrode may be a stamless-steel wire or, more conveniently, one of the cortical screws used for the surgery. Its location is not important, but it must be in contact with the fluid of the brain for electrical conductivity. 5.2.3. Working

Electrode

Working electrodes have been constructed from a variety of materials, including carbon paste, carbon epoxy, and carbon fibers. 5.2.3.1. CARBON PASTE AND CARBON EPOXY ELECTRODES Carbon paste electrodes have been constructed by sliding a 30 gage teflon sheath over a 29 gage stainless-steel wire so that the sheath extends about 0.3 mm beyond the wire. Carbon paste is packed mto the cavity and smoothed on a piece of paper. The carbon paste or carbon epoxy may also be forced mto a pulled glass capillary to form an electrode. These may be made with about a 50 pm tip diameter while the teflon sheath electrodes are about 200 km in diameter. Carbon paste electrodes provide a surface at which the oxidations of interest proceed slightly more reversibly than at a carbon epoxy surface, but the electrodes are not quite as robust. The carbon paste can be prepared m several ways. In general, a 1.9.1 ratio of UCP-1-M carbon powder (Ultra Carbon, Bay City, MI) to Nu~ol (mineral oil), silcone oil (Kissinger et al., 1973), or high vacuum grease is used. UCP-1-M is highly purified graphite with a particle size of 1 pm. As described by Conti et al. (1978), 0.9 g of Nu~ol is dissolved m 15 mL of carbon tetrachloride. This solution is vigorously stirred, and 2 1 g of graphite powder are slowly mixed in with continued stirring for 5 min. The slurry is then evaporated until the carbon tetrachloride is removed. Carbon-epoxy electrodes may be prepared with a commercially available material called Graphoxy (Dylon Industries, Cleveland,OH), or may be made by mixing 0.45 g of triethylene tetramme with 3.6 g of Shell Epon 815 resin and adding 1.05 g of this mixture to 1.35 g of the graphite-Nulol paste described above

219 (Conti et al., 1978). The commercial material does not give as low a background current as the latter preparation. It is convenient to prepare 20-30 electrodes at one time. 5.2.3.2. CARBON FIBER ELECTRODES Carbon fibers are formed from the high temperature pyrolysis of materials such as polyacrylomtrile or pitch and have diameters of 6-12 km. A single fiber can be sealed m a glass capillary to construct an electrode (Ponchon et al., 1979). A small amount of mercury or conducting epoxy is placed m the capillary to make electrical contact with the fiber. Those prepared by Ponchon et al. extend 0.5 mm beyond the seal to provide a larger surface area and therefore larger current These workers have also described an electrochemical treatment of these electrodes that improves the discrimination of catechols from ascorbic acid (Gonon et al., 1981b). Carbon fiber electrodes in which only the cross-section of the fiber is used have been described by Dayton et al. (1980a). Recently, Wightman and coworkers have demonstrated that very small electrodes (10 pm diameter) behave very differently from the larger 100-200 pm electrodes (Dayton et al., 1980a,b, Wightman, 1981) Because these electrodes are so small, linear diffusion to the surface is not the dominant mode of mass transfer to the electrode surface Instead, these electrodes have the characteristics of spherical electrodes, that is, material converges as it diffuses to the electrode surface, as illustrated in Fig 13. The appropriate form of the Cottrell equation for diffusion to spherical electrodes IS IIFAD”~C + nFADC 1= #2p2 Y where Y is the radius of the electrode; the other terms have been defined above. The first term is time-dependent, whereas the second term is independent of time. The second term becomes more

Fig. 13. A Dlffuslon to a large electrode IS ma&y planar as mdrcated by the parallel arrows Radial edge effects are mmlmal B. Dlffusion to a mlcroelectrode IS treated as spherlcal because radial contnbutions are significant.

220

Justlee,

Michael,

and Nell/

important as the electrode size decreases It is, at diameters of about 10 km, the dominant term m the equation. Galus et al. (1982) have recently examined the behavior of very small electrodes m solution and point out that the nonlinear component of the diffusion increases dramatically with decreasing electrode drameter. For a 100 Frn diameter electrode, after 1 s, the nonlinear contribution is only a few percent of the total By 10 Frn, it IS 50%, and by 1 km it is 10 times the linear contribution. The advantages of small electrodes over larger ones include the obtaining of more localized information, less perturbation of the neural environment because much less material is oxidized, and lower charging currents and therefore faster measurement times. An additional advantage is that chemical reactions that may follow the initial oxidation, such as reduction of oxidized DA by ascorbic acid, will have less effect on the current. This is so because at a large electrode the product of a chemical reaction near the electrode may diffuse back to some pomt on the electrode surface. The probability of this happening with, for example, DA, after being reduced by ascorbic acid, is much less with very small electrodes, given that the average distance the DA diffuses before reacting with the ascorbic acid in solution to again form the oxidizable DA is given by the square root of Dt, where t is the half-life of the reaction and D is the diffusion coefficient. In this case, the distance diffused IS larger than the diameter of the electrode, thus greatly reducing the catalytic current. An addmonal significant advantage of very small electrodes arismg from their diffusion properties is that, since the product of the reaction diffuses away so rapidly when the potential is stepped back, the resulting current is almost completely nonfaradaic. Galus et al. (1982) have calculated that for an 8 brn diameter disk-shaped electrode, the ratio of backward to forward current is 2 2%, m excellent agreement with Ewing et al. (1981b) Because the backward current contams almost no faradaic component, it is a good representation of the nonfaradaic current occurrmg during the forward step and may therefore be used to correct the forward current for the nonfaradaic component by subtraction. The response characteristics of carbon fiber electrodes with respect to diffusion in the extracellular environment of the brain have been described by Dayton et al (1983). It was shown that at short chronoamperometric potential steps (92 ms), the electrode apparently samples from a small pool at the electrode tip, whereas for longer pulses a change m concentration occurs, implying an alteration in diffusion properties. This observation sug-

Voltammetty

221

the current arises from material gests that at longer times, diffusing through brain tissue The fluid pool at the carbon fiber electrode is smaller than that at the larger electrodes, so that the smaller electrode more closely follows changing extracellular neurochemistry. This topic is discussed more fully in Section 5.4. The electrical characteristics of carbon fiber microelectrodes have been established (Fox et al., 1980) and their use m quantrfymg iontophoresis described (see Section 7 7)

5.3. Electrode Modification Considerable efort has gone into improving the resolution of the voltammetric signals obtained m viva. Various scan procedures developed for general electrochemical work have already been described. This section describes work that has been done to modify carbon paste, carbon epoxy, and carbon fiber electrodes to resolve the overlapping signals from ascorbic acid, DOPAC, and DA, all of which oxidize at similar potentials m viva at unmodified electrodes. The approach generally taken to change the selectivity of an electrode is to modify the electrode surface m some way to make a particular electrochemical oxidation either more reversible or more irreversible. Increasing the reversibility will sharpen the peak shape and move the oxidation potential to a less positive potential Increasing the irreversibility will broaden the peak shape and move the oxidation potential to a more positive potential. Increasing the reversibility will sharpen the peak shape and move the oxidation potential to a less positive value These two approaches have been accomplished either through electrochemical pretreatment of the electrode to improve the reversibility of ascorbic acid or through chemical “doping” of the surface to make ascorbic acid oxidation more u-reversible. These two approaches are discussed below.

5.3.1. Electrochemical Pretreatment A number of electrochemical pretreatments have been mvestigated (Falat and Cheng, 1982; Rice et al., 1983) since first reported for carbon fiber electrodes by Gonon et al., (1981b). The pretreatment varies widely among these authors. Typically the electrode is placed u-r phosphate-buffered saline and a potential waveform is applied. Several variations on potential range, amphtude, duration, and frequency have been used In Fig. 14 the Elj2 behavior of the carbon paste, epoxy and fiber electrodes is shown. As indicated in the figure, the untreated carbon paste and epoxy

Justice, Michael, and Nell/

222 -01

00

POTENTIAL 02 03

01

04

05

06

07

ELECTRODE

CARBON

AA DA

PASTE’

HVA 3-M-l

DOPAC

CARBON

EPOXY

b,c /

ELECTROCHEMICAL PRETREATMENT

CARBON

b

’

AA

HVA

DA

3-Ml

k!

\ b

AA

DA

FIBERdBe

DA

5-HT / A’

HVA DOPAC -‘5-HIAA ---AA

/ ELECTROCHEMICAL PRETREATMENT

AAe

--DA

&’ DOPAC

5-HT 5-HIAA

i(

Fig 14 Oxldatlon potentials of monoammes and metabolltes at carbon paste, carbon-epoxy, and carbon fiber electrodes References “Sternson et al , 1973, ” Falat et al , 1982, ’ Contl et al , 1978, ” Ponchon et al , 1979; ‘Gonon et al , 1981b

electrodes give similar results m vitro. The oxidation currents from ascorbic acid, DA, and DOPAC are unresolved, but occur at a different potential from that for the methoxylated metabolites, homovanillic acid (HVA) and 3-methoxytyramme (3-MT) Falat and Cheng’s treatment of Graphpoxy electrodes separated the ascorbic acid from the DA signal by several hundred millivolts, indicating that these compounds could be resolved. The untreated carbon fiber electrode (Ponchon et al., 1979) shows separation of the DA and 5-HT signals However, the HVA, 5-hydroxymdole-3-acetic acid (5-HIAA), DOPAC, and ascorbic acid signals are all very close together. Gonon’s electrochemical treatment (1981) of the carbon fiber electrode generated three separate signals. ascorbic acid, DA/DOPAC, and 5-HT/5HIAA. Although this treatment did not yield complete separation of all signals, it has proven particularly useful as there are individual signals for ascorbic acid, dopaminergic compounds, and serotonergic compounds. This treatment consists of a 70 Hz triangular wave from 0 to +3 V (vs Ag/AgCl) applied for 20 s, followed

Voltammetfy

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by a constant potential of +1.5 V for 20 s. Figure 15 shows the resolution of the ascorbate signal from the catechol signal. Note that the overall effect of the above treatments is to move the oxidation potentials of ascorbic acid, DOPAC, and 5-HIAA to lower values, reflecting the enhanced reversibility of these couples. Hutson and Curzon (1983) have recently summarized the oxidation potentials of endogenous electroactive compounds at the various types of electrodes. No definitive theory exists about the mechanisms by which electrochemical pretreatment changes the nature of a surface reaction Falat and Cheng (1982) have shown with electron micrographs that the surface of the electrode becomes roughened by the treatment Evans and Kuwana (1977) had previously correlated the Increase in surface oxygen functionalities with an improved ascorbic acid reversibility. Rice et al. (1983) demonstrated

in vivo

in vitro

12

39

ID

PBS

'E C

:

d, 0 +.2

-

15 Typical DP voltammograms recorded from the neostrlatum of unanesthetlzed freely moving rat (m VIVO) and recorded from standard solutions (in vitro) lust after the experiment The AB segment represents the measure of the peak 1 height m nA The CD segment represents the measure of the peak 2 height m nA DPV parameters. AV = 50 mV, V = 10 mV/s From F. Gonon et al (1981a), with permission Fig

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that a high anodic potential at a carbon paste electrode increased the electron transfer rate of some redox couples and increased the reversibility of DOPAC m particular. A loss of the hydrophobic pastmg compound from the electrode surface, resulting m improved hydrophilicity of the electrode, was considered to be a possible contributor to the change in electrode performance Carbon paste electrodes are reportedly not robust enough to withstand the typical electrode treatments and have therefore received less attention 5 3.2. Chemrcal

Modrficatlon

The surface state of an electrode is critical m determining the rate constant of an electrode reaction This dependence on the chemical nature of the electrode has been utilized to construct a carbon paste electrode with considerable selectivity for DA over ascorbic acid, DOPAC and 5-HT (Blaha and Lane, 1983). By mcorporatmg stearic acid into the carbon paste, a negatively charged surface is created that inhibits negatively charged molecules from reaching the electrode surface As DOPAC and ascorbic acid are negatively charged at physiological pH, this has the effect of making the electrochemical oxidation of these compounds more n-reversible Thus, these compounds oxidize at higher potentials at the modified electrode than at the unmodified electrode. Note that this approach IS complementary to the electrochemical pretreatment The effects of paste composition and surface states on electron transfer rates of carbon paste electrodes have been examined by Rice et al. (1983). In particular the effect of paste composition on the oxidation of DOPAC was examined. Using hydrocarbons of 8-21 carbon atoms m cham length as the pasting liquid, these workers showed that the rate constant for the electron transfer reaction decreased as a function of chain length, although the effect was only slight until the 18-carbon-atom length, when a more significant decrease m the rate occurred. The slowest rates were seen for Nu~ol. On the other hand, the electron transfer rate could be increased by chemical or electrochemical pretreatment of the electrode. It is suggested that the rate is enhanced through the formation of surface groups which modify the mterfacial properties of the electrode, but do not participate directly m the electrode reaction. An electrode that elimmates the ascorbic acid component of the oxidation current has been described by Nagy et al (1982), whose procedure involved covering a carbon paste electrode with

Vo/tammetry

225

a dialysis membrane A drop of ascorbic acid oxidase solution IS placed between the membrane and the electrode surface to oxidize any ascorbate before it can reach the electrode surface. The electrode has been used for chronoamperometric measurements on the effect of potassium ion stimulation on brain slice preparations. In a study that outlmes what appears to be a significant improvement m electrode selectivity, Gerhardt et al (1984) have reported a Nafion-coated carbon epoxy electrode that is simple to make and yet has excellent relection of negative ions such as ascorbate, DOPAC, and 5-HIAA at physiological pH. Nafron is a perfluorosulfonated derivative of teflon The principle of the electrode is that by coatmg the electrode surface with a negatively charged polymer, only positive ions reach the electrode surface for oxidation Thus, the electrode is primarily responsive to DA serotonin, and NA If the sensitivity of DA is taken to be 100, the relative sensitivities for the endogenous compounds are: serotonin, 65; NA, 35; 3,4-dihydroxyphenylethylene glycol and 5-HIAA, all 0 5 Al(DOPEG), 2; ascorbic acid , DOPAC though the electrode may have too slow a response for scan methods due to the membrane, it is very satisfactory for chronoamperometry. The electrode is prepared by dippmg a carbon epoxy electrode m an alcohol solution of Nafion (C. G Processing, Box 133, Rockland, DE) and evaporating the alcohol, leavmg a thin film of Nafion on the electrode. The electrode is then tested m 200 PM ascorbic acid, to which DA is added m pM amounts for precahbration

5.4. Model of Electrode Response in Wvo Voltammetry with microelectrodes m vivo produces data very drfferent from those obtained with voltammetry m solution. Figure 16 illustrates typical data obtained during the imtial period of data collection from 100 km diameter carbon paste or carbon epoxy electrodes implanted m CNS tissue. Chronoamperometric pulses of 1 s duration were applied every 3 min at 0.6 V vs Ag/AgCl reference electrode These recordings were obtained every other day over an 8-d period. Only the first 3 h of each 8 h experiment are shown The signal declmes over the mitral period of recording and eventually reaches a steady-state level This result 1s very different from that obtained if the same experiment is done m a beaker. Chronoamperometry m solution will give the same response on repeated measurements, that is,

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Fig 16 Chronoamperometrlc current at a 100 km diameter carbon-epoxy electrode m strlatum of freely moving rat recorded every other day in the order ABCD. One s pulses of +0 6 V vs Ag/AgCl applied at 3 mm intervals From J B Justice et al (1983), with permlsslon

showing no decline in current This effect is due to the relatively small amount of material consumed relative to that m the large volume of solution and rapid equihbration of the lowered concentration at the electrode tip with the surroundmg bulk solution through diffusion. A model of m viva voltammetry has been proposed to account for the difference (Cheng et al. 1979a, Cheng, 1982) It is suggested that a small pool of fluid exists at the electrode tip surrounded by tissue and extracellular fluid in contact with the pool. It IS this pool that 1s sampled by the voltammetric process However, the pool size is so small that the oxidation of components at the electrode tip depletes the pool of oxidizable material by an amount sufficient to make the next oxidation current notlceably smaller. As the depletion of material m the pool contmues, a concentration gradient develops between the pool Material diffuses mto the pool and the surroundmg medium. from the surroundmg tissue to replace the material oxidized. Eventually a steady-state is reached. Cheng et al have modeled this process with the followmg equation

Voltammetry

227

2D”zt c, = G-1 - @:3 cn-1+ ; L(G - G-1) 112

where C, is the concentration in the pool at the tip of the electrode when the nth pulse is applied; C,-i is the concentration at the previous pulse; D is the diffusion coefficient; K is a mass transfer coefficient; L is the thickness of the pool from the electrode surface; t, the duration of a single chronoamperometric pulse; t, is the time between pulses; and C, is the concentration in the surrounding tissue compartment. The effect of varying the time between pulses, the volume of the pool, and the pulse width have been simulated (Lindsay et al., 1980a). Figure 17 illustrates the effects on the chronoamperometric signal mentioned above for the initial period of decline and for a sudden, brief increase m extracellular material in the surroundmg tissue. Such a situation can be induced in the striaturn with electrical stimulation of the medial forebrain bundle, as shown m Fig 18. Perhaps most noteworthy m the above simulation data is the result of decreasing the pool size It is clear that the electrode can follow events m the surroundmg tissue more effectively as the volume of fluid at the tip is decreased. This result is taken advantage of with the 8p.m diameter carbon fiber electrodes, which, having a very small pool, are able to follow extracellular neurochemistry more closely (Dayton et al., 1983). The initial decline has been shown (Justice et al., 1983) to be due not to all the electroactive components of the ECF, but prmcipally to ascorbic acid. A possible explanation for this is that the other components present m significant concentration m the striaturn, the metabolites DOPAC and HVA of DA, and the serotonm metabolite 5-HIAA, have relatively high turnovers relative to the rate of removal by the sampling methods, and so are not appreciably depleted by samplmg. Ascorbic acid, on the other hand, may have a much slower turnover and, though present in relatively high concentration, be significantly depleted by the sampling process. Cheng has published a more general derivation of the original model that can account for these data (Cheng, 1982). The compartment model has recently been discussed by Albery et al (1983).

5.5. Calibration of Electrodes Aside from the qualitative question of what is bemg measured under different stimulus conditions, there is the problem of quantifying m units of concentration the current being measured Al-

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L

6

2 I

1

I 50

I

1

1

TIME

1

150

100

I

I

1

200

(mln)

Fig 17 Slmulatlon of the chronoamperometrlc response to a brief square wave change m concentration using Cheng’s equation The effect of increasing pool size IS shown, with A having the smallest pool size and C the largest From W S Lindsay et al (1980a), with permission. though electrodes are usually calibrated m 4-methylcatechol at pH 7.4, once they are placed in brain tissue the response may be very different. If possible, rt IS useful to retest the electrodes after the experiment. Even rf this IS done, however, rt only indicates whether the electrode has changed or not. It does not answer the questron of to what a given current at the electrode corresponds m extracellular concentration. The data are often expressed as observed concentratrons based on the calrbratron data, but this should be done with the knowledge that drffusron coeffrcrents may be altered in VIVO relative to homogeneous solution values

229

Voltammetry

13

12 OXIDATION CURRENT t7A

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9

7

6

5

!

24 I

4 0

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I

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4

5 I

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I

50 TIME,

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100 MINUTES

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Fig. 18. Chronoamperometnc result m the stnatum after electncal stlmulatrons of the medial forebrain bundle Seven stlmulatrons of varying intensity (10-40 PA) and duration (10-60 s, 300 ms trams, 1 tram/s, 100 pulses/s, 1 msipulse) were used Each ended 20 s prior to a chronoamperometrrc pulse (Dayton et al , 1983). Concentrations reported in this manner will be m error by the change m diffusion coefficient and any other parameters m the relatronshlp between current and concentra-

tion, such as active surface area of the electrode and the number of electrons mvolved u-r the oxidation step This latter point may be important with regard to the observed catalytic effect of DA on ascorbic acid oxrdatron (Dayton et al., 1980b, Freed and Echlzen, 1983). Adams and Marsden have recently discussed electrode testing and calibration (1982) A useful suggestion m regard to reducing the variance m

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voltammetric data has been made by Morgan and Freed (1981) who suggest administering acetaminophen as a standard that allows normalization of response among different electrodes. The sensitivity of each electrode is measured by a 75 mg/kg ip dose of acetaminophen, which generates a peak at +0 55 V vs Ag/AgCl using carbon paste electrodes and semidifferentlal voltammetry. The response of the electrode in an experiment is standardized by the response to acetammophen. This procedure assumes that the acetammophen is uniformly distributed at different electrodes, which is a reasonable assumption, particularly for comparisons of bilaterally implanted electrodes The procedure does not actually calibrate the electrode to the point of providmg knowledge of concentrations, since the acetammophen concentration is itself unknown, but it nevertheless seems very useful for normahzmg the response from different electrodes.

6. Interpretation 6.1. Introduction In 1973, Kissinger, Hart, and Adams implanted a carbon paste electrode m the striatum of an anesthetized rat and recorded cychc voltammograms, thereby demonstratmg the feasibility of voltammetry in vivo. The cyclic voltammetry Indicated that ascorbic acid was a malor contributor to the observed oxidation current. It was known, however, that DA, NA, serotonm, and many of their metabohtes are electroactive, that is, easily oxidizable, at low potentials. Accordmgly, work continued to monitor these compounds. McCreery et al. (1974a) demonstrated that DA, NA, and ascorbic acid could be monitored when mlected next to the tip of a recording electrode. The neurotoxm 6-hydroxydopamme was also exammed by this method (1974b) In one of the first approaches toward mterpretation of the obtained over a range of 0.5-5 mg/kg amphetamine (Huff et al , 1979). This change was attributed to DA because amphetamme is known to release DA, and other work (Gonon et al., 1978) had demonstrated that the amphetamine-induced changes in oxidation current are abolished by unilateral injections of 6-hydroxydopamme in the substantia nigra and are suppressed by pretreatment with alpha-methyl-p-tyrosme Further, it had been shown that there is no stimulated efflux of ascorbic acid from minced caudate tissue with concentrations of amphetamine

Voltammetly

231

as high as mM (Chey, 1978, Milby et al., 1981). Contributions from DOPAC could not be ruled out, however, as pointed out by the authors. Gonon et al. (1978) studied the effects of ip injections of amphetamine in the strlatum using untreated carbon fiber electrodes. The chronoamperometrlc data were very similar to those described above. The effect was eliminated by pretreatment with alpha-methyl-p-tyrosme or by prior (8 d) injection with 6-hydroxydopamine, a neurotoxin, in the substantia nigra The 6-hydroxydopamme effect was observed only ipsilateral to the site of injection These results seemed consistent with the mterpretatlon of the increase m signal being due to DA release. There was a confusing aspect to this interpretation of the increased OXIdation currents, however. Push-pull perfusion results generally indicated such low extracellular levels of DA that radioactivity methods were usually used to study DA release. This observation implied that extracellular DA levels were too low to be easily detected by voltammetry m the presence of higher concentrations of other electroactlve compounds. The early interpretations were brought into question (Gonon et al, 1980) when it was suggested that it was DOPAC and not DA that was the principal catecholamine species detected m the striatum, a suggestion based on data obtained with an electrochemlcally treated pyrolytic carbon fiber of 8 pm diameter that discriminated the catechols from ascorbic acid (see Fig 15). The electrochemical treatment shifts the ascorbate oxidation to more negative potentials, thereby resolving it from the catechol peak. Differential pulse voltammetry was used to generate a series of oxidation peaks, one of which could be attributed to ascorbic acid (-50 mV vs Ag/AgCl) and another to DA, DOPAC, or a combmatlon of the two (+lOO mV vs Ag/AgCl). Two peaks were obtained m vivo that corresponded with the potentials observed m vitro. The -50 mV peak in vivo was attributed to voltammetrlc signals observed in VIVO, differential pulse voltammetry m the strlatum of anesthetized rats at iodide-treated platinum electrodes yielded two distinct peaks (Lane et al., 1976). The peak occurrmg at higher potentials (0.2 V vs Ag/AgCl) was significantly increased by local injection of both DA and NA near the electrode. Because the concentration of NA is only 1% of the DA concentration m the stnatum, the peak was attributed to DA. The other peak, at about 0 V vs AgiAgCl, was increased by inlettlons of ascorbic acid. Inlectlon of 2 pg of d-amphetamine near the working electrode resulted m a 7-lo-fold increase m the apparent

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catecholamme peak No change m the ascorbate peak was observed. Wightman et al. (1976) used voltammetry in the cerebrosplnal fluid (CSF) to monitor transmitter metabolltes following stlmulation of the mgrostriatal pathway. Homovamllic acid was detected m the CSF following this stimulation, whereas 5-HIAA was detected followmg stimulation of the midline raphe To follow up this work, Wrghtman et al. (1978) implanted stimulatmg electrodes in the medial forebrain bundle (MFB) and carbon paste electrodes m the lateral ventricle. Followmg stimulation of the MFB, increased oxidation currents were detected m the ventricle. Samplmg the ventricular fluid and chromatographmg it by high performance liquid chromatography with electrochemical detection confirmed an increase in HVA m the ventricles. It remained to be shown that the voltammetrlc technique would work for momtoring neurotransmltter release in CNS tissue.

6.2. Dopamine, DOPAC, and Ascorbic Acid Because it is well known that amphetamine causes an increase in extracellular DA, this drug was used to test the voltammetric technique in the striatum (Conti et al., 1978, Huff et al., 1979). Conti et al. (1978) observed 2040% changes from baseline chronoamperometric signals at carbon paste electrodes following l-10 mg/kg ip doses of d-amphetamine. The signals remained elevated for l-2 h following injection The voltammetnc signals were well associated over time with the typical behavioral effects of amphetamine Additional work at this time supported the mterpretatlon of the signal as being caused by DA. Semidifferential linear sweep voltammetry at carbon paste electrodes showed distinct differences before and after amphetamine admmistration (Lane et al., 1979). A clear dose-dependent increase m oxidation current was ascorbic acid oxidation as it was shown that this peak was absent in scorbutic guinea pigs. Pharmacological manipulation of the catecho1 peak produced results consistent with DOPAC rather than DA For example, administration of pargyline, an MAO mhibitor, markedly reduced the peak, as did amphetamine, whereas haloperldol increased it. However, pretreatment with pargyline followed by amphetamine resulted m a small catechol peak estimated to be equivalent to 0.2 pM dopamme These results are illustrated in Fig 19.

Vo/tammetly

233 n=l

Control

Pargyllne

(75

mg

kg

‘)

n ’

-100~

n Amphetamine

(2mg

n=5

kg-‘)

Haloperldol

Chloral

(0.5mg

kg“)

n’4

II=4

hydrate

:::A

-

3001

50 0

-1

tlO0

flO0 ,2

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Time Fig 19 Evolution of the peak 2 herght measured from DP voltammograms obtained from the neostrlatum of unanesthetlzed freely moving rats Carbon fiber electrodes were implanted under brief (30 mm) and light halothane (0 7%) anesthesia. DP voltammograms were then recorded every 2.5 mm until the peaks were stable (about 1 5 h) Data show the peak 2 height evolution during the next 3 h (mean -+ SEM of n experiments) For each experiment the results were expressed as the percentage of the mean control value calculated by averaging the seven absolute values of the peak 2 heights obtained durmg the last 15 mm before the qectlon. From F Gonon et al. (1981a), with permission

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Using electrochemically-treated carbon fiber electrodes, Gonon et al. (1981a) demonstrated that it was ascorbate, rather than DA, that was a major contributor to the amphetamineinduced increase m oxidation current, but that the increase depended on an intact dopammergic system. This interpretation of the amphetamine-induced signal was later supported by further voltammetric evidence (Dayton et al., 1981; O’Neill et al., 1982b) and by push-pull perfusion data (Salamone et al., 1984; Justice et al., 1983). Further, using differential pulse voltammetry at an electrochemically treated carbon fiber electrode to detect ascorbic acid and DOPAC m the striatum of unanesthetized rats, these workers estimated extracellular concentrations of about 300 FM for ascorbic acid, 18 FM for DOPAC and <50 nM for dopamme. The half-life for DOPAC was also calculated for several DA-rich regions (Buda et al., 1981). With the improved electrochemical pretreatment (Gonon et al., 1981a), these mvestigators were able to quantitate the effects of amphetamine on the observed ascorbic acid peak. An approximately 170% increase in peak height was observed 1 h after ip mjection of 2 mg/kg amphetamine. Animals with a unilateral 6-hydroxydopamme injection showed a delayed and reduced response on the side ipsilateral to the mlection. Also, the effect was not observed during halothane anesthesia. Thus, the lack of change m ascorbic acid after amphetamine reported earlier (Lane et al., 1976) may have been related to the use of anesthesia m the experiment. In this regard, Clemens and Phebus (1983) recently reported that the use of anesthesia produced opposite effects from unanesthetized animals m voltammetric measurements m the striatum following admmstration of pergohde, a DA agonist. Additional evaluation of the voltammetric effects of ip admmistration of amphetamine was provided by Dayton et al. (1981) who used backstep-corrected normal pulse polarography at disk-shaped carbon fiber electrodes (Ewing et al., 1981b). They were able to construct oxidation profiles (voltammograms) that were similar to profiles of ascorbic acid rather than DA Unilateral 6-hydroxydopamme injections m these experiments also produced a delayed response, but no decrease m signal Ewing et al. (1982) used background subtraction with the above method to construct difference voltammograms that represented the change m signal before and after pharmacological or other stimulation. The difference voltammogram should therefore have properties similar to the voltammograms of the com-

Voltammetry

235

pound(s) causing the increase in oxidation current. The difference voltammogram observed with amphetamine resembled the voltammogram of ascorbic acid, while that following local application of potassium ion resembled the voltammogram of DA. Large systemic mlections of ascorbic acid resulted m difference voltammograms similar to those seen with amphetamme. To study further the effects of amphetamine, Ewing et al. (1983) combined the use of in vivo electrochemistry with single unit recordmg. The unit recording and electrochemical electrodes were placed approximately 500 km apart in the strratum. A 2.5 mg/kg ip dose of amphetamine resulted m a reduction of neostriatal firing rates and a concomitant increase m chronoamperometric signal With 7.5 mg/kg, the firing rate was accelerated and the electrochemical signal showed a dosedependent increase over baseline. In this case, however, the firing rate reached a peak and returned to baseline much faster than the electrochemical signals The sources of oxidation currents m the striatum were exammed by O’Neill et al. (1982a,b, 1983a,b) using linear sweep voltammetry at carbon paste electrodes. Under microprocessor control the signal was semidifferentiated to improve the resolution. Three peaks were observed in scans obtained from -100 to +500 mV vs Ag/AgCl at 5 mV/s. A fourth peak was observed when the scans were extended to 750 mV. The first of these was attributed to ascorbic acid through ip inlections of ascorbic acid and microinlections of ascorbate into the striatum DOPAC is obscured in the first peak and occurs at a potential about 50 mV more posmve than ascorbate. Because amphetamine Increased the height of the first peak without shifting its potential, these results support the interpretation that amphetamine increases extracellular ascorbic acid without a significant voltammetric contribution from the catechols. Schenk et al (1983) have recently used chronoamperometry in brain slices for quantitative evaluations of in vivo electrochemistry. It was concluded that the malor portion of the baseline signal m caudate chronoamperometric measurements comes from ascorbic acid and that, of the biogemc amine metabolites, DOPAC is the major compound detected. To provide nonvoltammetric verification of the sources of the in viva oxidation currents, several workers sampled extracellular fluid from the striatum and chromatographed it using high performance liquid chromatography with electrochemical detection

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(Saraswat, 1981; Justice et al., 1983; Salamone et al., 1984). It was demonstrated that ascorbic acid, DOPAC, 5-HIAA, and HVA are the malor electroactive constrtuents of the extracellular fluid m the striatum (Fig. 20) and that ascorbate increases, while DOPAC and HVA decrease, following systemic amphetamine. Using a dialyzed perfusion procedure, Blakely et al. (1984) showed that the DA receptor blocker haloperidol induces an mcrease m DOPAC and HVA, but not ascorbate or 5-HIAA. The relationship of these changes in extracellular concentration to observed oxidation currents has been described (Justice et al., 1983). In particular, for chronoamperometry the relation between relative current and relative concentration of electroactive components of the extracellular fluid 1s: z(t) = relative response X relative concentration (t) for all compounds electroactive at the potential of interest For a carbon-epoxy electrode at a potential of +0.6 V vs Ag/AgCl, this equation becomes: i(t) = 0 40AA(t) + 1 ODOPAC(t) + 0 53HVA(t) + 0 85[5-HIAA( t)]

o\ 2

3

4

5

6

7

8

HOURS

Fig 20 Major electroactlve components of extracellular fluid m the strlatum sampled by dlalysls over an 8-h period Note the difference in scale for ascorbic acid Ascorbic acid IS the only component showing slgmflcant decline From J B Justice et al (1983), with permlsslon

237

Voltammetry

Calculation of an absolute current would require absolute extracellular concentrations, the active area of the electrode, and diffusion coefficients m the extracellular fluid. Using this and a similar equation for carbon paste electrodes, it can be shown that the chromatographic data can account for the imtlal declme m chronoamperometric oxidation current, as shown m Fig. 21. Additional chromatographlc data on electroactive compounds in the strlatum have recently been reported by Zetterstrom et al (1983) who estimated basal extracellular concentrations of DA and DOPAC to be 50 nM and 5 pM, respectively. These investigators also found high levels of ascorbic acid and demonstrated the presence of uric acid, an addltional electroactive compound, m the extracellular fluid Sharp et al. (1984)

. .

CHROMATOGRAPHY CHRONOAMPEROMETRY

180

.

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3

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~HOURS)

Fig 21 Calculated oxldatlon current durmg the initial period of sampling compared to the observed current From J, B Justice et al (1983), with permlsslon.

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have used simultaneous DPV at carbon fiber electrodes and mtracerebral dialysis to verify the voltammetric signals with respect to measurement of DOPAC and 5-HIAA. The results strongly support the use of DPV for monitoring DOPAC. The carbon fiber electrode appeared less sensitive to increases m extracellular 5-HIAA than the dialysis method

6.3. Serotonin and 5-HlAA Although not as extensively studied as the nigrostriatal system has been dopaminergic system, the serotoninergic sufficiently examined to permit some fairly firm conclusions to be drawn about the chemical identity of voltammetric signals arismg from the serotonmergic components of the CNS. It was initially suggested that serotonin was the major source of the serotoninergic signal (Marsden, 1979a,b, 1980). Pharmacological agents and electrical stimulation have been used to manipulate the serotonin system, and it now seems fairly certain that the major species being monitored by in viva voltammetric methods is the serotonin metabolite 5-HIAA, although Crespi et al. (1983), using DPV at carbon fiber electrodes, have shown that uric acid oxidizes in the same potential range as 5-HIAA and may contribute as much as 30% of the peak height. Typically, the use of p-chlorophenylalanme (PCPA) to mhibit tryptophan hydroxylase and thus reduce tissue content of serotonm has served to mampulate the observed voltammetric peaks (Marsden, 1979b; 1980). In general three peaks are observed with scan methods such as differential pulse voltammetry. The potential at which each of these peaks occurs depends on the electrode treatment. Marsden, using carbon paste electrodes, reported peaks at +O.lZ, 0.20, and 0.35 V vs a Ag/AgCl reference electrode (Marsden et al., 1981). Intracranial mjection of ascorbic acid increased the first peak, whereas the third peak was increased by a similar mjection of serotonm or 5-HIAA. The second peak was increased by inlection of DA or DOPAC (Braze11 and Marsden, 1982a) With electrochemically treated carbon fiber electrodes, Cespuglio et al. (1981a,b,c; Cespuglio, 1982) observed three peaks m the differential pulse recordmgs performed over a scan range of -0 1 to +0.45 V vs Ag/AgCl. The first, attributed to ascorbic acid occurred at -0.05 V. The second, attributed to DA and DOPAC, occurred at +0 10 V. The third, at +0 3 V, contained possible contributions from 5-hydroxytryptophan, serotonm, and 5-HIAA. That the third peak is of serotonmergic origm IS indicated by the disappearance of the peak following mtraventricular mlection of the

239

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neurotoxm 5,7-dihydroxytryptophan (Braze11 and Marsden, 1981) This peak was not affected by intracerebral mlection of ascorbic acid oxidase (Braze11 and Marsden, 1982b). To determme the identity of peak three, Cespuglio et al. (1981a, 1981~) administered the MAO inhibitor clorgyline, which decreased peak three by 40%. This drug also decreased the tissue content of 5-HIAA (55%), but increased serotonin tissue levels by 51%, leading to the conclusion that peak three is 5-HIAA rather than serotonin. Additional evidence that the malor chemical source of voltammetric oxidation current from the serotonmergic system is 5-HIAA includes the observation that probenecid, which blocks outflow of 5-HIAA from the CSF, increased peak three m the striatum and lateral ventricle and mcreased 5-HIAA u-t ventricular CSF as measured by HPLC, while no detectable 5-HT was found. The increase followmg admmistration of probenecid was also observed by Kennett and Joseph (1982). Further, admmlstration of NSD-1015, a decarboxylase enzyme blocker, produced a steady decline m the voltammetric peak attributed to the serotonin system, which correlates with tissue assays by HPLC that showed a 50% decrease m 5-HIAA, and a slight increase m serotonin. Clorgylme, an MAO inhibitor that blocks the formation of 5-HIAA, decreased the signal by 40% The chromatographic data demonstrated a 55% decrease in 5-HIAA and a 51% increase m serotonin. Baumann and Waldmeier (1984) have used differentral pulse voltammetry at carbon fiber electrodes m conscrous rats to further support the contention that it is mainly 5-HIAA that is measured by DPV m the potential range m which hydroxymdoles are oxidized. The serotonmergic peak can be increased by electrical stimulation (Cespuglio et al., 1981~). Tissue assays under the same conditions show increased 5-HIAA, but not serotonin. In other work using electrical stimulation Wightman et al. (1976) used HPLC with electrochemical detection to demonstrate an increase m 5-HIAA in the ventricles following electrical stimulation of the raphe. Taken together, the above evidence strongly supports the interpretation that the malor source of voltammetric signal arises from the serotonm metabolite 5-HIAA

6.4. Neurotransmitter

Detection

The above discussion has stressed the contrrbution of neurotransmrtter metabolltes and other compounds to m vivo

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voltammograms. We do not want to leave the reader with the rmpressron that neurotransmrtter release itself cannot be monitored with voltammetry The research to date has more clearly defined the problem of momtormg neurotransmitter release as one of followmg a relatively small srgnal m the presence of larger, sometimes changing, background signals from other more concentrated electroactive compounds Two approaches have been taken to this problem. One, modrfrcatron of the electrode, has been described above. With thus approach, Gonon et al. (1984) have recently demonstrated that DA can be momtored rf the DOPAC peak is suppressed by mhlbmon of DOPAC synthesis Drfferentral normal pulse voltammetry was used Blaha and Lane (1984) use stearrc acid modified electrodes to monitor changes m DA release in response to DA receptor blockers, halopendol and chlorpromazme To demonstrate that the signal is due to DA, rt was shown that the signal was not reversed by pargylme, nor did the electrodes respond to admmrstratron of ascorbic acid The other approach IS based on the idea that the m vrvo voltammogram represents the summatron of several voltammograms from individual compounds. Voltammograms recorded prior to the onset of some stimulus are treated as a background or control set of voltammograms. Difference voltammograms are obtamed by subtracting the average of the background voltammograms from those recorded followmg the stimulus The difference voltammograms represent those components whose extracellular concentration IS changed as a result of the stimulus A successful applrcatron of this approach to the detection of neurotransmrtter release has been made by Wrghtman and coworkers (Dayton et al., 1981; Ewing et al , 1982, 1983a, 1984; Kuhr et al , 1984) A fast scannmg normal pulse waveform IS applied to a carbon fiber electrode m the strratum. In these studies, the difference voltammogram obtained by subtracting control signals from those followmg electrrcal strmulatron of the medial forebrain bundle mdrcated that DA was the compound causing a major portion of the change m signal Thus conclusron was reached by comparing the difference voltammogram with a DA curve obtained m vitro (see Fig. 22). The two curves were similar and clearly different from voltammograms for ascorbic acid and DOPAC The above work clearly demonstrates the utility of m VIVO voltammetry for mot-utormg raprd changes m extracellular levels of DA

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7. Applications 7.1, Introduction In viva voltammetry is a much newer field than the other methods reported m this volume For this reason, much of the work to date has been concerned with developing the method rather than applying it. The work described m the mterpretation section mvolved the use of pharmacological agents with known properties to learn about the nature of the voltammetric signals. The reverse is also possible, as shown in a series of papers in which voltammetry is used to investigate the action of a drug, HA-966, that depresses the firing rate of central dopammergic nerve cells (Broxterman and Mos, 1980, Broxterman et al., 1980; Mos et al , 1981). In this section, examples of the ways voltammetry can be applied m vivo are described indicating how new mformation about the functionmg of the central nervous system may be obtained

7.2. Clearance of Released Dopamine From Extracellular Fluid One of the main advantages of voltammetry is the speed of analysis. The work described m this example illustrates how in viva voltammetry may provide information about characterrstics of neurotransmitter dynamics not obtainable by other methods Using carbon fibers and normal pulse voltammetry, Wightman’s group (Ewing et al, 1983a, Kuhn et al., 1984; Ewing and Wlghtman, 1984) has monitored the release and disappearance of DA m the stnatum during electrical stimulation of the medial forebrain bundle Dopamine is detected at the electrode when the stimulation is initiated, and disappears almost immediately when the stimulation is terminated The left panel of Fig. 22 shows the time course of the extracellular increase. The stimulation lasts 10 s and the extracellular increase lasts for only 20 s. The right hand panel of Fig. 22 shows the fit of the voltammogram resulting from stimulation with the voltammograms for DA and ascorbic acid. The voltammogram is seen to be voltammetrically identical to DA. The mechanisms of clearance of the released DA were studied pharmacologically. In order to examme the metabolism of the released DA, several inhibitors of DA metabolism were adminis-

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Fig. 22. Oxidation current from a microvoltammetric electrode placed in the caudate nucleus during a 10 s, 130 kA, 60 Hz stimulation (horizontal bar) of the ipsilateral MFB. Left: Chronoamperometric current vs time at 0.5 V; 6 s intervals. Right: Difference voltammograms. Solid lines: DA (25 p,M) and ascorbic acid (200 PM) in pH 7.4 buffer after in vivo use; circles: in vivo result obtained by subtraction of a scan before the stimulation from that obtained at the peak of stimulation. Current scales: i = 17 pA, DA; i = 8 pA, ascorbic acid; and i = 34 pA, in vivo. From A. G. Ewing and R. M. Wightman (1984). tered. If metabolism occurs prior to extracellular uptake, the use of inhibitors should increase the time that DA is observed in the extracellular fluid. However, inhibitors of DA metabolism (pargyline or tropolone) did not significantly affect the rate at which DA disappeared from extracellular fluid. Similarly, inhibitors of neuronal uptake of DA (amphetamine and benztropine) also had no effect on the time course of the clearance. These results suggest that DA leaving the synaptic cleft (because of neuronal uptake mechanisms saturated as a result of the stimulation) is cleared by an extraneuronal uptake mechanism. The rapid clearance of DA suggests that the diffusion of DA over large distances is unlikely. These results tend to argue against a model of the DA system in which the released DA is free to diffuse and modulate activity far from the site of release.

7.3. Dopamine Release in the Median Eminence Dopamine release has been measured in the median eminence to study the dynamic regulatory role of this prolactin-inhibiting factor under conditions of simulated suckling (Plotsky et al., 1982; Plotsky and Neill, 1982). Carbon microelectrodes were implanted into the medial median eminence region among capillaries of the

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primary portal plexus of urethane-anesthetized lactating rats During electrical stimulatron (15 Hz, 5-30 V, 15 mm) of a surgltally isolated mammary nerve trunk, a transient (3-5 mm) 35% decline in electrochemrcally detectable catecholamme release was observed. Figures 23, 24, and 25 illustrate the voltammetrlc response to sham and electrical stimulation of the mammary nerve [3H]-Dopamme content of stalk blood IS also shown. These results demonstrate that mammary nerve strmulation induces a brief decrease m hypothalamic DA secretion that precedes or accompanies the early rise m prolactm release evoked by the same strmulus This work further demonstrates the utrlrty of m VIVO voltammetry for momtormg transient changes m neurotransmitter levels.

7.4. Catecholamines in the Locus Ceruleus Although most of the developmental work on in VIVO voltammetry has been done m the striatum, other regrons are begmnmg to be examined. Differential pulse voltammetry has been used for a pharmacologrcal and behavioral study of catecholamine metabolism m the rat locus ceruleus (LC) (Buda et al., 1983; Gonon et al., 1983) Using electrochemrcally treated carbon fiber electrodes, voltammograms were recorded every 2 min for 5 h from conscious freely moving rats. These mvestrgators concluded that the catechol peak recorded from LC is mainly due to DOPAC synthesized by LC noradrenergrc neurons. A 50 nM concentration of NA was estimated. The DOPAC concentration was varrable up to 23 pM An mterestmg observatron m this study was that the DOPAC concentration increased significantly in response to the stress of rmmobrlizatron or saline inJectron.

7.5. Catecholamines and Ascorbic Acid Although obviously not a monoamine, ascorbic acid seems to be intimately involved m the functroning of the catecholammes. High concentratrons of ascorbic acid exist u-t the brain, but its function is little understood The hrgh concentratron, and facile oxrdatlon, make its study by voltammetrlc methods relatively straightforward. Using mrcroprocessor-based voltammetry to monitor ascorbate and monoamine transmitter metabolites in the striatum of unanesthetlzed rats, O’Neill et al. (198213, 198313) were able to demonstrate that haloperidol did not affect extracellular ascorbate, whereas pargylme, an MAO inhibitor, caused a grad-

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Trne (mu-d Fig 23 In sztu electrochemical recording obtained from the medran eminence region of anesthetized lactatmg rats 5 mm before electncal stlmulatlon of the mammary nerve through end of 15-mm stlmulatlon period Rats A-G received nerve stimulation as described m the text, whereas rats H-K were sham-stimulated. The general pattern observed m the stimulated ammals mcluded a transient decrease m the electrochemical signal during the stlmulatlon period Arrows mdlcate stlmulatron interval A transient decrease (3 3 t 1 0 mm, x +SEM) of 31 -+ 6% was observed m the stimulated animals when comparing the lowest point with the mean value of the five measurements preceding stimulation. From P M Plotsky et al (1982), with permlsslon

Vo/tammet/y

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ual slight increase. Using lmear sweep voltammetry at carbon paste electrodes, they were further able to show that a circadian varratron m ascorbate levels exists, being highest durmg the dark phase, in concert with HVA. Peak concentration was reached at 0400 h while the mmrmum occurred at 1600 h. The effect of unilateral cortical lesions on the circadian changes m ascorbate and HVA has also been examined (O’Neill et al., 1983~). The height of the ascorbate peak was 55% smaller on the lesloned side compared with the intact side, whereas the HVA peak showed no significant change The lesion also reduced the circadian increase in ascorbate from 80% above baseline on the unlesroned srde to 31% on the lesroned side while leavmg the HVA varratlon unaffected, as shown u-r Fig. 26 Stamford et al. (1984) have reported a cychc voltammetrrc method for measuring ascorbic acid at a carbon fiber electrode based on the irreversible nature of the ascorbate oxrdation. The functional sigmflcance of changing ascorbic acid levels is unknown at present.

7.6. Serotoninergic Pathways Rlvot et al. (1982, 1983a,b) have applied voltammetry to the study of the serotonmergic systems of the brain. Using carbon fiber mlcroelectrodes with differential pulse voltammetry (-50 to +450 mV vs Ag/AgCl, 2-mm mtervals), these workers made recordmgs of a peak at 280-300 mV u-r the dorsal horn of the spmal cord of chloral hydrate-anesthetrzed rats (Rrvot et al., 1982) Strmulatron of the nucleus raphe magnus for 10 mm produced an immediate and sustamed Increase m peak amphtude The basal level of this 5-hydroxymdolammergtc srgnal IS strongly depressed after pretreatment of the animal with I’CPA Strmulatton of the nucleus raphe magnus under these condmons was totally meffrcrent. The strmulation used (biphaslc rectangular pulses of 0 5 ms duration and 150 FA peak to peak delrvered at 300 Hz m trams of 100 ms duratron) has been found to induce strong analgesra m freely moving ammals. These results Indicate that voltammetry can be used to study the relatlonshrp of serotonmergic activity to analgesra. The authors have gone on to show that electrrcal strmulatron of the hypothalamus or of the dorsal raphe nucleus induced an increase m the 5-hydroxyindole srgnal m the neocortex (1983a) and that the signal results from predominantly 5-HIAA (Rivot et al , 198313) They have also shown that voltammetry may be used to examine the laminar dlstrrbutlon of serotoninergrc innervation m rat somatosensory cortex (Lamour et al., 1983).

!

Fig 24 Comparison of catecholamme flux in the median emmence and stalk blood of urethaneanesthetized lactatmg rats The mammary nerve was Isolated and placed III the nerve chamber, but not stlmulated (sham stlmulatlon) Voltammetrlc measurements were obtamed from the median emmence at l-mm mtervals [3H]-Dopamme (3H-DA) content of stalk blood was measured tn consecutive 3-mm collections obtamed from severed stalk Both methods revealed similar patterns of catecholamme release it”’ (PA s”‘), measure of amount of current flowing per unit time From I’ M Plotsky and J D Nell1 (1982), with permlsslon

sham

iao

Istim

!

Fig 25 Comparison of catecholamme flux m the median eminence and stalk blood of urethaneanesthetized lactatmg rats before, during, and after electrical stlmulatlon of the isolated mammary nerve A slgmflcant 35 2 7% (mean ? SEM) decline m the voltammetrlc signal was observed during stlmulatlon (P < 0 05) [3H]-Dopamme (3H-DA) content of the stalk blood decreased 62 5 +5.9% (mean + SEM) during the same period (P < 0 005) Excellent correlation existed between the two methods ztli2 (PA + s”~), measure of amount of current flowing per unit time From P M Plotsky and J D Nell1 (1982), with permlsslon

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;

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Echlzen and Freed (1983) have used linear sweep voltammetry with semrdrfferentiation to momtor 5-HIAA in the dorsal raphe nucleus They have measured the serotonm turnover rate with the method and have excellent agreement with other methods (1984) McRae-Degueurce et al. (1984) used DPV with carbon fiber electrodes to monitor 5-HIAA in a serotoninergic denervated striatum before and after transplantation of mesencephalic raphe nuclei mto the lateral ventricle m the rat In all the transplanted anrmals who had surviving grafts, a 5-HIAA signal could be detected The signal was comparable to normal levels and could not be detected 1 mm above or below the transplant.

7.7. Voltammetry and Iontophoresis Voltammetry at carbon fiber microelectrodes has been used to assay iontophoretically applied DA at the tip of multibarreled microelectrodes that can also be used for unit recording (Millar et al., 1981). The authors show that the concentratron of DA produced at the tip can be accurately measured, and the spike activity resulting from the rontophoresis can be recorded with a high signal-to-noise ratio from the same electrode. These investigators have also described a method for etching the tips of the electrode (Armstrong-James et al , 1980a), the quantrfrcation of NA (1980b), catecholamme rontophoresis (1981a), and the detection of enkephalms followmg iontophoresis (1981b) 7.8. Catecholamines and Behavior One of the most exciting applications of in vivo voltammetry 1s the monitormg of neurotransmitter systems durmg behavior The data can be obtained on a fast time scale relative to other methods of chemically momtormg the CNS, such as push-pull perfusion, and multiple sites can be monitored about as easily as one site Most of the voltammetric work m behaving animals has been done m rats, but it has also been shown to be applicable to prrmates (Lindsay et al , 1981). A primary reason for working with unanesthetized freely moving animals IS to study the relatronshrp of neurotransmissron to behavior. However, rt is also important to note that results from tissue slice experiments or even experiments using anesthesia may give results that are not mdicatrve of a functronmg central nervous system. For example, it has recently been reported (Clemens and Phebus, 1983), that m viva voltammetric mom-

249

Voltammetry Circadian

6

changes

in

striatal

ascorbate

Lesioned

2

24

12

24 Time

12 of

24

12

h

day

Fig. 26. Changes in the height of peak 1 (striatal ascorbate concentration, seeFig. 12) over a 36-h period in a rat with a unilateral cortical lesion. Measurements every 12 min. Ordinate is the absolute peak height in nA; abscissais the time of day in hours. From R. D. O’Neill et al. (1983c), with permission. changes in anesthetized vs toring revealed opposite unanesthetized rats with respect to pergolide, a DA agonist. Using semidifferential pulse voltammetry at carbon paste electrodes located in the striatum, it was shown that pergolide decreased the ascorbate/DOPAC peak and the HVA peak in anesthetized animals, but increased the ascorbate/DOPAC peak in unanesthetized animals. In one of the first reported applications of voltammetry to behavior, Curzon et al. (1979) studied the effect of stressful manipulations on the caudate. Linear sweep voltammetry (O-l V, 40 mV s-i) at carbon paste electrodes at 5 min intervals was used. Tail pinch induced an increase in the ascorbate/DOPAC peak occurring at +0.35 V vs Ag/AgCl within 1 min. The extent of increase following tail pinch was attenuated after treatment with the catecholamine synthesis inhibitor alpha-methyl-p-tyrosine. These authors have also presented voltammetric evidence to sug-

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gest an involvement of 5-HT m tall pinch-induced gnawing (1980). Caudate DA has been known to be involved in movement for some time. Most studies on the relationship have used lesion techniques such as the admu-ustratlon of the selective neurotoxm 6-hydroxydopamme to cause a neurochemical deficit that could then be related to a behavloral deficit. It has been difficult to study the dynamic relatlonshlp between DA release and movement m unlesloned, normal animals. Voltammetry may offer a way to overcome this dlfflculty. Yamamoto et al. (1982) have used m vlvo voltammetry to examme lateralized dopammerglc activity in rats tramed to circle. A sucrose-water reward was used to train the rats. Bilateral stearlc acid-modlfled carbon paste electrodes were Implanted m the left and right caudate to monitor asymmetric dopaminergic-related activity in the tramed circling rats. As dlscussed in the electrode modification section, this electrode 1s more responsive to DA than to DOPAC or ascorbate. One day after electrode implantation, rats were placed m a circular drum of identical dlmenslons to the trammg apparatus and a 60 mm baseline established Animals were then transferred to the trainmg drum where reinforced turning was started. Reinforcement continued for 70 min and recording contmued an addltlonal 135 mm. Scans were made from 0.0-1.0 V vs Ag/AgCl at 10 mV/s and the resulting signal processed by semldlfferentiatlon. Scans were made every 5 mm alternating between electrodes. When turning began, the signal increased rapidly in the caudate contralateral to the turning direction, as shown m Fig 27 A 33% greater increase m the signal at +0.2 V occurred contralateral to the directlon of rotation relative to the signal m the ipsilateral caudate. Signal maximum and the time of maximum rate of turning closely comclded To verify that DA metabolism was affected by the circling behavior, trained animals were sacrificed for measurement of caudate DA and DOPAC at selected times during the turning behavior. Baseline tissue concentration ratios of DA and DOPAC were not different from umty whereas ratios at the time of peak turning intensity were 1.33 1 0.09 and 1.24 +- 0 06, respectively. These analyses confirm a lateralized caudate DA metabolism during the turning behavior. Chronoamperometry in rat strlatum has been used to examme the neurochemlcal correlates of behavioral responses assoclated with various types of stlmulatlon (Keller et al., 1983). It was

Voltammetfy

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REINFORCEMENT 1

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IN TRAINED

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27. Mean standardized caudate signal responses contralateral and ipsilateral to the trained turning direction for six animals. Three animals had been randomly chosen for training to the left and three to the right. Turning behavior in the trained direction during the entire recording period is depicted in the lower graph. Elevated signal persists after turning has ceased. Signal units are relative to the peak produced by 75 mg/kg acetaminophen ip. From B. K. Yamamoto et al. (1982) with permission. observed that homeostatic challenges, including abrupt decreases in glucose utilization, blood volume, or arterial blood pressure, were ineffective in altering the signal. Electric shock or placing the animals in a shallow ice-water bath, however, generated large and abrupt increases that decayed rapidly. Smaller and more long-lasting increases were produced by rats eating after 24-h fast, drinking after a period of dehydration, or presented with novel olfactory or visual stimuli. Pretreatment with alpha-methylp-tyrosine or gamma-butyrolactone markedly attenuated the large increases in signal, which suggests that the observed signal is associated with an increase in the activity of the nigrostriatal dopaminergic system. Chronoamperometry has also been used with stearic acidmodified carbon paste electrodes by Broderick et al. (1983) to provide evidence for an enkephalinergic modulation underlying ster-

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eotyped behavior. Ikeda et al (1984) measured DOPAC and 5-HIAA by differential pulse voltammetry at a carbon fiber electrode to examme the effects of stress on release of DA and serotonin m spontaneously hypertensive rats. The relationship of the voltammetric signal to drug-induced stereotypy and locomotor activity was examined recently by Salamone et al. (1982). Behavioral observations were made while recording chronoamperometric signals from the ventral anterior striatum and nucleus accumbens followmg admuustration of 1, 4, and 8 mg/kg amphetamine in rats Data from an mdividual animal are shown in Fig. 28. Onset of the change m voltammetric signal paralleled the onset of activity or stereotypy, but the subsequent declmes in signal and behavior were only weakly correlated The percent increase over baseline for striatal electrodes did not change significantly across the dose range. The response at electrodes in the nucleus

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the nucleus of 8 mg/kg amwere applied to Three hours of Salamone et al

Voltammetry accumbens showed increases similar to those at ventral anterior striatum at 1 and 4 mg/kg, but yielded significantly smaller mcreases at 8 mg/kg At the 4 mg/kg dose, the magnitude of signal increase from striatum was positively correlated with locomotor counts and negatively correlated with indices of stereotypy In other words, animals who demonstrated a large locomotor response at this dose also had a large increase m signal whereas animals who showed primarily focused, nonlocomotor stereotypy had a small increase in signal. As has been discussed previously, the signal increase under these conditions is arising largely from increased extracellular ascorbic acid (Salamone et al., 1984), but unilateral destruction of the nigrostriatal DA system with 6-OHDA attenuates the amphetamme-induced increase in signal on the lesloned side (Gonon et al., 1981a), so that one is followmg a dopaminergic-related signal. This behavioral study then suggests that there IS less dopammergic activity m focused stereotypy than in locomotor activity. Regional differences m dopaminergic activity withm the striatum and m the nucleus accumbens occurring during deprivation-induced feeding have been demonstrated by Salamone et al. (in prep). Usmg carbon epoxy electrodes in the ventral anterior striatum, central striatum, and the nucleus accumbens of rats, these researchers obtained chronoamperometric recordmgs durmg deprivation-induced feeding. These recordings showed clear regional differences m the increases in oxidation currents The onset of the increases generally paralleled the initiation of feeding. Latency of the signal to increase 3% above baselme was significantly correlated with latency of the animal to initiate feedmg When the animals showed intrameal breaks m feeding, the signal usually remained above baseline, as shown m Fig 29 The declme of the signal back to baseline generally lagged behind the termination of feeding. Duration of the signal increase correlated with the total time spent eating. The greatest Increase in signal came from the central striatum, as shown m Fig. 30. In the above experiment, one source of the chronoamperometrrc signal in the region showing the largest increase, the central striatum, has been identified by repetition of the deprivation-induced feeding experiment with the replacement of the voltammetric electrode with a dialysis membrane for dialyzed perfusion of the extracellular fluid (Wages et al., m prep.) The fluid is analyzed for electroactive components by HPLC with electrochemrcal detection (Justice et al., 1983) Baseline electroactive

60

TIME (minutes) Fig. 29. Temporal comparrson of feeding and central strratum from smgle experrment Top Time 2-mm interval between voltammetrlc data points. current as percent of prefood chronoamperometrrc components metabolites

present

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DOPAC and HVA, the serotonm metabolite 5-HIAA, and ascorbic acid Dopamine and serotonm were not present m detectable concentrations under the conditions of the experiment, which means they were present at no more than 5% of the DOPAC concentration. Other work has shown DA to be present at about 1% of the DOPAC concentration (Zetterstrom et al., 1983, Wages and Justrce, 1984). The results of thus experiment mdrcate a signrfrcant Increase m the DA metabolrte DOPAC (12 = 9,

p < 0.05), but not m ascorbic acid, the serotonm metabohte 5-HIAA, or HVA Although there was an increase m HVA, it was not statistically significant. The results of the perfusion experiment described above seem to indicate that voltammetry at plam carbon epoxy or carbon paste electrodes may be used to follow DOPAC as an index of DA released during behavior. These results were significant to us because they meant that the neurochemistry associated with ordi-

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2 4 6 8 IO 12 TIME AFTER FEEDING ONSET (min) Fig. 30. Chronoamperometric oxidation current (?SEM) for first 12 min of feeding expressed as a percent of mean of the 12 min prior to feeding. Data are averaged over 3 d of deprivation-induced feeding. CS, central striatum; VAS, ventral anterior striatum; ACC, nucleus accumbens. nary behaviors could be studied with voltammetry. It was not clear to us prior to this whether or not the amphetamine, haloperidol or electrical stimulation-induced signals were going to turn out to be far larger than any behaviorally related release. With the modified electrodes or signal processing described earlier, it seems possible to monitor DA release itself in behaving animals as a function of behavior. To test the feasibility of using voltammetry to examine the more complex behavioral repertoire available in primates, voltammetric recordings have been obtained from the neostriatum of a behaving rhesus monkey (Lindsay et al., 1981). Chronoamperometry was performed in the caudate and putamen using carbon paste electrodes. It was shown that signal amplitude increased during emotional excitation and feeding. Figure 31 is a record of the signal obtained at 2 min intervals using a 1 s pulse of

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m which a glove used m the animal’s capture from her home cage was visually presented at point A and removed at point B. During the 6 mm the glove was in the animal’s

0 6 V vs Ag/AgCl, view,

the monkey

showed

piloerectlon,

grimacing,

squlrmlng,

and squealing. The animal remained aroused for several minutes after removal of the glove, while the signal remained elevated for an extended period.

Acknowledgments Aspects of the research reported in this chapter have been supported

by

Neurobiology, thors would

the

National

Science

Foundation,

Division

of

and the Emory University Research Fund. The aulike to thank

M

Flllenz,

C. R Freed,

F. Gonon,

I’.

Plotsky, and R M. Wightman for kindly supplying figures from their work.

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References Adams R N (1969) Appllcatlons of modern electroanalytlcal techmques to pharmaceutical chemistry 1. Phavrn. Scl. 58, 1171-1184. Adams R N (1969) Electrochemzsfry at Soled Electrodes, Dekker, New York. Adams R. N. (1976) Probing brain chemistry with electroanalytlcal techniques. Anal Chem 48, 1128A-1138A Adams R N and Marsden C. A. (1982) Electrochemical detectlon methods for monoamme measurements m vitro and m VIVO, m Hundbook of Psychopharmacology, Vol 15, (Iversen L L., Iversen S D., and Snyder S H , eds ) pp l-74, Plenum, New York Adams R. N., Contl J , Marsden C A. and Strope E. (1978) The measurement of dopamme and 5-hydroxytryptamme release m CNS of freely moving unanesthetlzed rats. Br. 1 Phurmac 64, 47OP-471P Albery W J., Flllenz M , and O’Nelll R. D (1983) The compartment model for chronically implanted voltammetrlc electrodes m the rat brain. Neuroscz Left. 38, 175-180 Armstrong-James M and Mlllar J. (1979) Carbon flbre mlcroelectrodes J Neuroscz

Methods 1, 279-287.

Armstrong-James M , Fox K , and Mlllar J. (1980a) A method for etching the tips of carbon flbre mlcroelectrodes 1 Neuroscl. Methods 2, 431432. Armstrong-James M., Mlllar J , and Kruk 2 L. (1980b) Quantlflcatlon of noradrenalme lonophoresls. Nufwe (Lond.) 288, 181-183 Armstrong-James M , Fox K., Kruk Z. L , and Mlllar J (1981a) Quantltatlve lonophoresls of catecholammes using multlbarrel carbon flbre mlcroelectrodes 1 Neuroscl Methods 4, 385406 Armstrong-James M , Fox K , Kruk Z. L , and Mlllar J. (1981b) The electrochemlcal detection of enkephalms m bulk solution and followmg lontophoresls. J Physrol (London) 313, 38 Bard A. J and Faulkner L R (1980) Electrochenztcal Methods Wiley, New York. Baumann I’ A. and Waldmeler I’. C (1984) Negative feedback control of serotonm release m vlvo Comparison of a 5-hydroxymdoleacetlc acid levels measured by voltammetry m conscious rats and by blochemical techniques Neuroscz. 2, 195-204 Blaha C D and Lane R F (1983) Chemically modified electrode for m vlvo momtormg of brain catecholammes Bratn Res Bull 10, 861-864 Blaha C D. and Lane R F (1984) Direct m vlvo electrochemical momtormg of dopamme release m response to neuroleptlc drugs Eur j Phurmacol 98, 113-117 Blakely R. D and Duvarney R C (1983) A microcomputer controlled system for momtormg multiple voltammetrlc electrodes m vlvo Bram Res Bull 10, 315320 Blakely R D., Wages S A , Justice J B., Herndon J G , and Nell1 D B

JustIce,

258

Michael,

and Nell/

(1984) Neuroleptlcs increase stnatal catecholamme metabohtes but not ascorbic acid m dialyzed perfusate Brazn Res. 308, 1-8. Braze11 M. P. and Marsden C A. (1981) Identlflcatlon by differential pulse voltammetry of 5-hydoxymdoleamme oxldatlon peak m the strlatum and frontal cortex of the anaesthetlzed rat Br 1 Pharmacol 74, 219P. Braze11 M. P and Marsden C A (1982a) Differential pulse voltammetry m the anaesthetlzed rat. ldentlflcatlon of ascorbic acid, catechol and mdoleamme oxldatlon peaks m the strlatum and frontal cortex Br 1 Pharmacol

75, 539-547.

Braze11 M. I’ and Marsden ascorbate oxldase-effect

C A (1982b) Intracerebral on m vlvo electrochemical

mlectlon of recordings.

Bratn Res 249, 467472.

Broderlck P. A., Blaha C. D., and Lane R. F (1983) In vlvo electrochemical evidence for an enkephalmerglc modulation underlying stereotyped behavior reverslblllty by naloxone. Bralrz Res 269, 378-381 Broxterman H J and Mos J (1980) Dopamme hypoactwty measured by m vlvo voltammetry Eur ] Pharmncol 68, 389-391 Broxterman H J., Van Valkenburg C F M and Noach E L (1980) Different changes m dopamme metabolism m the strlatum and olfactory tubercle of the rat after HA-966 Br 7, Pharmacol 70, 130~ Buda M., Gonon F , Cespugllo R , Jouvet M , and Pqol J. F. (1981) In vlvo electrochemical detection of catechols m several dopammerglc brain regions of anaesthetlzed rats. Eur 1 Pharmacol 73, 61-68. Buda M., De Slmom G , Gonon F , and Pqol J (1983) Catecholamme metabolism m the rat locus coeruleus as studied by m vlvo dlfferenteal pulse voltammetry I Nature and origin of contributors to the oxldatlon current at +O.l V. Bram Res. 273, 197-206 Cespugllo R (1982) In vlvo measurement by differential pulse voltammetry of 5-hydroxymdole compounds. 1 Hlsfochem. Cyfochem 30, 821-823 Cespugho R , Farad11 H , Ponchon J L , Rlou F , Buda M , Gonon F , I’u~ol J -F , and Jouvet M. (1981a) In vlvo measurement by dlfferenteal pulse voltammetry of extra-cellular 5-hydroxymdoleacetlc acid m the rat brain ] Phystol (Pans) 77, 327-332. Cespugllo R , Farad11 H , Ponchon J L , Buda M , Rlou F , Gonon F., Pu~ol J.-F., and Jouvet M. (1981b) Dlfferentlal pulse voltammetry in brain tissue I. Detection of 5-hydroxyindoles m the rat stnatum. Brain Res. 223, 287-298

Cespugllo R., Farad11 H., Rlou F., Buda M , Gonon F , Pu~ol J -F., and Jouvet M (1981~) Differential pulse voltammetry m brain tissue. II Detection of 5-hydroxymdoleacetlc acid m the rat strlatum Bram Res. 223, 299-311 Cheng H.-Y (1982) Compartment model for chronoamperometrlc measurement m vwo 7 Elecfroanal. Chem 135, 145-151. Cheng H.-Y , Schenk J 0 , Huff R. M , and Adams R N (1979a) In vlvo

259

Vo/tammetfy electrochemrstry. Electruanal.

behavror

of micro

electrodes

m bran-r trssue J

Chem 100, 23-31

Cheng H.-Y., Strope E and Adams R N. (197913) Electrochemrcal studies of the oxldatron pathways of apomorphine. Anal. Chem 51, 2243-2246 Cheng H.-Y., White W , and Adams R. N (1980) Mlcroprocessorcontrolled apparatus for m VIVO electrochemrcal measurement Anal

Chem 52, 2445-2448

Chey W. M (1978) The apphcatron of high performance liquid chromatography m bram slice release studies. MS Thesis, Umversrty of Kansas, Lawrence, KS Clemens J. A. and Phebus L A (1983) Changes m brain chemistry produced by dopammergrc agents m vrvo electrochemical momtormg reveals opposrte changes m anesthetized vs unanesthetrzed rats Brazrz Res. 267, 183-186. Contr J C., Strope E., Adams R N., and Marsden C. A (1978) Voltammetry m bram tissue: chronic recordmg of stimulated dopamme and 5-hydroxytryptamine release. Life Scl 23,2705-2716 Crespr F , Sharp T , Maldment N and Marsden C (1983) Drfferentral pulse voltammetry m vrvo-Evidence that uric acid contrrbutes to the mdole oxrdatron peak Neuroscz. Lett 43, 203-207. Curzon G and Hutson P H. (1981) Momtormg strratal dopammerelated circadian changes by automated m vrvo voltammetry J PhysX?l. (Lonlion) 317, 3op-31p Curzon G , Hutson P H., and Knott P J. (1979) Voltammetry m vrvo effect of stressful mampulatrons and drugs on the caudate nucleus of the rat. Br ] Pharmacol 66, 127P-128P Curzon G., Hutson I’. H., and Knott P J. (1980) Behavioral and voltammetrrc evidence for mvolvement of 5-hydroxytryptamme m tail pmch-Induced gnawing. Br J Pharmacol 70, 132P--133P Dayton M A , Brown J C., Stutts K J , and Wrghtman R. M. (1980a) Faradarc electrochemrstry at mrcrovoltammetrrc electrodes Anal. Chem 52, 946-950.

Dayton M. A., Ewing A G., and Wrghtman R. M. (1980b) The response of mrcrovoltammetrrc electrodes to homogeneous catalytic and slow heterogeneous charge transfer reactions. Anal Chem 52,2392-2396 Dayton M. A , Ewing A. G., and Wrghtman R. M (1981) Evaluatron of amphetamme-induced m vivo electrochemrcal response. Eur \ Pharmacol 75, 141-144. Dayton M. A , Ewing A G., and Wightman R. M. (1983) Drffusron processes measured at mrcrovoltammetrlc electrodes m brain tissue J Electroanal Chem 146, 189-200 Echrzen H and Freed C R. (1983) In VIVO electrochemrcal detection of extraneuronal 5-hydroxymdoleacetrc acid and norepmephrme m the dorsal raphe nucleus of urethane-anesthetized rats Bram Res 277, 55-62

260

Justlce,

Michael,

and Nell1

Echizen H. and Freed C R. (1984) Measurement of serotonm turnover rate in rat dorsal raphe nucleus by m viva electrochemistry I Neurochem 42, 1483-1486 Evans J F and Kuwana T. (1977) Radiofrequency oxygen plasma treatment of pyrolytic graphite electrode surfaces. Anal Chem. 49, 1632-1635

Ewing A. G , Withnell R and Wightman R M (1981a) Instrument design for pulse voltammetry with microvoltammetric electrodes Rev. Scl lnstrurrz 52, 45 Ewing A G., Dayton M A , and Wightman R. M (1981b) Pulse voltammetry with microvoltammetric electrodes. Anal Chem. 53, 1842-1847.

Ewing A G., Wightman R M , and Dayton M. A (1982) In viva voltammetry with electrodes that discrimmate between dopamme and ascorbate Brain Res 249, 361-370 Ewmg A G , Bigelow J -C , and Wightman R M (1983a) Direct m viva momtormg of dopamme released from two striatal compartments m the rat Sueme, 221, 169-171 Ewing A. G , Alloway K. D , Curtis S. D , Dayton M. A., Wightman R. M , and Rebec G V (1983b) Simultaneous electrochemical and unit recordmg measurements Characterization of the effects of d-amphetamine and ascorbic acid on neostriatal neurons. Beam Res 261, 101-108. Ewmg A G and Wightman R M. (1984) Momtormg the stimulated release of dopamme wrth in viva voltammetry II Clearance of released dopamme from extracellular fluid 1, Neurochem 43,570-577. Falat L. and Cheng H -Y (1982) Voltammetric differentiation of ascorbic acid and dopamme at an electrochemically treated graphite/epoxy electrode. Anal Chem 54, 2108-2111 Fox K., Armstrong-James M., and Millar J, (1980) The electrical characteristics of carbon fibre microelectrodes I Neuroscl Methods 3, 3748.

Freed C. R and Echizen H. (1983) Factors affecting m vivo electrochemistry Electrode modification by brain tissue and amplification of catecholamme responses by ascorbic acid Sot Neuroscz Abstr , 9, 999

Galus Z., Schenk J 0 , and Adams R N (1982) Electrochemical behavior of very small electrodes m solution. Double potentral step, cychc voltammetry and chronopotentiometry with current reversal 1 Electroanal Chem 135, l-11 Gerhardt G. A and Adams R. N. (1982) Battery-powered apparatus for chronoamperometric measurements Anal Chem 54, 1888-1889 Gerhardt G A , Oke A F , Nagy G , Moghaddam B , and Adams R. N (1984) Nafron-coated electrodes with high selectivity for CNS electrochemistry, Bram Res 290, 390-395 Gonon F., Cespuglio R , Ponchon J -L , Buda M , Jouvet M , Adams R

Voltammetry

261

N , and Pu~ol J.-F (1978) Mesure electrochimique continue de la hberation de dopamme real&e in VIVO dans le neostriatum du rat. C R Acad Ser. Pans, 286, 120&1206 Gonon F , Buda M , Cespugho R , Jouvet M , and Pu~ol J -F (1980) In viva electrochemical detection of catechols m the neostriatum of anaesthetized rats dopamme or DOPAC? Nnt~rr (Lond.) 286, 902-904

Gonon F., Buda M , Cespuglio R , Jouvet M , and Pu~ol J -F (1981a) Voltammetry m the striatum of chronic freely movmg rats. detection of catechols and ascorbic acid. Bra~rz Res 223, 69-80 Gonon F , Fombarlet C M , Buda M. J , and Pu~ol J -F (1981b) Electrochemical treatment of pyrolytic carbon fiber electrodes Anal Chem 53, 1386-1389

Gonon F , Buda M , de Simoni G , and I’u~ol J -F (1983) Catecholamme metabolism m the rat locus coeruleus as studied by m viva differential pulse voltammetry II Pharmacological and behavioral study Bram Res 273, 207-216

Gonon F , Navarre F , and Buda M J (1984) In viva momtormg of dopamme release m the rat brain with differential normal pulse voltammetry Allal Chcm 56, 57%575 Goto M and Ishu D (1975) Semidrfferential electroanalysis. Electroanal. Chem. and lnterfanal

Elecfrochem

61, 361-365.

Hawley M D , Tatawawadi S V , Piekarski S., and Adams R. N. (1967) Electrochemical studies of the oxidation pathways of catecholammes I Am Chem Sot 89, 447-450. Hefti F and Melamed E (1981) Dopamme release m rat striatum after admuustration of I-DOPA as studied with m viva electrochemistry Bram Res 225, 333-346.

Huff R. M. and Adams R N (1980) Dopamme release n-t N accumbens and striatum by clozapme: simultaneous momtormg by m viva electrochemistry. Neuropharmacology 19, 587-590 Huff R. M., Adams R N., and Rutledge C 0. (1979) Amphetamine dose-dependent change; of in vivo electrochemical signals m rat caudate Bram Res 173, 369-372. Hutson I’ H and Curzon G (1983) Monitoring m viva of transmitter metabolism by electrochemical methods. Bzochem 1, 211, 1-12 Ikeda M., Hirata Y , Fulita K , Shmzato M , Takahashi H , Yagyu S. and Nagatsu T (1984) Effects of stress on release of dopamme and serotonm in the striatum of spontaneously hypertensive rats An m viva voltammetric study. Neurochem Inf 6, 509-512. Justice J. B. Jr, Lindsay W. S , Kizzort B. L , Neil1 D. B , and Salamone J D (1980) Neurochemical momtoring with a microcomputercontrolled electrochemical system Proc. 2nd Ann Conf E~~~~nec~rt~g Med

Bloi Sot

of 1EEE

2, 4650

Justice J B Jr , Wages S. A , Michael A C , Blakely R D , and Neil1 D B (1983) Interpretations of voltammetry in the stnatum based on

262

Justice,

Michael,

and Nerll

chromatography of striatal dialysate ] Lupzd Ckromafogr 6, 187%1896 Keller R W Jr., Stricker, E M , and Zigmond M J (1983) Envuonmental stimuli but not homeostatic challenges produce apparent mcreases m dopammergic activity m the striatum an analysis by m viva voltammetry. Brum Res 279, 159-170. Kennett G A and Joseph M H (1982) Does m VIVO voltammetry m the hippocampus measure 5-HT release’ Brum Res 236, 305-316 Kissinger I’. T , Hart J B and Adams R N (1973) Voltammetry m brain tissue-a new neurophysiological measurement Brum Res 55, 209-213. Kruk Z L., Armstrong-James M and Millar J. (1980) Measurement of the concentration of 5-hydroxytryptamme elected during iontophoresis using multibarrel carbon fibre microelectrodes Lrfe Sa 27, 2093-2098 Kuhr W. G , Ewmg A G and Wightman R M (1984) Monitoring the stimulated release of dopamme with m viva voltammetry. I Characterization of the response observed m the caudate nucleus of the rat ] Newockem 43, 560-569 Lake D M and Marsden C A (1980) Electrochemical detection of 5-hydroxytryptamme and tryptamme Br ] Pkurmacol 69, 334P Lamour Y , Rivet J I’ , Pomtis D , and Ory-Lavollee L (1983) Lammar distribution of serotonergic mnervation m rat somatosensory cortex, as determined by m viva electrochemical detection Branz Res 259, 163-166 Lane R F and Hubbard A T (1976) Differential double pulse voltammetry at chemically modified platinum electrodes for m viva determmatton of catecholamines Anal Ckem 48, 1287-1293 Lane R F , Hubbard A T , Fukunaga K , and Blanchard R J (1976) Brain catecholammes detection m viva by means of differential pulse voltammetry at surface-modified platmum electrodes Bruzn Res 114, 346-352 Lane R F , Hubbard A T , and Blaha C D (1978) Brain dopammergic neurones. m VIVO electrochemical mformation concernmg storage, metabolism, and release processes Btoelectrockem Bmeneqefzcs 5, 504-525. Lane R. F , Hubbard A T , and Blaha C D (1979) Application of semidifferential electroanalysis to studies of neurotransmitters m the central nervous system ] Elecfroanul Ckem 95, 117-122 Lindsay W S , Justice J B Jr , and Salamone J D (1980a) Simulation studies of m viva electrochemistry Comp Gem. 4, 19-26 Lindsay W S., Kizzort B L., Justice J. B , Salamone J.D , and Neil1 D B. (1980b) An automated electrochemical method for m viva momtormg of catecholamme release ] Neuroscz Methods 2, 373-388 Lmdsay W. S , Kizzort B L , Justice J. B Jr , Salamone J D , and Neil1 D. B (1980~) Microcomputer controlled multielectrode system form

263

Voltammetry

viva electrochemistry C/rem , Bmned., Envlron lnstrumen 10, 31 l-330 Lmdsay W S , Herndon J, G Jr , Blakely R D , Justice J 8. Jr , and Neil1 D B (1981) Voltammetnc recording from neostnatum of behavmg rhesus monkey Brain Res 220, 391-396 Marsden C A. (1979a) Functional aspects of 5-hydroxytryptamme neurones Application of electrochemical monitormg m wvo Trends Neuroscz

2, 230-234.

Marsden C A (1979b) Evidence for the release of hippocampal 5-hydroxytryptamme by cY-methyltryptamine Br 1 Pharrnacol 67, 438P439P. Marsden C A , Conti J. C , Strope E , Curzon G , and Adams R N. (1979~) Momtormg 5-hydroxytryptamine release m the brain of the freely moving unanaesthetized rat using m viva voltammetry. Bram Res 171, 85-99

Marsden C A. (1980) Involvement of 5-hydroxytryptamine and dopamme neurones m the behavioral effects of a a-methyltryptamme Neuropharmacology 19, 691-698. Marsden C A , Bennett G. W , Braze11 M , Sharp T , and Stolz J, F (1981) Electrochemical monltormg of 5-hydroxytryptamme release m vitro and related m viva measurements of mdoleammes ] Physzol (Paris) 77, 333337 McCreery R L., Dreilmg R., and Adams R. N. (1974a) Voltammetry in bram tissue quantrtative studies of drug mteractions. Brain Res 73, 23-33. McCreery R L., Dreilmg R , and Adams R N (197413) Voltammetry m brain tissue the fate of mlected 6-hydroxydopamme Bram Res 73, 15-21 McRae-Degueurce A , Serrano A., Sandrllon F , Pnvat A and Scatton B measurement of extracellular (1984) In VIVO voltammetric 5-hydroxymdoleacetic acid m the denervated stnatum after transplantation of mesencephalic raphe neurons. Neurom Lett 48, 97-102. Milby K H., Mefford I. N , Chey W., and Adams R N (1981) In vitro and m viva depolarization-coupled efflux of ascorbic acrd m rat brain preparations Brain Res Bull 7, 237-242 Millar J , Armstrong-James M , and Kruk Z L (1981) Polarographic assay of iontophoretically applied dopamme and low-noise unit recording using a multibarrel carbon fibre microelectrode. BraIn Res. 205, 419424 Morgan M E and Freed C. R. (1981) Acetammophen as an internal standard for cahbratmg m viva electrochemrcal electrodes. 1 Pharmacol

Exp Ther 219, 49-53

Mos J , Broxterman H J., and van Bennekom W P (1981) In viva voltammetric mvestigations mto the action of HA-966 on central dopammergic neurons. Bram Res 207, 465470

264

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Michael,

and Nell1

Nagy G , Rice M. E , and Adams R N (1982) A new type of enzyme electrode. the ascorbic acid elimmator electrode. Lrfe Scz 31, 2611-2616. Nicholson R. S. and Sham I (1964) Theory of stationary electrode polarography. Anal. Chem 36, 706-723. Oldham K. B. (1969) A new approach to the solution of electrochemical problems mvolvmg diffusion. Anal. Chem 41, 1904-1905 Oldham K B (1973) Semuntegral electroanalysis analog implementation Anal Chem 45, 3943. Oldham K. B (1981) An algorithm for semnntegration, semidifferentiation, and other instances of differentegratlon ] Electroanal

Chem 121, 341-342

O’Neill R D., Fillenz M , and Albery W. J (1982a) Circadian changes m homovamllic acid and ascorbate levels m the rat striatum using microprocessor-controlled voltammetry Netrroscz Lett 34, 189-193 O’Neill R D , Grunwald R. A., Fillenz M , and Albery W J (1982b) Lmear sweep voltammetry with carbon paste electrodes m the rat striaturn Neuroscf 7, 19451954. O’Neill R D , Fillenz M , and Albery W J (1983a) The development of linear sweep voltammetry with carbon paste electrodes m VIVO. ] Neuroscl.

Methods 8, 263-273

O’Neill R D , Flllenz M , Albery W. J., and Goddard N J (1983b) The momtormg of ascorbate and monoamme transmitter metabolltes m the striatum of unanaesthetized rats using microprocessor-based voltammetry Neuroscz 9, 87-93 O’Neill R D , Grunewald R A , Flllenz M and Albery W J. (1983~) The effect of unilateral cortical lesions on the circadian changes u-r rat striatal ascorbate and homovanillic acid levels measured m vlvo using voltammetry Neuroscrence Lett 42, lO.!+llO Papouchado L , Petne G , and Adams R N (1972) Anodic oxidation pathways of phenolic compounds Part I Anodic hydroxylation reactions 1 Electroanal. Chem 38, 389-395 Plotsky P M. and Neil1 J D (1982) The decrease in hypothalamic dopamme secretion mduced by suckling comparison of voltammetnc and radioisotopic methods of measurement Endocrrnology 110, 691-696 Plotsky I’ M , DeGreef W J , and Neil1 J D (1982) In situ voltammetric microelectrodes application to the measurement of median emlnence catecholamme release during simulated sucklmg Bvuln Res 250, 251-252 Ponchon J -L , Cespugho R , Gonon F , Jouvet M , and Pu~ol J.- F (1979) Normal pulse polarography with carbon fiber electrodes form vitro and in viva determmation of catecholammes Anal Chem 51, 1483-1486 Rice M E , Galus Z , and Adams R N (1983) Graphite paste electrodes Effects of paste composition and surface states on electron-transfer rates J Elecfroanal Chem. 143, 89-102 Rivet J P , Chlang C Y and Besson J M (1982) Increase of serotonm

Voltammetry

265

metabolism within the dorsal horn of the spinal cord during nucleus raphe magnus stimulation, as revealed by m VIVO electrochemical detection Bram Res 238, 117-126 Rivet J. I’ , Lamour Y , Ory-Lavollee L., and Pomtis D. (1983a) In viva electrochemical detection of 5-hydroxyindoles m rat somatosensory cortex. effect of the stimulation of the serotonergic pathways m normal and pCPA-pretreated animals. Buarn Res. 275, 164-168. Rivet J I’., Ory-Lavollee L , and Chiang C Y (1983b) Differential pulse voltammetry m the dorsal horn of the spinal cord of the anesthetized rat are the voltammograms related to 5-HT and/or to 5-HIAA7 Bran Res 275, 311-319 Salamone J D., Lindsay W S., Neil1 D B , and Justice J B (1982) Behavioral observation and mtracerebral electrochemical recording followmg admmistration of amphetamine m rats Pharmncol Bzochem Behav. 171, 445-450 Salamone J. D , Hamby L S., Neil1 D. B , and Justice J B. (1984) Extracellular ascorbic acid Increases m striatum followmg systemic amphetamine Pharmacol Brochem Behav 20, 609-612. Salamone J D , Lindsay W S , Neil1 D. B , and Justice J, B (1985) Voltammetric recording m neostriatum and nucleus accumbens during deprivation-Induced feeding m rats, m preparation. Saraswat L D. (1981) Chromatographic analysis of dopamme and its metabohtes m neostriatal tissue and extracellular fluid of the rat PhD Dissertation, Emory University, Atlanta, GA. Schenk J. 0 , Miller E., Gaddis R , and Adams R. N (1982) Homeostatic control of ascorbate concentration m CNS extracellular fluid Brain Res 253, 353-356

Schenk J. 0 , Miller E , and Adams R N. (1983) Chronoamperometry m brain slices Quantitative evaluation of m viva electrochemistry Bram Res 277, l-8 Sharp T , Maidment N. T , Braze11 M I’., Zetterstrom T., Ungerstedt U., Bennett G W and Marsden C A. (1984) Changes m monoamme metabolites measured by simultaneous m viva differential pulse voltammetry and mtracerebral dialysis Neuvoscl 12, 1213-1221 Stamford J A., Kruk Z L , and Millar J. (1984) A double-cycle highspeed voltammetric technique allowmg direct measurement of irreversibly oxidised species: characterization and application to the temporal measurement of ascorbate m the rat central nervous system 1. Neuroscl Methods 10, 107-118 Sternson A W , McCreery R , Femberg B., and Adams R. N (1973) Electrochemical studies of adrenergrc neurotransmitters and related compounds J Electroarral Chem. 46, 313-321 Tse D C S , McCreery R L , and Adams R N (1976) Potential oxidative pathways of brain catecholammes. 1, Med. Chem 19, 37. Wages S. A and Justice J B Jr (1984) Detection of dopamme m extracellular fluid of the striatum using dialyzed perfusion 1984 LCEC Symposium Abstr , 24-27.

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Wages S. A , Nell1 D. B., and Justice J B. DOPAC increases in striatum during feeding, m preparation Wightman R M (1981) Microvoltammetrx electrodes Anal Chem 53, 1125A-1134A Wrghtman R. M., Strope E , Plotsky I’. M , and Adams R. N. (1976) Momtormg of transmitter metabolites by voltammetry m cerebrospmal fluid followmg neural pathway stimulation Nature (Lond ) 262, 145. Wightman R M., Strope E , Plotsky P , and Adams R N (1978) In vlvo voltammetry. momtormg of dopamme metabolites m CSF followmg release by electrical strmulation Brm Res 159, 55-68. Yamamoto B K., Lane R F , and Freed C. R (1982) Normal rats tramed to circle show asymmetric caudate dopamme release L@ Scz 30, 2155-2162 Zetterstrom T., Sharp T , Marsden C A., and Ungerstedt U (1983) In vivo measurement of dopamine and its metabolites by mtracerebral dialysis. Changes after d-amphetamine. J Neurochem. 1769-1773

Chapter 6

Immunohistochemistry and Radioimmunoassay of Brain Amines GREGORY

M. BROWNANDLEEJ.GROTA

1. Introduction Immunologic techmques are bemg used extensively m mvestrgatron of brain transmitters and there are prospects for significant advances m the the use of this approach. A variety of techniques has been developed for production of antisera with characterrstics that make them suitable for use m radioimmunoassay or m immunohrstochemistry For radioimmunoassay, rt is desirable that the antiserum have a high degree of specrflcity for the substance itself or for a derrvative that can be formed readily For immunohistochemistry m fixed tissue, it IS necessary that the antiserum recognize the substance frxed to trssue protems. Radioimmunoassays have been developed for several of the amme neurotransmitters and their derrvatives. For the most part, these assays have been applied to these substances u-r the circulation As yet, the application of radroimmunoassays to the examination of brain tissue has been limited Immunohistochemistry has been applied widely to the exammation of mdoleammes m brain tissue, with much less work being done on the catecholammes and on other substances

2. Antigens 2.1. Coupling

Reactions

The brain ammes and their derivatives are not rmmunogemc To make them rmmunogemc, they must be conlugated (coupled) to 267

268

Brown

and Greta

an antigemc protein. In a conlugate, the small molecular weight substance (neurotransmitter or derivative) is called a hapten. The injection of a hapten-protein comugate stimulates antiserum containing a variety of antrbodies, some of which bmd sites on the protein and others of which bmd the hapten or the hapten plus a portion of the adjacent antigemc protein. Small molecular weight amme haptens have only a few antigemc determinants or bmdmg sites for the antibody relative to the number of determinants for the large molecular weight protem. Various chemical procedures have been used to couple ammes and their derivatives to protein (Fig 1) The protein is frequently albumm because it contains many free carboxyl and amino groups (Erlanger, 1973) It is clearly advantageous to have either a carboxyl or an ammo function, but not both, on the hapten because these permit the use of the well known carbodiimlde-couplmg reaction (Kennedy et al., 1976, Williams and Chase, 1967) that conveniently can be carried out m either aqueous or organic solutions at approximately neutral pH. Another approach for amme-contammg haptens is to use glutaraldehyde (Richards and Knowles, 1968, HaIdu and Friedrich, 1975). For phenolic derivatives, the ortho-pava directmg formaldehyde (Manmch) reaction has been used (Burckhalter et al,, 1946, Thompson, 1968, Grota and Brown, 1976, Grota et al., 1983) This reaction forms a methylene bridge between an active (H) on the hapten and a free ammo group on the protein. Ovthopudyudirecting reactions couple at a position ortho to a phenolic hydroxy group. If the ortho position IS occupied, by a substitutmg group, the couplmg will be at the WUYQ sue. If both ortho and yara positions are occupied by nonreacting groups, the hapten ~111not couple to the protein The diazonmm functional group is often used to couple phenohc, tyrosyl, histidyl, ammo, and carboxyl groups of proteins (Wllhams and Chase, 1967) Speclfrcity studies of antisera generated with steroid-protein conlugates have shown that antisera are not able to discriminate structural changes in the hapten that are at, or immediately adlacent to, the site of conjugation (Baummger et al., 1974, Lmder et al., 1972, Parker, 1971) Hence the choice of the conlugahon reaction should be determined by the type of discrimmation that 1s necessary 2.2. Indolealkyfamines

and Their Derivatives

2.2. I. Formaldehyde Condensation Our approach to increasing specificity of an&era that bind mdolealkylamme derivatives has been to focus upon the structure

Fig

R-N”‘+CH30rNHCOCH3

1

Glutaraldehyde

Carbodltmlda

H

Examples

RI

-

-

~

-

conlugatlon

3 M sodwm acetate room temperature neutral pli

Cti.20

3 M sodium acetate room temperature neutral pn

CHZO

0 021 M Glutaraldehyde pH 7 5. room lemperature

d~methylammopropyl) carbodnmlde (ECDI) pH= 6 5. room temperature overntght mcuballon

l-eihyl-3(3

of hapten-protem

R-NH2+H2N-I?,

Reaction

R-NH2+ti0

0

R-&Oti+H2N-R,

Reactnon

reactions

__)

-

-

-

0

kn,

R-NH

0

RI

Ii

H

< R-NH

See text for references

WJ~

HO

Ii

R-l;r-&C”,)3-\C-~-R,

0 R-<-N-R, k

N”CO%

Brown

270

and Grota

of the antigens used to stimulate the antisera The structures of the mdole nucleus and some of the indolealkylamine derivatives that are of interest are shown m Fig. 2. Differences among the haptens occur at R1, RZ, and R3. Our initial studies followed from earlier work by Ranadive and Sehon (1967a, 196713) on melatonm and 5-hydroxyindoleacetrc acid and by Strahilevitz et al. (1971) on 5-methoxy-N-N’-dimethyltryptamme coupled to antigeruc protem with formaldehyde We determined that N-acetylserotonm (NAS) could also be conlugated to bovine serum albumin (BSA) with formaldehyde (methylene bridge, M) Inlection of these hapten-protein conlugates produced antisera that bound ‘251-labeled antigen The antiserum stimulated by serotonm-MBSA bound both 5-hydroxytryptamme (serotonm; 5-HT) and whereas the antiserum stimulated by 5-methoxytryptamme, NAS-M-BSA bound both NAS and melatonin (5-methoxy-N-acetylserotonm). Based on the assumption that antisera will not discrrmmate analogs that have substitutrons at or near the site of coup@ of hapten to protein in the antigen, these data indicate that 5-hydroxymdolealkylamme derivatives were coupled to antigemc protein at or near the 5-position. The formaldehyde reaction couples ortho to phenollc hydroxy groups on phenols, and since 6-hydroxymelatonin did not bmd to the NAS antisera it is

H

H

H

tryptamme

H

OH

H

serotonin

COCH3

OH

H

N-acetylserotonln

COCH3

OCH3

H

melatonin

H

OCH3

H

5-methoxytryptamine

OCH3

OH

6-hydroxymelatonln

COCH3

Fig 2

Structures of some mdolealkylammes

Immunologic Studies of Bra/n Amines

271

likely that the couplmg occurred at the 4 position (Grota and Brown, 1974) Other workers have replicated these crossreactrvrty findings (Kennaway et al., 1977). We subsequently found that Manmch coupling of melatonm to BSA occurs at a much slower rate than with 5-HT or NAS and that the cross-reactivity of the resulting antiserum is markedly dissimilar. The most striking difference between the melatonmM-BSA induced antiserum and the NAS-M-BSA induced antrserum IS that melatomn-M-BSA antiserum binds melatonm (Pang et al , 1977; Lemaitre and Hartmann, 1980), but does not bmd NAS, whereas NAS antiserum binds both NAS and melatonm These data mean that the conjugation of melatonm to antlgenic protein IS at a drfferent site for melatonm than for NAS. Moreover, since the melatonin-M-BSA-stimulated antiserum bound only melatonin, it is likely that other mdolealkylamine haptens coupled to BSA at the same site as melatonm would stimulate antisera with bmdmg specific for that mdolealkylamme. Studies to define the locus of the site of conjugation of melatonm to antlgemc protein using formaldehyde therefore were done (Grota et al., 1983). Model reactions of melatonm, formaldehyde, and the glycme ethyl ester or prperidine were subjected to conditions similar to those used to generate melatoninM-BSA. The structures of the reaction products were analyzed by nuclear magnetic resonance and infrared spectroscopy. This analysis Indicated that couplmg of melatonin to BSA likely occurred at posmon 2. To gain further insight into the exact locus of conjugation of melatonm to protein m the formaldehyde reaction, we examined cross-reactivity of the intermediate reactron products and other llgands that mrght approximate the hapten bridge m the antigen The rationale for this analysis was that the affinity of the antisera for these lrgands would be greatest for the structural conflguratlon that most closely duplicated the antigen. The highest cross-reaction occurred with C-2 substituted melatonin derivatrves. These data confirmed our previous conclusion that the methylene brrdge conjugatmg melatonin to BSA occurred at the 2 posmon of the mdole nucleus.

2.2.2. Couphng

at the 1 Posltlon

Based on our analysis of the above antigens and antisera that bmd melatonin, a generalized strategy for producing antisera that bmd mdrvldual indolealkylammes IS to employ antigens with couplmg at or near the mdole N. We have implemented this strategy using 1-(p-carboxybenzyl)-melatonm coupled to BSA (melatonm-pcb-

272

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BSA) as antigen The denvatrve was synthesized by a phasetransfer catalysis method described by de Srlva and Srueckus (1978) and coupled to BSA with carbodnmrde The conlugate was u-qected mto rabbits and resulting antisera from several recipient animals were found to bind melatonm specrfrcally (Brown and Grota, 1980; Grota et al., 1981) A similar strategy was used by Blair and Seaborn (1979) to produce mdole N-substrtuted hapten-protein corqugates of melatonm, this was confu-med by us. For example, melatonm-1-propromc acid coupled to BSA strmulated antiserum that bound melatonm, but did not bmd other brologrcally synthesized indolealkylamme derrvatrves (Grota et al , 1981). Srmrlarly, 1-(p’-carboxybenzyl)-N-acetylserotonm coupled to BSA-stimulated antrserum m several different recipient animals that bound NAS speclflcally, 1 e , other naturally occurrmg mdolealkylammes and then derrvatrves, did not bmd to the antiserum (Pang et al , 1981). Thus the strategy of couplmg via the 1 positron IS a reliable method for productron of specrfrc antisera against these mdolealkylammes A similar approach has been used by Kawashima and Nagakura (1982) who coupled 1-(4-carboxybutyl)-melatonm to protein

2.2.3. Coupling at the Side Cham Several other approaches have been used to produce antigens capable of mducmg antisera which bmd mdolealkylammes, including N-acetyl-5-methoxytryptophan coupled to protein using carbodumrde (Arendt et al., 1977), succmyl-5-methoxytryptamme coupled to protein (Rollag and Nrswender, 1976), and mdomethacm coupled to protein (Levine and Rrceberg, 1975) Although the latter three approaches mvolve couplmg via the side chain and would be expected to be msensmve to changes m structure on the side chain, they all produced useable melatonm antisera. Delaage and Puzrllout (1981) coupled the N-hemrsuccrnate of serotonm to protein m order to produce antiserum to serotonin

2.2.4. Diazot/zatlon Antiserum to serotonm has been produced using as antigen, 5-HT coupled to protein via a drazo lmkage (Peskar and Spector, 1973, Kellum and Jaffe, 1976) Thus antiserum shows crossreactrvrty srmrlar to that produced wrth 5-HT coupled to protem wrth formaldehyde condensatron, that IS, the antiserum IS msensrtrve to changes m structure at or near the 5 posrtron of the mdole rmg The same approach has been used for melatonm (Wurzburger et al , 1976) wrth Improved specrfIcrty

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2.3. Catecholamines and Their Derivatives 2.3.1. Coupling

at Terminal

Amino

Function

An early strategy was to stimulate antisera with conlugates coupled through the terminal amino group. Peskar et al (1972) conlugated normetanephrme (NMN) to an antigenic carrier using glutaraldehyde. The resultmg antiserum bound NMN, but also bound metanephrme (MN) and 3-methoxytyramme with about equal affmlty and the 3-methoxy-4-hydroxyphenylethanol and phenylacetic acid derivatives with somewhat less affmlty. The same group has also reported antisera to mescaline and DOM (2,5-dimethoxy-4-methylphenyhsopropylamme) (Riceberg et al., 1974) and to DMPE (3,4-dimethoxyphenylethylamine) (Riceberg and Van Vunakis, 1975) By couplmg dopamme (DA) to antigemc carrier through a dimethoxylated intermediate, Spector et al (1973) produced antiserum that bound DA. Another couplmg approach was used by Knoll and Wrsser (1973), who made the hemisuccinate of 3,4-dimethoxyphenethylamme and subsequently of octopamme and NMN (Diener et al., 1981) and coupled the Intermediates to antigemc protein usmg carbodiimide In all of these cases couplmg was via the termmal amino function and cross-reactivity studies of the resulting antisera showed that they were not able to discrimmate changes in the structure of the hapten near the terminal amme 2.3.2. Formaldehyde

Condensation

In an alternative strategy the 4-hydroxyphenethylamme analogs of DA and adrenaline (A), p-tyramme (PTA) and synephrme, respectively, were coupled to protein at position 3 usmg the formaldehyde reaction (Fig. 3) (Grota and Brown, 1976) These hapten-protein conjugates produced antisera that also bound the corresponding 3,4-dihydroxy and 3-methoxy-4-hydroxy substances The resultmg antisera bound the 3-methoxy derivatives with highest affmrty, the 3 unsubstituted analog with less affinity, and the dihydroxy substituted compounds with further reduced affinity. Substances with substrtutions at position 5 or 6 on the phenolic ring, the beta position, or the terminus of the side chain showed very low levels of binding. The same pattern of crossreactivity was observed for synephrine-M-BSA-stimulated antiserum the 3-methoxy derivative had the highest affinity, followed by the unsubstituted and the 3-hydroxy derivatives with reduced binding. Derivatives with the N-methyl substituent absent had less than 0.1% cross-reactivity. Although these antisera did not

Brown and Grota

274 Catecholamines

CH2-CH2-NH2

Dopamrne

CHOH-CH2-NHCH3

Epmephrme

Fig 3 (adrenaline)

Structural relatronshlps with the correspondmg

p-Hydroxyphenethylamtnes

CH2-CH2-NH2

p-Tyramine

CHOH-CH2-NHCH3

Synephrine

of dopamme and epmephrme p-hydroxyphenethylammes

specrfically bind individual catecholammes, they were more specrfrc than antisera strmulated by conjugation at the terminus of the side chain. Slmrlar cross-reactrvrties have been reported by others (Lam et al., 1977, Raum and Swerdloff, 1981a, Drener et al., 1981) The fact that the hapten-protein conjugates coupled at posrtion 3 on the phenohc rmg strmulated antisera that were relatively insensitive to 3-substituted ligands prompted a reevaluation of the strategy for producing antisera that would speclfrcally bmd the catecholammes Since bmdmg sites with the highest degree of drscrimmatron are those most distant from the site of conjugation of the hapten to protein, antisera with the highest specrflclty will be produced from antigens in which the hapten is coupled at a site distant from all metabolrcally active sites on the molecule For catecholammes, the metabolically active sites are posmons 3 and 4 on the phenolic ring, the beta posrtron, and the terminus of the side chain These consrderatrons as well as the fact that positron 6 IS in the pava position to 3, suggest that corqugatron at posmon 6 should stimulate the most specrfrc antisera for catecholammes. Furthermore, no naturally occurring metabolrtes with reactive groups at posrtlon 6 have been isolated. The ortho-para directing formaldehyde reaction (Burckhalter et al., 1946) can be used to couple the 3,4-dihyroxy catecholammes to protein at the 6 posrtion. In studies with the model compound 4-methylcatechol,

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Miwa et al. (1978a) showed that couplmg occurred in the paya position. Dopamine and noradrenaline (NA) contam ammo groups at the terminus of the side chain, and haptens containing both phenolic and amino groups form alkaloids m the presence of formaldehyde (Cohen and Collms, 1970) In order to use the formaldehyde reaction for couplmg at the 6 position it is necessary to reversibly block the termmal ammo function of the side chain. In addition, the relative instability of the catecholammes requires that the blocking group be removed under very mild conditions after conlugation We have used N-phthaloyl protection (Barton, 1973), removing the N-phthaloyl group with hydrazine after conlugation, whereas Mlwa and coworkers (1977) have employed N-maleyl protection and removed the protective group with hydrolysis in dilute acid. In our hands, antiserum to a DA conlugate failed to bmd 3H-DA However, Mlwa et al (1977) produced antisera to DA, NA, A, and L-DOPA and Shn-ahata et al (1980) produced antisera to MN that were relatively specific for the hapten. Detailed cross-reactivity studies for antisera to A (Miwa et al., 197813)and MN (Shirahata et al., 1980) show a high degree of specificity of these sera. Coupling to position 6 on the phenohc ring of catecholammes appears to offer the best strategy for the production of antisera that specifically bind mdlvidual catecholammes. 2.3.3. Coupbng

to Diazotlzed

Protein

Fara) and coworkers (1975) produced a specific antiserum to TA by coupling it to diazotized protein. Subsequently they used a similar approach for 3-0-methyldopamme (Faral et al., 1977) and produced an antiserum that had highest affinity for 3-0-methyldopamme followed by 3,4-dimethoxyphenethylamme and N-methyl-DMPEA. Wisser et al. (1978) used this approach, adding a nitrate to positron 6 of DMPEA and couplmg to protein at this position They succeeded m producing highly specific antisera of low titer

2.4. Other Antigens Antisera have been raised against histamine (Mulder and Steinbusch, 1983), glutamate (Strom-Mathisen et al., 1983) and gamma-ammobutyric acid (Strom-Mathisen et al., 1983, Sequela et al., 1983) coupled to protein using glutaraldehyde Absolute specificity of the resulting antisera remains to be established.

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2.5. Production and Characterization of Antisera No specialized immunization technrques are required. Several different injection schedules and various species have been used successfully to produce antisera. One issue to bear in mind IS that the protein used for conjugatron must be from a species other than that being rmmunrzed 2.5.1. Antiserum

Affinity

To compare antrsera it is essentral to define the binding characterrstrcs of the antisera quantitatively by estimatmg the average assocratron constant for an antigen-antibody reaction. It is desirable that lmmunrzatron lead to the productron of high affmrty antrbodies that will possess high avidity for the substance of Interest Antibody affinity may be estimated by examining the abrlrty of varying concentrations of the antigen to displace the labeled antigen bound to antiserum. The inhibition data may then be plotted as described by Scatchard (1949) to obtain the affmrty constant Alternatrvely, the assocration constant may be expressed by the following. K = h?

Abl

b%l [Abl

at equrhbrrum

and

K=

(n T r)C

where r = number of antigen molecules bound per antibody molecule c = free concentration of antigen n = maximum number of antigen that can be bound per antrbody molecule (valence of antibody). Values for Yand c may be obtained by using equrlrbrmm dralysrs with a constant concentration of antiserum but different amounts of antigen. Substitutron of Y = n/2 m the above equatron produces: K,, = l/C Where K, IS the average associatron constant based on the K value when one half of the antrbody sites are occupied (Werser et al., 1970)

Immunologic 2.5.2. Binding

Studies

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277

Capacity

The concentration of antibody may also be estimated from Scatchard analysis of displacement data (Scatchard, 1949). If one assumes that each antibody molecule binds one antigen molecule, then the antibody concentration is equivalent to its bmdutg capacity.

3. Radioimmunoassay 3.1. Basic Considerations Antisera are capable of binding both native and labeled antigens. Competition of unlabeled antigen (Ag) and radioactive labeled antigen (Ag’) for bmdmg to a fixed amount of antibody IS the theoretical basis for radioimmunoassay (RIA) (Brunswick, 1980) In a typical RIA, a standard displacement curve is employed m which the quantity of Ab and Ag* is kept constant and the quantity of unlabeled antigen is varied As the amount of unlabeled Ag (e.g., NAS) m the sample/standard increases, the binding of Ag* to Ab decreases For an RIA to be valid, a number of criteria must be met (Table 1) Neurotransmitters are small molecules that have numerous structural analogs m the biological matrix Hence specificity is particularly important m the RIA of these substances. It is therefore essential that particular attention be paid to three of the criteria listed: Cross-reactivity, chromatographic identity of immunoactivity with the authentic substance, and cross-validation with another assay method. Preliminary separation to elimmate crossreacting or mterferring substances is one strategy that has been employed to enhance specificity Another strategy that has been used is to convert the substance to a derivative for which a specific RIA is available

3.2. lndolealkylamines

and Related Substances

As mentioned above, to be useful an antiserum must show an ability to discrimmate different mdoleammes Cross-reactivities of compounds m an RIA are calculated by comparmg concentrations of the ligands that displace 50% of specific bmdmg of labeled indoleamine in the test tube Cross-reactivity must be determined both for substances similar in structure to the ligand of interest and also for those likely to be present m significant amounts m the tissues assayed Cross-reactivity data for antisera stimulated

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and Grota

TABLE 1 Crrterla for a Valid Radlormmunoassay 1

Satisfactory cross-reactivity with other substances at concentrations encountered m the sample 2 Parallelism of serial drlutrons of the sample with a standard curve 3. Consrstent recovery of added radloactrve and nonradroactlve standard 4 Acceptable mtra- and mterassay varlatlon 5 Demonstration of chromatographrc identity of lmmunoradroactlvlty with authentic standard 6. Cross-valrdation with another specific assay method 7. Physrologlc valldatlon e g , for melatonm, low or undetectable levels followmg short term pmealectomy by NAS-M-BSA, mela tonm-M-BSA, and NAS-pcb-BSA are shown m Table 2. Two drfferent antisera stimulated by melatoninM-BSA are shown Cross-reactrvrtres of the two antrsera are slmllar, further documentmg that cross-reactivity IS largely determined by the site of couplmg and the nature of the bridge. Two different antisera stimulated by NAS-pcb-BSA also show crossreactlvitles that are quite similar To determine whether the brrdge might have an effect on antiserum formation, we have rmmumzed four rabbits with melatonm-pa-BSA and four other rabbits with melatonm-pcbBSA and compared the titer and affinity of the resultmg antisera over a 6-mo period. Cross-reactivity and affinity were comparable m all antisera. Antisera stimulated by melatonm-pcb-BSA had a slgnifrcantly greater titer, suggesting that a more rrgrd bridge may result m a more potent rmmunogemc material (Brown et al., 1983).

On the basis of these studies and others m our laboratory, rt is evident that rmmunizatron with antigens consisting of mdoles coupled at positron 1 or 2 will reliably produce antisera capable of speclfrcally bmdmg melatonin or NAS Generalization of this approach should permit productron of antisera specific to other mdolealkylammes. 3.2.1. Melatonin

Radlolmmunoassay

The melatonm RIA developed by us has been validated for deer, hamster, human, rat, and sheep serum, hamster and rat pmeals, and human and sheep cerebrospmal fluid The method 1s rmproved over an earlier one (Pang et al , 1977) and IS relatively srm-

“0 cross-reactmty

100 13 10 0 0 0 0 0 0

Mel-M-BSA R139 July 4175 100 0 01 0 95 0 0 0 0 0 0

0 12 100 0 50 0 0 0 0 0 0

NAS-pcb-BSA R228 June 91’80

sera

0 08 100 0 05 0 0 0 0 0 0

NAS-pcb-BSA R238 June l/81

20, 1972 and less than 0 01% for the other

100 100 10 0 0 60 0 0 0 0 0

NAS-M-BSA R69 Nov 20172

Indolealkylammes”

Mel-M-BSA R158 Aug 13176

IS less than 0 1% for R 139 July 4, 1975 and R 69 Nov

Melatonm N-Acetylserotonm 6-Hydroxymelatonm 5-Methoxytryptamme 5-Hydroxytryptamme 5-Methoxytryptophol 5-Hydroxymdoleacetlc acid N-Methyltryptamme 5-Methoxy-N, N’-dlmethyltryptamme

Antigen serum

Cross-Reactlvlty

TABLE 2 of Representatwe

5 5

;

8

0-l

280

Brown and Grota

ple and effective The current method 1s detailed m Brown et al. (1983); essentially 1 mL of serum 1s extracted with 5 mL of methylene chloride and the phases separated by slow speed centrifugation The aqueous layer IS aspirated and the organic phase placed on dry Ice for 20-40 mm to freeze the remammg water and lipids to the walls of the tube The orgaruc phase 1s decanted into clean tubes and dried under N2 or m a Savant concentrator The residue 1s reconstituted m buffer (pH = 6.8, 0 01M phosphate buffer contammg 0 1% gelatin) and 3H-melatonm (2000 cpm) and melatonm antiserum (R-158, Aug. 13, 1976, l/39,000-l/48,000 final dilution) are added to a total volume of 0 65 mL Tubes are incubated at 4°C for 5-7 d After mcubatlon, bound llgand is preclpltated by the addition of 0 65 mL of saturated ammonium sulfate, followed by gentle shakmg, mcubatlon at 4°C for approximately 30 mm, and centnfugatlon. The supernatant 1s decanted and the precipitate redissolved m water and then counted m a llquld scintillation spectrometer The mass of melatonm m samples 1s estimated from the relationship between the amount of labeled llgand bound and the amount of authentic standard added to the assay tubes Six samples of rat sera were assessed by this radlolmmunoassay and by gas chromatography/negative chemical lomzatlon mass spectrometry (GCMS-NCI) There were no differences between the assay methods m the amounts assayed and the correlation between the two methods was Y = 0 983 (Grota et al , 1981) The use of GCMS-NC1 IS currently the best test of validity because of its great specificity Studies using GCMS-NC1 have demonstrated the virtual disappearance of serum melatonm m the rat (Lewy et al , 1980) and of urinary 6-hydroxymelatonm in the rhepmealectomy Slmlsus monkey (Tetsuo et al , 1982) followmg larly, melatonm levels m serum from pmealectomlzed rats are at or below the limits of sensitlvlty of our radloimmunoassay (Harvey et al , unpublished) These data support the conclusion that the pineal gland 1s the major source of cu-culatmg melatonm m the rat and that the disappearance of melatonm from the serum of pinealectomlzed rats should be one criterion for a valid assay. Currently, several other laboratones have workable RIA systems for the quantlflcatlon of melatonm m blologlcal &sues, pmeal (Geffard et al , 1982a, Wurzburger et al , 1976, Rollag and Nlswender, 1976), serum (Kennaway et al., 1977; Arendt and WIlkinson 1979; Wetterberg et al , 1978); and urine (Ozakl and Lynch 1976). These assays employ different techniques, m terms

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of mode of antiserum productron, the radlohgand employed (3H, i3’I or i2iI), type of extraction, and method of separation of gzund from free melatonm. They all have m common, though, the essential requisites of sensitivity, specificity, and precision. A major problem with one of these assays (Rollag and Niswender, 1976) is that levels reported m serum are generally higher than with other assays (Wetterberg and Eriksson, 1981; Lewy et al., 1978) and that nonparallelism has been reported for hamster serum (Tamarkm et al , 1979). Hence this assay system appears to require a prelimmary purification step before RIA when used for serum 3 2.2 N -Acetylserotonm

(IVAS) Radiormmunoassay

Parallelism between standard NAS and rat, hamster, and rabbit serum, rat and hamster serum extract, and rat brain, pmeal and retmal extract has been demonstrated m a specific RIA which has been described m detail by Pang et al (1981). It is summarized here. 50-100 PL of serum is added to appropriate amounts of 0.1% gelatin phosphate buffer, pH = 6 5, to give 500 PL To the solution are added 50 FL of 3H-NAS (200 cpm) and 100 PL of antibody to NAS (R-238, June 1, 1981, 1.4500 final dilution; R-228, June 9, 1980), the mixture is incubated at 4”C, for 40 h. Equal volumes (0.65 mL) of saturated ammonium sulfate are added, the tubes gently shaken, incubated for 1 h at 4”C, and then centrifuged The supernatant is decanted and the precipitate redissolved m water and counted by liquid scmtrllatlon spectrometry. The mass of NAS m samples 1s estimated from the relatronship between the amount of authentic NAS added to standard tubes and the amount of labeled ligand bound. The within- and between-assay variability, based on 10 assays of a rat serum pool, were 6.1 and 13 5%, respectively. Assay sensitivity under optimal conditions is less than 10 pg per tube. For validation, ex tracted samples of rat serum were chromatographed on HPLC and fractrons of the eluate assayed for NAS by RIA. A single peak correspondmg to authentic NAS was obtained This same HPLC system is used to purify the labeled NAS m order to improve performance m the RIA. In rat serum 1.0 ng/mL is a typical leve 1 during the daytime and 3 0 ng/mL is the approximate level during the latter half of the dark period Two other radroimmunoassays for NAS have been described. One uses an antiserum bmdmg both melatonm and NAS and requires a differential extraction procedure prior to the assay (Pang

Brown

282

and Grota

et al., 1977). The other uses chemical conversion of NAS to melatonm and subsequent estimation of melatonm (Geffard et al., 1982a). Neither of these procedures has as yet been fully validated for serum. 3.2.3, 5-HT

Rad/oimmunoassay

Several different radioimmunoassays for 5-HT have been described. The assay of Peskar and Spector (1973) utilized antiserum produced by 5-HT coupled to protem via a diazo lmkage and showed significant cross-reactivity with analogs substituted at the 5 position (such as 5-methoxytryptamme) and tryptamme Kellum and Jaffe (1976) modified this system and then validated it extensively as applied to serum. Cross-reacting analogs of serotonin were present m sufficiently low amounts to make mterference negligible Engbaek and Volby (1982) have further modified this approach and validated their assay for CSF, plasma, and serum. The assay of Delaage and Puizillout (1981) utilized antiserum to 5-HT coupled to protein via a succmyl bridge and chemical conversion of 5-HT m serum or tissue samples to N-succmyl5-HT. In combmation with an lodmated label, this approach resulted in an appreciable gain in specificity as well as mcreased sensitivity An assay described by Geffard et al (1982a) mvolves chemical conversion of 5-HT m the tissue sample to melatonm followed by melatonin radioimmunoassay using a system similar to that of Rollag and Niswender (1976) but utilizmg a more soluble lodmated label. Although the assays of Delaage and Puizillout (1981) and Geffard et al (1982a) have considerably enhanced sensitivity and selectivity, full validation data have yet to be published 3.2.4. Radioimmunoassay

of Dimethylindolealkylamine

An assay sensitive to 200-700 fmol of N,N-dlmethylmdolealkylamines has been described m urine, plasma, and whole blood and is based on high-performance liquid chromatography followed by radioimmunoassay This assay uses an antiserum that does not discrimmate between N,N-dimethyltryptamme (DMT), 5-hydroxy-DMT, and 5-methoxy-DMT (Riceberg and Van Vunakis, 1978) 3.2.5. 5-Hydroxymdoleacetlc

Acrd Radioimmunoassay

Radioimmunoassy of 5-hydroxymdoleacetic acid (5-HIAA) m blood, cerebrospmal fluid and brain tissue has been described using antiserum to 5-HIAA coupled to protein with carbodnmide (Pulzillout and Delaage, 1981, Delaage and Puizillout, 1982a).

Immunologic

Studies of Brain Amlnes

283

Iodmated 5-HIAA-glycyl-tyrosine was used as a label and biological samples were converted chemically to make the antigen more similar to the lmmunogen. The sensitivity of this system is very good (80 pg), but full validation has yet to be published. 3.2.6. 5-Methoxytryptophol

Radiolmmunoassay

Kennaway et al (1983) have developed and validated a RIA for 5-methoxytryptophol and utilized it for assay of pineal and serum levels The antigen used was 5-methoxytryptophol coupled to protein via formaldehyde condensation. Significant crossreactivity with melatonm was found so that a preliminary isolation step IS required prior to assay. 3.3. Catecholamines 3.3.1. Dopamine

and Related Substances

Radiolmmunoassay

An enzyme radioimmunoassay for dopamine (DA) in human plasma and urine has been developed and validated by Faral et al. (1978, 1981). The assay utilizes enzymatic conversion of DA to 3-0-methyldopamme (MD) followed by RIA for MD (see below), 3.3.2. Noradrenalrne

Radioimmunoassay

Raum et al. (1981b) used antiserum specific for metanephrine (MN) (Grota and Brown, 1976) with radioiodmated synephrine for assay of noradrenalme (NA) and adrenaline (A) m both tissues and serum The strategy was to enzymatrcally convert NA and A to MN prior to assay. Adrenalme and NA were separated from other interfering substances by adsorption on alumina. Two aliquots were then assayed. one was incubated with catechol-omethyl transferase (COMT) and appropriate cofactors to convert adrenaline to MN and the second was incubated with phenylethanolamme-iV-methyltransferase and then with COMT to convert both NA and A to MN. The amount of NA in the sample was calculated by subtraction. The limits of sensitrvity of the assay are approximately 10 pg for A and 30 pg for NA. Within-assay coefficients of variation ranged from 6 7 to 15 3% and between-assay variation from 11 8 to 36.4%. The assay has been used to determine A and NA levels m human and dog plasma and rat hypothalamic tissue. 3.3.3. Adrenaline

Radioimmunoassay

Our laboratory has investigated use of the synephrine-M-BSAstimulated antibody (Grota and Brown, 1976) and 3H-MN m an assay of A taking advantage of the cross-reactivity of A to assay adrenal tissue homogenates that contam very little, if any, MN

284

Brown

and Grota

There was a lmear relationship between the volume of homogenate (1 mg/mL m 0.1 HCl) added and the amount of A. There was no evidence of specific interference and parallelism was clearly evrdent. It IS well known that insulin treatment results m a release of A from the adrenal gland (Hokfelt, 1951; Outshoorn, 1952). We have found that, consistent with earlier published results, adrenal A levels in the rat are reduced 3, 6, and 24 h after msulm treatment (0.25 U msulm/lOO g body wt) (5.2 -+ 0.8; 4.3 -+ 2 0, 3.8 + 1.4 pg/gland + SE, N = 3) relative to control levels (21.0 + 6 4 pgigland, N = 4) These data indicate that the synephrine antibody can be used to assay A levels m adrenal tissue homogenates. It must be kept m mind that this is possible because there is lrttle or no MN and synephrine present m the gland to interfere in the assay Miwa et al (1978b) have developed an A radioimmunoassay using a specific antiserum that is sensitive to 0 1 pmol of A. Validation for serum and other tissues was not published. 3.3.4, 3-0-Methyldopqmine

(3-Methoxytyramme)

Radroimmunoassay

A RIA sensitive to O-5 ng of 3-0-methyldopamme (MD) has been developed and validated for use m urme and plasma (Faraj et al., 1977, 1978, 1981). The assay employs antiserum generated using MD coupled to protein vra a p-aminohippuric acid bridge. Only 3,4-dimethoxyphenethylamine and Its N-methyl derivative exhrblted significant affinity for the antiserum In combination with enzymatic conversion of L-dopa and DA to MD, the RIA for MD has been used to measure plasma DA (Faral et al., 1978) and urinary levels of all three substances (Faral et al., 1981). 3.3.5. Dimethoxyphenethylamine

Radioimmunoassay

Assays for the determination of urinary levels of P-3,4-dimethoxyphenethylamme (DMPEA) have been described using antisera stimulated by N-succmyl-DMPEA coupled to poly-L-lysine (Riceberg and van Vunakrs, 1975) or to bovine serum albumin (Knoll and Wisser, 1973) by means of carbodiimide As expected, the resulting antisera cross-reacted with substances with various structural changes at the termmus of the side cham. Riceberg and van Vunakls (1975) used an [1251]-DMPEA derivative, and Knoll and Wisser (1976) used [3H]-DMPEA as labeled hgand. Preliminary chromatography was essential to provide specrfrcity m these assays. Subsequently, Wisser et al (1978) produced a considerably more specific antiserum using as an antigen DMPEA coupled on the benzene ring at a site intermediate between the methoxy

Immunologic

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of Braln Amlnes

groups and the side chain. As yet, this antiserum has not been used m radioimmunoassay. 3.3.6. Metanephnne

Radioimmunoassay

A radioimmunoassay for free and conlugated urmary MN was developed by Lam et al. (1977) using antisera stimulated by synephrine-M-BSA (Grota and Brown, 1976). Our laboratory mdependently investigated radioimmunoassay of urmary MN using antisera stimulated by synephrine-M-BSA. Both assays are remarkably similar, used 3H-MN as a label, and gave essentially similar results: 16 9 -+ 11.4 pgid, N = 15 (Lam et al., 1977) and 9.1 + 1.5 k/d, N = 4 (Grota and Brown, unpublished) total MN in healthy adults The 5-10% cross-reactivity of A with synephrine antibody is msigmficant m the assay for MN because the A IS oxidized under the conditions used for the assay Resrdual nonspecific interference m the assay of MN was controlled by Lam et al (1977) by use of standards m catecholamme-free plasma. Raum and Swerdloff (1981a) developed an RIA using the same type of antiserum with extracted urine and radioiodmated synephrme as a label. This assay is considerably more sensitive than the previous RIA and gives results in volunteers that are comparable with those of the calorimetric assay (94.2 vs 87 6 kg/d). These results are higher than those of Lam et al. (1977), but are fully corrected for recovery and are not sublect to nonspecific interference. A more specific radioimmunoassay for MN was developed by Shirahata et al. (1980) using antisera specific to MN that showed mmimal cross-reactivity with synephrine or A. 3 3.7. 3-Methoxy-#-Hydroxyphenylethyleneglycol Radioimmunoassay

(MOPEG)

A radioimmunoassay for MOPEG has been developed and vahdated (Keeton et al., 1981) utihzmg antiserum produced by MOPEG coupled to protein via a 5-carboxypentoxy bridge at the 4 position. The radioimmunoassay has high specificity, a sensitivity of 0.5 ng, and has been applied to brain tissue. Validation for other tissues or fluids remams to be published 3.3.8. Mescaline

and DOM

Radio/mmunoassay

One of the earliest radioimmunoassays for catecholammes was that of Riceberg et al (1974) for 3,4,5-trimethoxyphenethylamme (mescalme) and 2,5-dimethoxy-4-methylphenyhsopropylamme (DOM). Since the antigens were coupled to protein through the ammo terminal of the side chain, binding of the resultmg antisera

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was relatively msensitive to than es at the terminus of the side chain. The labeled ligand was [ 925I]-N-(3,4,5-trimethoxyphenethyl)-4-hydroxyphenacetamide. Intravenously infused mescaline was rapidly metabolized to the acid moiety. Thus serum and urine concentrations of immunoreactive mescaline m rabbits were due to the bmdmg of 3,4,5-trimethoxyalmost entirely phenylacetic acid derivative. 3.3.9. Tyramine RadIoimmunoassay A radroimmunoassay for the phenolic aromatic amme tyramme (TA) has been developed and validated by Faral et al. (1975) Coupling of TA to protein was via a p-aminohippuric acid bridge and tritiated TA was used as a label. The assay showed good specificity, and sensitivity was 200 pg This assay was used to measure TA m plasma, urine, and tissues in rabbits

4. Immunohistochemistry 4.1. Basic Considerations Immunohistochemical procedures have been widely used to localize neurotransmitters m the CNS. Two approaches have been used. One approach has utilized antisera to the enzymes that synthesize or metabolize the transmitter, the other has utilized antisera to the neurotransmitter itself. Each of these approaches has problems of validation that are somewhat different. Locahzation of enzymes depends on the specificity of the antisera to the enzyme, but testmg specificity is difficult since the antigen is isolated from biological tissue and no structurally related analogues are available for testmg. Specificity is essentially based on the definable purity of the antigen Validation of immunohistochemical procedures for the transmitter itself depends on crossreaction of the antisera, keeping the hgand in the tissue and on a variety of tests that support conceptual validation without any single one of them providing absolute validation Some of these tests for conceptual validation include replacing the primary antisera with preimmune sera to result in decreased stammg, reducing the staining by increasing amounts of neurotransmitter or of coupled neurotransmitter added to the antisera (saturation), and finally, performing pharmacologic studies with predictable effects to provide “physiological” validation, In the immunohistochemistry of small neurotransmitter molecules, the fixation methods employed are extremely important

lmmunologlc

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287

For immunohistochemistry of frozen unfixed tissue, the antigen in the tissue is the native neurotransmitter. In contrast, fixation procedures may chemically couple the substance to tissue protein at a specific position on the neurotransmitter molecule. Furthermore, the fixation procedure may chemically alter the neurotransmitter molecule itself. Hence the nature of the antigen can be dramatically altered by fixation. Many of the immunohistologic techniques take advantage of this process by employing an antiserum that recognizes the coupled neurotransmitter.

4.2. Indolealkylamine 4.2.1. Melatonin

tmmunohistochemistry

lmmunohistochem&y

In our early studies, fresh tissue was frozen on dry ice and cryostat sections (10 km) at -20°C were obtained (Bubemk et al., 1976a,b; Bubemk et al., 1977, Vivien-Roels et al., 1981) The tissue section was dried and then rehydrated m excess aqueous buffer (pH = 7.2) f or about 10 mm. During this rehydratron period, haptens, such as catecholamines or mdoleamines, could be washed from the section or moved from their exact location within the tissue. Therefore, frozen cryostat sections give only approximate hapten localization. The general method (Coons et al., 1955; Nakane and Pierce, 1966) incubates the tissue section with primary antisera, washes the sections to remove antibody, incubates with labeled second antibody, and then measures the label. Second antibody conlugated to fluorescein is often used because it can be easily visualized with UV light Second antibody labeled with peroxidase that is visualized with diammobenzidme has also been used. Qualitative assessment of the ligand can be done based on the assumption that increased presence of the ligand will result in increased binding of the primary antisera. Measurement of the amount of fluorescence induced with the fluorescein-labeled double antibody method has been used to semiquantitately determine the relative amounts of melatonin in tissues. Immunohistochemically active melatonin has been observed m the digestive system of the rat (Bubenik et al., 1977) and this tissue was chosen for quantitative analysis (Holloway et al., 1980). Increasing dilution of the primary antiserum resulted m a quantitative decrease u-t measurable fluorescence. In addition, affinity chromatography of melatonm antiserum on melatonm-sepharose columns reduced both the ability of the antiserum

288

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(eluate) to bind [3H]-melatonm and the amount of fluorescence intensity. This intensity of fluorescence reflected the amount of primary antibody bound to the tissue section. Decreasing melatonin synthesis with p-chlorophenylalanine treatment resulted m reduced immunofluorescence m the colon. For this tissue, mtra- and mterassay reliabilities are 10% (CV). This approach is only a semiquantitative method because the relationship between fluorescence and the absolute mass of melatonm IS unknown. This approach has been used to study 24-h rhythms of melatonm immunohistofluorescence m retma and hypothalamus from albino rats, Norway rats, and hamsters (Grota et al , 1982 and unpublished data) The semiquantitative immunohistochemical method was used to study the 24-h rhythm of pmeal melatonm, characterized by radioimmunoassay as having a crest late m the dark period (Brown et al., 1981) Rats were killed at various times during a 24-h period and pmeal tissue evaluated by radioimmunoassay and by immunohistofluorescence. The radioimmunoassay analysis confirmed a pineal melatonin 24-h rhythm with a peak late in the dark period. However, the immunohistofluorescence data mdicated a crest at the end of the light period To insure that this pattern was not due to some unique property of this antiserum, the experiment was replicated usmg four drfferent antisera from sheep or rabbits given melatonm protein conlugates produced by different conlugation reactions These data confirmed that the rmmunohistochemically determined melatonm rhythm in pineal was different from that determined by radioimmunoassay Immunohistochemistry can identify ligands bound to receptors (Sternberger and Petrali, 1975), so we hypothesize that in pineal tissue, immunohistochemically reactive melatonm using any of our antisera is assessing a melatonin receptor with a 24 h rhythm of occupancy reflected by the fluorescence pattern. These data point out one of the problems in using a physiological conditron to validate a immunohistochemical procedure Additional studies to determine what the immunohistochemical method is measuring may be necessary Saturation studies of pmeal immunohistofluorescence revealed that the pmeal receptor for melatonm also bound NAS but did not bmd serotonm or 5-methoxytryptophol One study has described melatonm immunoreactivity m fixed tissue. Photoreceptor cells of the teleost were examined with both light and electron microscopy (Falcon et al., 1981)

Immunologic Stud/es of Brain Amlnes

289

4.2.2. 5-Hydroxytryptamme (5-HT Serotonin) lmmunoh~stochemlstry Immunohistochemical localization of 5-HT in the nervous system has utilized primary antisera stimulated by 5-HT coupled to bovme serum albumin with formaldehyde (Ranadive and Sehon, 1967a, 1967b; Grota and Brown, 1974; Steinbusch et al., 1978) Essentially a double antibody immunofluorescent microassay aim, 1956) or a peroxidase-antiperoxidase system (Stern(N berger, 1974; PAP) has been used on tissues that have been fixed with formaldehyde. Immunoreactive serotonm was found in olfactory bulb, brain stem raphe, and spinal cord (Stembusch et al , 1978; Hokfelt et al , 1978), and later studies confirmed and extended these early data to include thalamus (Stembusch, 1981), hypothalamus (Stembusch and Nieuwenhuys, 1981), cerebellum (Takeuchi et al., 1983) and cortex (Lidov et al., 1980; Kohler et al., 1982). These studies using immunohistochemistry generally fmd wider localization of 5-HT throughout the brain than the histofluorescent method used earlier (Fuxe et al., 1968). In addition to the CNS, 5-HT has also been localized m the adrenal medulla by means of immunohistochemistry (Holzwarth and Brownfield, 1983) These authors report that the 5-HT was localized to the cells that also contam A. For the most part, various laboratories have observed widespread localization of 5-HT m otherwise untreated animals. However, studies m which fme details of nerve processes are sought have used animals pretreated with pargylme, malamide, colchitine or tryptophan to enhance 5-HT levels before fixation. Alternatively, treatment with 5,7-dihydroxytryptamine or p-chlorophenylalanine has been used to reduce 5-HT levels m brain. Manipulation of DA and NA levels with 6-hydroxydopamine has been used m validation studies. In general, studies in which one expects pharmacologic treatment to increase 5-HT have shown enhanced staining and pharmacologic reduction of 5-HT results m reduced stammg. Pretreatment with 6-hydroxydopamme has no effect on staining. A caution should be noted with respect to the widespread distribution of 5-HT. Both NAS and melatonm have been identified in brain, so 5-HT may be present m structures other than those m which 5-HT functions as a neurotransmitter. For 5-HT immunohistochemistry, animals are perfused with 4% paraformaldehyde and 5% glutaraldehyde and sections cut on

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a cryostat or vrbratome. The tissue sections are washed and reacted with excess primary antiserum that has been purrfred by carrier protein precipitation and affmrty column chromatography and contains 0 l-0.2% Triton X-100. Followmg incubation, the slides are washed to remove the antiserum and then processed further for mduect rmmunofluorescence or for the PAP technique The success of an rmmunohrstochemrcal approach depends m large part on the relatronshrp between the chemical reaction of the hapten with antlgenic protein and the method that is used to frx the tissue to be studred. In the mmal studies wrth 5-HT, this amme was coupled to bovine serum albumin with formaldehyde and the CNS was perfused and fixed with formaldehyde m the form of paraformaldehyde. The actual hapten m the antigen is probably a P-carbolme formed by the reactron of 5-HT with formaldehyde (Mrlstem et al., 1983, Brusco et al., 1983; Saavedra et al., 1983). Other data show that the conlugate (5-HT-M-BSA) IS bound to antisera with a much greater affinity than 5-HT conmgated to albumin with glutaraldehyde, a procedure that does not result m P-carboline formatron 5-HT not conlugated to protein or peptrde 1s bound to antiserum wrth much lower affuuty than the conmgate 5-HT, 5-methoxytryptamme and tryptamme not conjugated to protein have a high degree of binding to the antiserum whrch increases dramatrcally followmg paraformaldehyde treatment (Milstein et al., 1983). However, when linked to protein by formaldehyde, only the 5-HT derrvatrve 1s active The -CHz-CH2-NH2 group IS probably modified by formaldehyde to produce a cyclic derivative to form the antibody recognitron site. A different formaldehyde reactive site is required for linking to trssue. Thus 1s provided by the presence in 5-HT of 5-OH absent m the other cross-reacting substances. 4.2.3. N-Acetykerotonin

lmmunohlstology

The pattern of distribution of rmmunoreactive NAS m brain has been studied under a variety of experimental condrtrons (Pulido et al., 198313) A combmation of tests has been used to evaluate specificity m each region where positive immunoreactrve NAS was observed. These tests have included cross reactivity of the antiserum m radrormmunoassay, use of nonimmune serum, saturation tests using varrous concentratrons of NAS, mhlbrtron tests using potential cross-reacting substances, pharmacologrc mampulatrons, topographic distrrbutron comparrsons with serotonm and other ammergic systems. Other aspects of the distribution of

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291

NAS as measured by immunohistochemistry are found m various publications (Psarakis et al , 1982, Pulido et al., 1983a) Essentially, cerebellum, brain stem, hypothalamus, and hippocampus contam immunoreactive NAS. For the most part, these studies used cryostat sections obtamed from frozen tissue at -10°C with a double antibody system using fluorescem-labeled second antibody. For quantitative studies, the slides were examined m an incident-light fluorescent mlcroscope with a sensitive photometer. To standardize the procedure, each set of slides included samples from experimental and control animals and slides from each tissue were exposed to normal rabbit serum, NAS antiserum, or NAS antiserum saturated with ligand. Readings were taken 5 s after exposure to UV light The value obtained with the NAS antiserum minus the normal rabbit serum background is assigned to the brain region This procedure has been used to evaluate the effects of tryptophan hydroxylase inhibitors and adrenergic drugs on immunoreactive NAS m rat cerebellum It has proven to be a reliable and useful technique for the cerebellum It is generally advantageous to use fixed tissue for immunohistology because it provides excellent tissue preservation. But the fixatives may act on haptens and proteins in cells to form insoluble matrices Should the fixatives couple the hapten of interest at a different site on the hapten than that used in the antigen stimulatmg the primary antiserum, then the antiserum may not bind to the hapten and the immunohistochemical method will provide negative data Another potential problem with fixation methods is that the fixative and haptens do not react and the hapten is diluted and washed out by excess volumes of fixative. For the most part, useful fixation techniques have been established by trial and error. An ideal fixation should preserve the antigen during fixation and prevent its extraction or displacement during subsequent processing, preserve the antigen-antibody reaction, and retam good morphology after processmg and embedding in the supporting medium (Berod et al , 1981, McLean and Nakane, 1974, Samte-Marie, 1962) We have recently succeeded m the use of fixed tissue for NAS immunohistology Animals are anesthetized with Nembutal and the brain fixed by transcardial perfusion In our hands, best results are obtained by washing the blood for 10 mm with ice-cold Tyrode’s solution (Ca2 ’ free and 10% dextran) and perfusing for 20 mm with ice-cold 2% paraformaldehyde in phosphate buffer-saline, pH = 7.4, and 2% dextran Post-fixation is for 5 h

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usmg the same fixative followed by washing at 4°C for 18 h with 10% sucrose in PBS. The tissue IS paraffin embedded and prepared for stammg wrth the PAP method (Taylor, 1978). The drstributlon of lmmunoreactive patterns of NAS m cerebellum and hrppocampus as well as m retma has been found to be the same m paraffin-fixed sections stained with PAP as that obtained m unfixed frozen sections stained with the immunofluorescence method The major advantages of the PAP method m fixed tissues are low background, higher speclfrclty because antibody dllutlon of as much as 12000 can be used, and much better morphology, permitting more precrse locahzatlon of the hapten m the tissues.

4.3. Catecholamine Immunohistochemistry 4.3 1. Dopamrne lmmunohlstochemistry Porietis et al. (1977) used antiserum stimulated by TA-M-BSA m rmmunohlstochemrcal studies of DA m unfixed CNS. Stammg was eliminated by saturatron with either p-TA or DA. To further document that the method was staining for dopamine, various pharmacologic mampulatlons with measurable effects on specific cross-reacting substances were studied The major cross-reacting substances and the changes in the endogenous levels of these substances effected by pharmacologic treatments IS summarized m Table 3. Combined treatment with a-methyl-p-tyrosme and reserpine, which depletes the CNS of all cross-reacting substances except octopamme and J3-phenethylamme (Boulton et al., 1977, Harmar and Horn, 1976) ehmmates staining with the p-TA antiserum, suggesting that positive staining IS not caused by octopamine or beta-phenylethylamine. Treating animals with pyrogallol, a catecholmine-O-methyl transferase inhibitor (Axelrod and Laroche, 1959), results in decreased 3-MTA and NMN levels without affecting the levels of p-TA, DA, or NA Staining was unaltered m the strratum, dorsolateral septum, and median emlnence. Haloperidol treatment, effective m reducing p-TA levels but not dopamme levels, (Juorio, 1979), did not change stammg These studies as well as observations of mtense stammg m the strratum, dorsolateral septum, cmgulate gyrus, and median emlnence that are known to be 1oc1 containing dopamine (Ungerstedt, 1971; Versteeg et al , 1976) support the inference that the antiserum stimulated by p-TA-M-BSA can be used in a hlstochemical method for the detection of DA m the CNS Geffard et al. (1982b) used antiserum against DA coupled to

Immunolog/c Effects

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293

TABLE 3 Agents on the Concentration of Llgands with /I-Tyramlne-M-BSA Antisera Treatments

% Crossreactivity”

Analog 3-Methoxytyramme p-Tyramme Normetanephrme Dopamme Octopamme @Phenethylamme Noradrenalme Observed stammg

Reserpme plus (xMPT’

1 nc

213 100 40 22 9 38 16

Pyrogallol

1 nc”

1 nc -

Haloperldol i nc

17C

J none

nc

1lC

normal

normal

“nc = no change I 1 , decrensed

cor~crrztrntror~

‘-, ellmmated “Determmed by radlolmmunoassay by comparison maxlmal displacement of labeled hgand *cu-MI’T = a-methyl-)I-tyrosme

of quantltlcs

causmg

half

by formaldehyde m immunohistologic studies on goldfish brain fixed m 4% paraformaldehyde. Specificity as assessed using DA analogs coupled to protem suggested that this approach was valid Antiserum against DA or TA coupled to protein by glutaraldehyde was used by Geffard et al (1983) in immunohlstochemrstry Conlugated NA and DA were bound by the DA antiserum, whereas conjugated TA and DA were bound by the TA antiserum.

protein

4.32. IYoradrenahne /mmunohstochem/stry Immunohistochemlstry of NA using NA coupled to protein by formaldehyde as an antigen was reported by Verhofstad et al (1980) In pons and medulla oblongata, cell bodies containing NA were readily seen Fme NA-containing fibers were seen m several parts of the brain and the peripheral nervous system One cell type m the adrenal medulla showed intense stammg whereas a second type showed weak stainmg. Coffe et al (1983) have recently demonstrated the colocallzation of NA with vasopressm or neurophysin in cells of the locus ceruleus by immunohistochemistry.

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4.3.3. Adrenaline lmmunohlstochemistry Harvey (1980) used an antiserum against synephrine-M-BSA to study the immunohistochemical localization of A in the unfixed rat CNS This antiserum binds A, synephrme, and 3-methoxysynephrine (MN), but since brain levels of synephrine and MN are low or nonexistent, any staining should reflect the presence of A. Premcubation of antiserum with A or A bitartarate ehmmated staining but mcubation with the vehicle or bitartarate also ehminated staining. Antisera to various indolealkylammes produced the same pattern of staining. A series of tests were, therefore, done to establish the reason for the nonspecificity. Dialysis of the antiserum did not alter stammg. The redissolved precipitate produced by half-saturation of antiserum with ammonium sulfate was able to stain, mdicatmg the stammg pattern was probably caused by IgG An affinity column contammg synephrme was then prepared and used to treat antisera. The eluate was unable to bmd 3H-MN, but was capable of producing stammg, mdicatmg that the staining was not related to synephrme antibody. One remaming possibility was that the antibody to synephrme was saturated with endogenous hgand and that the stammg reflected the binding of the A-antibody complex to receptors (Sternberger and Petrah, 1975; Kurzon and Sternberger, 1978). Various procedures for the removal (strippmg) of ligand from antisera were used, resulting in a slight increase m stammg and indicating that a small amount of endogenous hgand was present m the antisera. Finally, normal rabbit serum was precipitated with ammonium sulfate and aliquots of the solubrlized pellet added to the normal rabbit serum used m the stammg procedure The stammg mimicked that seen with antisynephrme antiserum, indicatmg that excess IgG m the antiserum was responsible for the stammg pattern observed. Smce all buffers m these studies contained excess BSA, the stammg was not caused by cross-reaction with carrier antrbodres. These data indicated that further immunochemistry using this antiserum under such conditions was mappropriate because of nonspecific interference Verhofstad et al (1980), using antiserum produced by A coupled to protein by formaldehyde condensation as an antigen, were able to demonstrate intense stammg m some adrenal medullary cells. A second type of cell that failed to stain with antiserum to A stained with NA antiserum. This group was unable to detect any A m the CNS by immunohistochemistry

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4.4. Immunohistochemistly Neurotransmitters 4.4.1. Gamma-Aminobutyc

of Other Amine Acid (GABA)

lmmunohlstochem&y

GABA-like immunoreactivrty (GABA-LI) has been demonstrated in the CNS using antiserum produced using GABA coupled to protein with glutaraldehyde (Strom-Mathisen et al , 1983) GABA-LI localization corresponded to that of GABA uptake sites and to the marker enzyme glutamic acid decarboxylase. Extensive validation was accomplished and preliminary electron microscopic studies suggested a srgmficant concentration in synaptic vesicles 4.4.2. Glutamate lmmunohrstochemrstry Immunohistochemistry of glutamate (Glu-LI) using antiserum to glutamate coupled to protein by glutaraldehyde shows a Glu-LI distribution m nerve terminals m those regions m which Glu is a strong transmitter candidate (Strom-Mathison et al., 1983) The pattern of Glu-LI corresponds to known high-affinity Glu uptake sites. Extensive validation was done A vesicular localization was demonstrated in prehmmary electron microscopic studies. 4.4.3. Histamine

/mmunohistochem&ry

Using antiserum to histamme coupled to protein with glutaraldehyde, histamine-like rmmunoreactivity (HI-LI) has been mapped m the rat CNS (Mulder and Steinbusch, 1983). Cross-reactivity assessed against coupled 5-HT and catecholamines was negligible. HI-L1 positive cells were seen m several areas notably in the hippocampus.

5. Conclusions Immunologic techniques have been applied widely m the study of brain amine neurotransmrtters and there IS the prospect of increasing use of these techniques. Radioimmunoassay techniques have great sensitivity and are highly efficient, allowing the processing of large numbers of samples To this point, radioimmunoassays have been used principally for substances m the circulation or m the urine and to a considerably lesser extent for substances m CSF or m brain tissue. As these techniques are validated for use on brain tissue and CSF, expanded usage is to be expected The single most important requirement for a radioim-

Brown

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munoassay is a highly specific antiserum that has good affmity Antisera that meet these requirements have now been produced for several of the amme neurotransmitters or their derivatives This development has entailed the imagmative use of couplmg reactions, and, in many cases, the synthesis of specific analogs capable of being conlugated m the appropriate fashion Similar strategies are applicable to the remaining amme transmitters Thus, the prospect is for the development of highly specific and efficient radioimmunoassays for the malority of these substances Immunohistochemistry of the amme transmitter substances also has shown a malor expansion recently. This approach is uniquely capable of mapping endogenous amme transmitters m the nervous system. It is efficient and can be applied at both the light and electron microscopic level. The key factor m this approach has been the development of antisera capable of binding amine neurotransmitters m fixed tissues Smce the transmitter substance may be coupled to structural proteins m the tissue and may also be chemically altered by the fixation process, the antisera must be uniquely capable of recogmzmg this altered structure. The successful development of methods for additional amme neurotransitters can be predicted with confidence

References Arendt J , Wetterberg L , Heydon T , Sizonenko I’. C , and Paunierx L (1977) Radioimmunoassay of melatonm in human serum and cerebrospmal fluid. Hormone Res 8, 65-75 Arendt J and Wllkmson M. (1979) Melatonm, m “Methods of Hornzorzc Radzozmmzmoassay” (Jaffe B M and Behrman H R , Eds, 2nd ed.) pp 101-119 Academic Press, New York Axelrod J and Laroche M J (1959) Inhlbltlon of 0-methylatlon of eplnephrme and norepmephrme in vjtro and I?? ‘UIUO Scze)?ce 130, 800 Barton J. W. Protection of N-H bonds and NR3 (1973) Protectwe Groups III Orgum Chemzsfry, (McOmle J F. W , ed ) pp 43-93 Plenum Press New York Baummger S , Kohen F , and Lmdner J R (1974) Steroids and haptens

Optimal

design of antigens for the formation

of antibodies

to steroid

hormones ] Steroid Blochem 5, 739-747 Berod A , Hartman B K , and Pu~ol J F (1981) Importance of flxatlon m lmmunocytochemlstry use of formaldehyde solutions at variable pH for the locallzatlon of tyrosme hydroxylase ] Hlsfochem Cyfochem 29, 884850 Blair S A and Seaborn C J (1979) The synthesis of melatonm antigens Amt. J Chem 32, 399-403.

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Boulton A. A , Juorio A V., Philips S. R , and Wu I’. H (1977) The effects of reserpme and 6-hydroxydopamme on the concentration of some arylalkylammes m rat brain. Brat J PIlannaco2 59, 209-214. Brown G M and Grota L. J (1980) Use of immunologic techniques m the exammation of neurotransmitters and neuromodulators. Physzcochem Metkodol. Psycklatr. Res (Hanm I and Koslow S H , Eds ), pp. 65-81, Raven Press, New York Brown G. M., Grota L. J , Bubemk G , Niles L., and Tsui H. W. (1981) Physiologic regulation of melatonm. Adv Btoscl 29, 95-112 Brown G. M., Grota L J , Pulido 0 , Burns T G , Niles L I’, and Srueckus V (1983) Application of immunologic techniques to the study of pmeal mdolealkylamines. In heal Research Revzews Vol I (Reiter R J., eds ), pp 207-246 Liss, New York Brunswick D (1980) Prmciples of Radioimmunoassay, m Pkyslockcm Metkodol Psyckzatr Res (Hanm I. and Koslow S H , eds.), pp. 37-63. Raven Press, New York Brusco A., Peressml, and Saavedra J P (1983) Serotonm-like immunoreactivity and anti 5-hydroxytryptamme (5-HT) antibodies ultrastructural application m the central nervous system J Hzstockem

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31, 52P.530

Bubemk G. A , Brown G M , and Grota, L J (1976a) Differential locahzation of N-acetvlated mdolealkylammes m CNS and the Hardenan gland using immunohistology Brar~~ Res 118, 417427. Bubemk G. A , Brown G M , and Grota, L. J, (1976b) Immunohistochemical localization of melatonm m the rat Harderian gland 1, Hzstockem Cytockem 24, 1173-1177 Bubemk G A, Brown G M , and Grota L. J (1977) Immunohistochemical localization of melatonm m the digestive system of the rat Experzentfa 33, 662-663 Burckhalter J H , Tendick F H., Jones E M., Halcomb W. F., and Rawlms H L (1946) Ammoalkyphenols as antimalarials I Simply substituted alpha-ammocresols. J Am Ckem Sot. 68, 1894-1901 Coffe A. R., van Leeuwen F. W., and Stembusch H W M (1983) Colocalization of vasopressin, neurophysm and noradrenalm immunoreactivity m subpopulations of rat locus coeruletls and subcoeruleus

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9, 575.

Cohen G. and M Collms (1970) Alkaloids from catecholammes m adrenal tissue possible role m alcoholism. Science 167, 1749-1751 Coons A H., Leduc E H , and Connely J M (1955) Studies on antibody production I A method for histochemical demonstration of specific antibody and its application to study of the hypenmmune rabbit. ] Exp Med

102, 49-59

Delaage M A and Puizillout J J (1981) Radioimmunoassays for serotonm and 5-hydroxyhndoleacetic acid ] PkysroI Purls 77, 339-347.

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de Silva S. 0 and Smeckus V. (1978) Indole-N-alkylation of tryptammes via diamon and phtalimido intermediates New potential mdolealkylamme hapten Can I Chem 56, 1621-1628 Dlener V , Knoll E , and Wrsser H. (1981) Preparation of antibodies to catecholammes and metabolrtes-synthesis of various rmmunogens and characterization of the resultmg antibodies Clan. Chum. Acta 109, l-13. Engback F , and Voldby B (1982) Radioimmunoassay of serotonm (5-hydroxytryptamme) m cerebrospmal fluid, plasma and serum Clm Chem 28, 624-620 Erlanger B F (1973) Prmciples and methods for the preparation of drug protein conlugates for immunological studies Plzarlnacol Rev 25, 271-280 Falcon J., Geffard M , Juillard M , Delange M., and Collm J (1981) Melatonm-like immunoreactivity m photoreceptor cells. A study of the pmeal organ and the concept of photoneuroendocrme cells Biol Cell. 42, 65-68 Faral B A , Mu J Y , Lewis M S., Wilson J P , Isram Z H , and Dayton I’. G. (1975) Determmation of plasma and tissue levels of tyramme by radioimmunoassay Proc Sot Exptl Blol Med 149, 664-669 Faral B. A., Camp V M , I’rultt A. W., Isaacs J W , and Ah F. M. (1977) The measurement of 3-0-methyldopamme m urme and plasma by a rapid and specific radioimmunoassay 1 Nucl Med 18, 10251033 Faral B. A , Walker W R , Camp V M , All F. M., and Cobbs Jr W B (1978) Development of an enzyme-radioimmunoassay for the measurement of dopamme m human plasma and urine 1 Nucl. Med 19, 1217-1224 Faral B. A., Lawson, D A , Nixon D W , Murray D. R., Camp V M , Ali F. M., Black M., Stacciarmr W., and Tarcan Y (1981) Melanoma detection by enzyme-radioimmunoassay of L-dopa, dopamme and 3-0-methyldopamme in urine Clan Chem. 27, 108-112 Fuxe K, Hokfelt T , and Ungerstedt U (1968) Localization of mdolalkylammes m CNS Adv Pharmacol 6, 235-251 Geffard M R , Puizillout J J , and Delaage M L (1982a) A single radioimmunological assay for serotonm, N-acetylserotonm, 5-methoxytryptamme and melatonm I, Neurochem 39, 1271-1277 Geffard M , Chambolle P , LeMoal M., and Delaage M L (1982b) Premier application immunocytochimlque d’un anticorps antidopamme a l’etude du systeme nerveux central C R Acnd Scr Parts 295, 797-802 Geffard M., Sequela I’ , Bulls R M , and LeMoal M. (1983) Demonstration of dopamme and p-tyramme with specific antisera against these catecholammes Sot Neuroscl Abstr 9, 1149. Grota L J and Brown G M (1974) Antibodies to mdolealkylammes serotonm and melatonm Cat? ] Bzohenz 52, 196202. Grota L J and Brown G M (1976) Antibodies to catecholammes Etzdocnnolojy, 98, 61.5622

Immunologic Studies of Brain Amlnes

299

Grota L J,, Smeckus V., de Silva S. O., TSUI H W , Holloway W. R., and Brown G M (1981) Radloimmunoassay of Lewy A J, melatonm in rat serum Prog. Neuropsychopharnzacol 5, 523-526 Grota L. J , Holloway W R , and Brown G M. (1982) 24-Hour rhythm of hypothalamic melatonm immunofluorescence correlates with serum and retinal melatonm rhythms. Neuroendocnnology 34, 363368 Grota L. J , Smeckus V , de Sllva S 0 , and Brown G M. (1983) Antlbodies to mdolealkylammes II. site of conlugation of melatonm to protein using formaldehyde Can ] Blochem 61, 1096-1101. Hadlu J and I’. Friedrich (1975) Reaction of glutaraldehyde with NH1 compounds Aual B~ochem 65, 273-280 Harmar A. J and A S Horn (1976) Octopamme m mammalian brain rapid post mortem increase and effects of drugs. 1 Newochem 26, 987-993 Harvey L D (1979) An lmmunohistochemical study of the distributron of epmephrme m the rat bram. Unpublished M S thesis, McMaster University Harvey L. D , Brown G. M , and Grota L J. (1983) Serum melatonm and photoperrod induced reproductive changes m the rat Unpublished Hokfelt B (1951) Noradrenalme and adrenaline m mammalian tissues Acta Physzol Stand 25, Supplement 92 Hokfelt T , Llungdahl A , Stembusch H , Verhofstad A , N&son G., Brodm E , Pernow B , and Goldstein M (1978) Immunohistochemical evrdence of substance P-like immunoreactivlty m some 5-hydroxytryptamme-containing neurons m the rat central nervous system Neurosclelzce3, 517-538 Holloway W , Grota L J , and Brown G M (1980) Quantitative determination of lmmunoreactive melatonm m the colon of the rat 1 Hzstochem Cytochew 28, 255-262 Holzwarth M A and Brownfield M. S (1983) Serotonmimmunoreactivity m adrenal medulla of the rat Sot Netrroscr Abstr 9, 388. Juorio, A V (1979). Drug-mduced changes m the formation, storage, and metabolism of tyramme m the mouse Br 1 Phamacol 66, 377-384 Kawashlma K and Nagakura A. (1982) Improvement of radloimmunoassay for serum and tissue melatonm, J PhnrJfl D~JI 5, 5-26 Keeton T K Krutzsch H and Lovenberg W (1981) Specific and sensitive radioimmunoassay for 3-methoxy-4-hydroxyphenylglycol (MOPEG) Scle)zcc211, 586588 Kellum J M. and Jaffe B M (1976) Valrdation and application of a radlolmunoassay for serotonm Gastroent 70, 516-522 Kennaway D J , Froth R G , and Philllpou G (1977) A specific radroimmunoassay for melatonm m biological tissue and fluids and its vahdation by gas chromatography-mass spectroscopy Endocn~ology 101, 119-127 Kennaway D. J (1983) Radioimmunoassay of 5-methoxytryptophol m

Brown

300

and Grota

sheep plasma and pmeal glands. Life Sa 32, 2461-2469 Kohler C. S. and Stembusch H W M. 1982 Identificatron of serotonm and nonserotonm contammg neurons of the entorhmal area and the hippocampal formation A combined immunohistochemical and fluorescent retrograde tracing study m the rat brain Neurosczence 7, 951-975. Knoll E. and H Wisser (1973) Gewmnung und Charakterisierung von antikorpern gegen, 3,4-dimethoxyphenylathylamm Clan Chum Acta 48, 183192 Knoll E and H. Wisser (1976) Radioimmunologiche bestimmung von 3,4-dimethoxyphenlathylamm im urm Clr~ Cklm Acta 68,327-332 Kurzon R. M and Sternberger L A (1978) Estrogen receptor immunocytochemistry 1 Hlstochem Cytochem 26, 803-808 Lam R W , Artal R , and Fisher D A (1977) Radioimmunoassay for free and conlugated urinary metanephrme C11n Ckem 23, 12641267. Lemaitre B and Hartmann L (1980) Preparation of anti-melatonm antibodies and antigemc properties of the molecule J lmmunol Methods 32, 339-347 Levine L and Riceberg L J (1975) Radioimmunoassay for melatonm Res Commun Ckem Path01 Pharmacol 10, 693-702 Lewy A J and Markey S I’ (1978) Analysis of melatonm m human plasma by gas chromatography negative chemical ionization mass spectrometry Scrence201, 741-743 Lewy A. J , Tetsuo M , Markey S I’, Goodwin F K , and Kopm I (1980) Pmealectomy abolishes plasma melatonm in the rat 1 Clan Endocrmol. 50, 204-205 Lidov H G. W , Grzanna R , and Molliver M E (1980) The serotonm cerebral cortex in the innervation of the rat-An immunohistochemical analysis Neurosczence5, 207-227 Lmr H R., Perel E , Friedlader A , and A. Zeitlm (1972) Specificity of antibodies to ovarian hormones m relation to the site of attachment of the steroid hapten to the peptide carrrer, Sterords 19, 357-375. McLean I W and Nakane I’ K (1974) Periodate lysme paraformaldehyde fixative. A new fixative for immunoelectron mlcroscopy J Hrstochem Cytochem 22, 1077-1083 Milstem C , Wright B , and Cue110A C (1983) The discrepancy between the cross-reactivity of a monoclonal antibody to serotonm and its immunohistochemical specificity Mel lmmunol 20, 113-123 Miwa A., Yoshioka M , Shirahata A , and Tamura A (1977) Preparation of specific antibodies to catecholammes and L-3,4-dihydroxyphenylalanme I Preparation of the conlugates Ckem Pkarm. Bull (Tokyo) 25, 1904-1910. Mlwa

A , Yoshloka

M , and

Tamura

Z

(1978a)

Preparation

of speclflc

antibodies to catecholammes and L-3,4-dihydroxyhenylalalme II. The site of attachment on catechol moiety m the conlugates Ckem Purm Bull (Tokyo) 26, 2903-2905

Immunologic Studies of Bra/n Amlnes

301

Miwa A., M Yoshioka A, and Tamura Z. (1978b) Preparation of a specific antibody to catecholammes and L-3,4-dihydroxyphenylalarune III Preparation of antibody to epmephrme for radioimmunoassay Chn?? Plznrnz Bull (Tokyo) 26, 3347-3352 Mulder A H. and Stembusch H W. M (1983) Mappmg of hrstammeimmunoreactivity m the central nervous system of the rat Sot. Neurosct. Abstr , 9, 83. Nairn R. C (1956) Fluorescent Protem Tracing, Churchill-Livingston Edinburgh Nakane P K. and Pierce G B (1966) Enzyme labelled antibodies. preparations and application for the localization of antigens J Hlstochem Cytochem. 14, 929-931 Outschoorn, A S , (1952) The hormones of the adrenal medulla and their release Brat 1 Pharmacol 7, 605-615 Ozaki Y and Lynch H J (1976) Presence of melatonm m plasma and urine of pmealectomized rats Endocrmology 99, 641-644 Pang S F Brown G M , Grota, L. J , Chambers J W , and Rodman R L (1977) Determmation of N-acetylserotonm and melatonm activeties m the pmeal gland, retma, harderian gland, brain and serum of rats and chickens Neuroendocrmolugy 28, 1-13 Pang S. F , Brown G M., Cambell L , Smeckus V., de Silva, S O., Young L. M , and Grota L. J (1981) A radioimmunoassay for N-acetylserotonm m biological tissues 1 Immunolzssay 2, 263-276, Parker C. W (1971) The nature of immunological responses and antigen-antibody mteractlons, in Prrnciples of Cornpetztrve ProteinBmdzng Assays, (Ode11 W K. and Daughaday, W H Eds), pp 2548 Lippmcott. Philadelphia. Peskar B , Peskar B. M , and Levine L (1972) Specificities of antibodies to normetanephrme Eur J Bzochem 26, 191-195 Peskar B and Spector, S (1973) Serotonm. Radioimmunoassay Scrcrzce 179, 134&1341 Porietis A. V , Brown G M , Lloyd K G., Grota L J., and Friend W (1977) Immunohistochemical localization of dopamme m the rat CNS Pm Cm Fed Bzol Sot 20, 160 Psarakis S , Pulldo 0 , Brown G M., Grota L J , and Smith G K. (1982) Identification and quantification of N-acetylserotonm (NAS) m developmg hippocampus of the rat Pro8 Neuropsychopharmacol & Blol Psychlatr 6, 439-442 Puizillout J J and Delaage M A. (1981) Radioimmunoassay of S-hydroxymdoleacetlc acid usmg an iodmated derivative 1 Phannacol Exp Ther 217, 791-797 Pulldo 0 , Brown G M , and Grota L J (1983a) Beta-adrenerglc regulation of N-acetylserotonm (NAS) synthesis m the rat cerebellum Life Scz 33, 1081-1089 Pulldo O., Brown, G. M , and Grota L J. (198310) An immunohlstochemical method for the localrzatlon of N-acetylserotonm

302

Brown and Greta

(NAS) m the central nervous system Descnptton, valldatron and applrcatron of the technique. ] Hzstochem Cyfochem , 31, 1343-1350 Ranadrve N. and Sehon A (1967a) Anttbodles to serotonm Can J Blochem , 45, 1701-1710 Ranadrve N. and Sehon A (1967b) Antlgemcrty of 5-hydroxymdole-3acetrc acid, a derrvattve of serotonm Can ] Bmhem 45,1681-1688 Raum W and Swerdloff R (1981b) A radrormmunoassay for epmephrine and norepmephrme m &sues and plasma. L$e SCI 28, 2819-2827 Raum W. and Swerdloff R (1981a) Urmary metanephrme radrolmmunoassay Comparrson with the colonmetrtc assay C/III

Chem 27, 43-47 Rrceberg L J and Van Vunakrs H (1975) Esttmatlon of B-3,4dlmethoxyphenethylamme and related compounds m urine extracts by radrolmmunoassay B~ochem Pharmacol 24, 259-265 Rlceberg L J., Van Vunakrs H , and Levme L (1974) Radrormmunoassays of 3,4,5-tnmethoxyphenethylamme (mescaline) and 2,5-dlmethoxy-4-methylphenyllsopropylamme (DOM) Aual B/oclzeln 60, 551-559 Rrceberg L J and Van Vunakls H (1978) Determmatron of N,Ndlmethylmdolealkylammes m plasma, blood and urine by radlormmunoassay and high pressure llquld chromatography ] Pharmacol Exp They 206, 158-166 Richards F. M and Knowles J F. (1968) Glutaraldehyde as a protein cross-hnkmg reagent. J Mol B1o1 37, 231-233 Rollag M D and Nlswender G D (1976) Radrolmmunoassay of serum concentratron of melatonm m sheep exposed to different llghtmg regimes EttdocrznoloRy 98, 482-489 Samte-Mane G (1962) A paraffin embedding technique for studies employing rmmunofluorescence 1 Hzstochem Cytochem 10, 250-256 Saavedra J I’., Brusco, A , Peressuu S , and Oltva D (1983) Anti-5-HTlike antlbodres and their rmmunoreactrvlty to beta-carbolmes an rmmunocytochemlcal study Sot Neuroscr Abstr 9, 1182 Scatchard G (1949) The attractions of protems for small molecules and ions Ann N Y Acad Su 51, 660-672 Seguela P , Geffard M , Bur~rs R M , and LeMode M (1983) Antrbodres agamst small molecules An Applrcahon to GABA Sot Neuroscr

Abstr , 9, 404. Shlrahata A , Yoshroka M , Matsushita M , and Tamura Z (1980) Studies on radlormmunoassay of metanephrme Chefn Phann. Bu12 (Tokyo) 28, 29943001 Spector S , Dalton C , and Felix A M. (1973) Development of antibodies against catecholammes, m Frontlevs uz Catecholamme Research (Usdm, E and S Snyder, eds ), pp 345349 Pergamon Press, New York Stembusch H W , Verhofstad A A , and Joosten H W (1978) Locahzanon of serotonm m the central nervous system by lmmunohrsto-

Immunologic

Studies

of Brain Amines

303

chemistry. descrlptlon of a specific and sensmve technique and some appllcatrons Neuroscience 3, 811-819 Stembusch H W. M (1981) Drstrrbutron of serotonm-rmmunoreactlvlty m the central nervous system of the rat-cell bodies and termmals Neuroscmce

6, 557-618

Steinbusch H W M and Nleuwenhuys R (1981) Locahzatron of serotonm-lrke rmmunoreactrvlty m the central nervous system and pmutary of the rat with specral reference to innervation of the hypothalamus Adv Med Blol. 133, l-35 Sternberger L. A (1974) Infnlu?rocyfochemzsfry, Prenhce-Hall, Englewood Clrffs, N J rmmunoSternberger L A and Petralr J P (1975) Quantrtatlve cytochemrstry of pltmtary receptors for lutemlzmg hormonereleasing hormone Cell Tlsslle Res 162, 141-176 Strahllevrtz M , Narasrmhacharr N., FrJrmorr M , and Hlmwlch H. E. (1971) Blocking of 5-methoxy-N-drmethyltryptamme-induced EEG alerting m the rabbit by previous admmlstratron of anhserum to this compound Blol Psychafr 3, 227-236 Strom-Mathrson, J , Lekness A K., Bore A T , Vaaland J L , Edmmson P , Haug F M , and Ottersen 0 I’. (1983) First vlsualrzatlon of glutamate and GABA m neurons by rmmunocytochemrstry. Nnt~~e (Lond ) 301, 517-520 Takeuchl Y , Klmura H., Matsuura, T., Yonezawa T and Sano Y (1983) Drstrlbutlon of serotonergrc neurons in the central nervous system a peroxidase-antlperoxldase study with anti-serotonm antrbodres 1 H&o&em. Cytochem 181-185. Tamarkm L , Reppert S M., and Klein D C (1979) Regulation of pmeal melatonm m the Syrian hamster Endocrr~zology 104, 385-389 Taylor C. R Immunoperoxldase techniques (1978) Arch Patkol. Lab Med. 102, 113-121 Tetsuo M , Perlow M J , Mlshkm M , and Markey S P. (1982) Light exposure reduces and pmealectomy vrrtually stops urinary excretion of 6-hydroxymelatonm by rhesus monkeys Endocrinology, 110, 997-1003 Thompson B. B (1968) The Manmch reaction, mechanistrc and technical consrderatrons / Pkarm Scl 57, 715-733 Ungerstedt, U (1971) Stereotaxlc mapping of the monoamine pathways m the rat brain Acfa Physrol Stand. 82, supplement 367 Verhofstad A A J , Stembusch H W J., Penke B , Varga J , and Jooster H. W. J (1980) Use of antibodies to norepmephrme and epmephrine u-r lmmunohrstochemrstry Adv Brockem Psyckopkarnmcol 25, 185-193 Versteeg D H G., Van der Gugten J., DeJong W , and Palkovrts M (1976) Regronal concentratrons of noradrenalme and dopamine u-r rat brain Bralrl Res 113, 563-574 Vlvlen-Roels, B , Pevet P., Dubors M P , Arendt J , and Brown, G. M

304

Brown and Grota

(1981) Immunohlstochemlcal evidence for the presence of melatonm m the pmeal gland, the retma and the HarderIan gland. Cell Tissue Res 217, 105-115 Wetterberg L , Erlksson 0 , Frlberg Y , and Yangbo B (1978) A slmpllfled radlolmmunoassay for melatonm and its appllcatlon to blologlcal flulds Prellmmary observations on the half life of plasma melatonm m man CIln Chum Actn 86, 169-177 Wetterberg L and Erlksson 0 (1981) Melatonm m human serum-a collaboratlve study of current radlolmmunoassays Adz) Bmscmces 29, 15-20 Wllllams C A and Chase M W (Eds) (1967) Methc~Is 111Znrr~~ur~ologymzd Immu~~ochenrzstu~, Vol 1 Preparation of Antigens and Antibodies Academic Press, New York Wlsser H , Herrmann R , and Knoll E (1978) Methodical mvestlgatlon of the productlon of antibodies towards 3,4-dlmethoxyphenylethylamme CIIM C/ZUTI Actor 86, 179-185 Wurzburger R J , Kawashlma K , Miller R L , and Spector S. (1976) Determmatlon of rat pmeal gland melatonm content by a radlolmmunoassay Life SCI 18, 867-868

Chapter 7

Combined Gas Chromatography-Mass Spectrometry in the Analysis of Biogenic Amines in Humans FAROUKKAROUM 1. Introduction The so-called trace ammes or noncatechollc brogemc amines have recently attracted a consrderable amount of attention among neuropharmacologrsts and psychratrrsts. Some of these amines, e.g., phenylethylamine (PEA) and tyramme, are suspected to play some yet undetermined role in the etiology of schizophrema (Sandler and Reynolds, 1976; Karoum et al., 1980) and depression (Karoum et al , 1982) The behavioral effects of these ammes, although not clearly understood, are believed to result from their abrlrty to interact with central putative neurotransmitter systems For this reason, studres related to these ammes are best conducted m conlunctlon with brogemc ammes such as catecholammes and serotonm This strategy IS partrcularly Important m the clmical mvestrgatron of the role of brogenic amines m mental Illnesses. Of the varrous analytical tools that are available for the srmultaneous assay of ammes m brologrcal materials, gas chromatography (GC) and hrgh pressure liquid chromatography (HPLC) are perhaps the most convement and practical. Unfortunately, the use of these two techmques wrth conventronal detectors does not offer good specrficrtres unless coupled to a mass spectrometer (MS). The apphcatron of combmed HPLC-MS, n-rcontrast to combined CC-MS, has not been well studred. GC-MS has been used extensively during the last 15 yr to Identify and quantrfy brogenic 305

306

Karoum

ammes m various blologlcal media m man and the experimental animal. Quantification by GC-MS is normally performed by momtormg the MS to measure the mtensltles of preselected speclflc ions or fragments m the mass spectra of the compounds of mterest This technique 1s commonly referred to as mass fragmentography (MF). In this chapter, modifications of previously reported MF methods (Karoum and Neff, 1982, Karoum, 1983) will be described. These methods were speclhcally developed to enable the routine analysis of blogemc amines and metabolltes m human biological materials They are simple, reliable, and, above all, capable of analyzmg 30-50 samples per batch

2. Materials and Methods Stationary phase SE 54 and chromosorb G, 80/100 mesh were obtamed from Pierce Chemical Company, Rockford, IL. Fused slhca capillary columns were purchased from Supelco Inc., Bellafonte, PA. All deuterated isomers of blogemc ammes were either synthesized (Karoum et al., 1975) or bought from Merck, Sharp, and Dohme, Canada Ltd, Quebec or Kor Inc., Cambridge, MA. MIcroflex tubes (1 mL) were purchased from Kontes, Vmeland,

NJ.

Ethyl acetate used for derlvatlzatlon was obtained from Regls Chemical Co , Morton Grove, IL. Crude sulfatase was purchased from Sigma Chemical Company, St. LOWS, MO. All chemicals and reagents used were of the highest purity commercially available.

2.1. Mass Fragmentography Models 3200 and 4000 Fmmgan gas chromatograph-quadrupole mass spectrometers (Finmgan Corp , San Jose, CA) were used Assays of catecholammes and metabolltes were performed on a 10 ft, 3 mm od stainless-steel column packed with 3% SE 54 on chromosorb G (Analabs, North Haven, CT) A 30-m fused-silica capillary column, bonded wall-coated with SPB-5 (methylphenylvinyl silicone gum), was used for the assay of phenylacetic acid For the assay for phenylethylamme and m- and p-tyramme, a 30-m fused slllca column bonded wall coated with 15% SP 2250 (methylphenyl silicone), 60% SE 54, and 25% SP 2401 (tnfluorpropyl slhcone) was used The capillary columns had an inner diameter of 0 32 mm and a coating film thickness of 1 km.

K-MS

and Amlne

307

Analysis

Packed GC columns were maintained isothermally at temperatures ranging from 190 to 2OO”C,and m capillary column analysis the followmg parameters were used: temperatures ranging from 160 to 18o”C, head pressure around 10 psi and split ratios rangmg from 1 50 to l-80. The m/z of the fragments employed for MF are summarized m Table 1

2.2. Derivatization Amines and alcoholic metabolites are acylated to their pentafluoroproplonate derivatives while the acidic metabolites are esterlfled to the ethyl ester then acylated to their ethyl ester/ pentafluoroproplonate derivatives. Phenylacetlc acid IS converted to its pentafluoropropionate ester

2.3. Pen tafluoropropiona te Deriva ties (PFP) To the dry residues obtained from the biological materials (m 1 mL microflex tubes) are added 10 PL ethyl acetate (dried over CaH*) and 100 PL pentafluoropropionic anhydride (Pierce Chemical Co.). The tubes are tightly capped and heated at 80°C for 10 min m a heating block. After cooling to room temperature, the mixture is evaporated to dryness under a gentle stream of N2 and the residue is reconstituted in 10 or 25 FL of “dry” ethyl acetate. One or 2 PL of the solution is injected into the GC column.

2.4. EthylestedPentafluoropropionate

Derivatives (EWPFP)

Acidic metabolites m microflex tubes are first esterifled with 100 PL of 20% HCl in dehydrated ethanol; the tubes are capped and left at room temperature for 5 mm. The HCUethanol in the microflex tubes is evaporated to dryness under N2 (care should be taken to make sure that the residue is very dry or the next derivatization step will not be successful). The HCUethanol IS prepared by mixing dropwise 1 mL acetyl chloride into 5 mL dehydrated ethanol The temperature of the mixture should rise to around 45°C. Failure of the mixture temperature to rise will indicate the presence of moisture in either the acetyl chloride or ethanol. Presence of moisture will result in poor yield of products The ethyl esters of phenolic and catechollc acids are next acylated to their PFP derivatives by reacting them with 10% pentafluoroproplonyl imldazole (Pierce Chemical Co.) m ethyl acetate at 70°C for 10 min. The final product should be clear and

308

KCl~OU7l

TABLE 1 Mass to Charge Ratio (m/z) of Approprrate Fragments of Derwatrzed Ammes and Metabolltes Used m Mass Fragmentography Molecular ion (M’ )

Name

Derrvatwe

Phenylethylamme Phenylethanolamme Octopamine (o-, nr, p-) Tyramme (o-, m-, ~7-) Amphetamine Methamphetamme

PFP PFP PFP PFP PFP PFP

267 429 591 429 281 295

Noradrenahne Dopamine Adrenaline Normetanephrme Metanephrme 3-Methoxytyramme Epmme Phenylacetrc Acid Phenylalanme p-Tyrosme 3-Methoxy-4-hydroxymandehc acid p-Hydroxyphenylacetrc acid p-Hydroxymandek Homovanlllrc acid 3,4-Dlhydroxyphenylacetic acid 3-Methoxy-4-hydroxyphenylglycol Tryptamme 5-Hydroxytryptamme Indoleacetrc acid 5-Hydroxymdoleacetrc acrd

PFP PFP PFP PFP PFP PFP PFP PFP ester PFP ester PFP ester EEJPFP

acid

“163 = NH$2OC2F;, 190 COOCH2C2F5, 73 = COOCzHq

Structure of fragment’

Brogenrc m/z of fragment 104 253 428 266 118 204

753 591 767 621 635 459 605 268 443 605 518

M’ -163 M’-176 M’ -163 M’ - 163 M+-163 CHCH3-NCHTCOC2F5 M + -163 M’ -163 M+ -163 Ml-163 M+ -177 M’ -163 M’ -177 M+ M’-163 M+-163 M’ -73

EEIPFP

326

M’

326

EEIPFP EEIPFP EEiPFP

488 356 488

M’ -73 M+ M+

415 356 488

PFP

622

M’

622

PFP PFP EEIPFP EE/PFP

452 614 349 511

M’-147 M’ -163 M’ M+-73

305 451 349 438

=

CH2NCH&IOCrFS,

177

=

590 428 604 458 458 296 428 268 280 442 445

NCH-,COC2F;

or

309

GC-MS and Amine Analysis

slightly yellow. A colorless product indicates poor derivatization due to the presence of moisture. On the other hand a dark yellow or brown color indicates excessive heating or an excessive amount of protein, One or 2 mL of the mixture is mlected into the GC column. The derivatization reactions of a phenolic acid with the general structure of (HO)RCOOH proceed as follows: HO-R-COOH -H20

+ C2Hs0H -+ HO-RCOOC2Hs + HZ0 EE HO-RCOOC2H5 + C2FSC0 C3H3N2 --) C2F5CO0 -RCOOC2HS EEIPFP

2.5, Pentahoropropionate

Ester

This derivative IS prepared for phenylacetic acid, phenylalanine, and tyrosme. The biologrcal residue in a microflex tube is mixed with 100 PL of pentafluoropropanol and 25 PL pentafluoropropionic anhydride, capped, and heated at 70°C for 10 mm. After coolmg to room temperature, excess acylating reagents are evaporated off under N2 and the solution volume is reduced (takmg care not to completely dry the residue) The final volume m the vial should be about 2 J.LL.The derivative is mixed with 10 or 25 PL of ethyl acetate One or 2 ~J,Lof this solution is inlected into the GC column (Karoum et al., 1983)

2.6. Quantification In contrast to animal studies where five measurements per group IS often sufficient to establish a finding, m human studies it is often necessary to analyze a large number of samples. This is especially so in clinical mvestigations where the changes expected are moderate and therefore a large “n” is needed Besides, rt 1s also frequently necessary to combme or compare data obtained from different analyses In order to achieve these capabilities, it is mandatory that the analytical methods employed are consistent and highly reproducible. To assure good reproducibility and to control for any mconsistency that may influence reproducibihty we have adopted the followmg precautions (1) Concentrated solutrons of deuterated and nondeuterated standards, 100 pg/mL, are prepared in a large volume, divided mto small aliquots, 0.2 mL, and stored at -16°C m screw-capped

310

Karoum

vials. A new vial 1sused m every batch to prepare the appropriate standard solutrons and then discarded. This approach reduces variabilities introduced by preparation of standard solutions from solid compounds or from stock solutron by frequent thawing and freezing. (2) Whenever possible, triplicates of the same sample (reference sample) are included m every batch of analyses For urine analyses, a normal 24-h urine is divided mto 1 mL aliquots and stored in glass vials at -16°C. One vial is used as the urine reference sample. The mean of the results of the triplicate reference sample is compared with the mean obtained the first time the sample was analyzed and proper correction or normalization made. Thus, if m a batch of analyses the mean for the triplicate assay of a substance m the reference sample is 90% of that determined the first time the reference sample was analyzed, the appropriate normalization will be by increasing all the values obtamed in this batch of analyses by 10%. This approach provides a method by which correctron can be made for minor day-to-day variation m setting up the electronic adlustments m the mass spectrometer. This latter variation is not related to the specificity of the assay nor to poor reproducrbility of the method. It is solely related to minor variations in the quadropole electronics when selecting the appropriate fragment for MF (mass marker adlustments). Once an adjustment is made, the multiple ion detection of a substance in the biological sample and its authentic standard are always comparable, mdrcatmg good specificrty. Further, repeat analyses of the same biological samples (urine, CSF, and brain tissues) on different days employing the normalization procedure described above always give an mtraclass correlation (ICC) over 0 95, indicating good reproducibility (a perfect ICC is 1 00) (3) Appropriate deuterated isomers of the compound of mterest are added to the samples at the begmnmg of the assay and carried through the whole procedure. The deuterated isomers are used to measure the concentration of the nondeuterated compounds by comparison. For added accuracy, a standard curve for the nondeuterated compound is also constructed, primarily to check the amount of deuterated standard added (4) Whenever an unexpectedly high concentration of a compound is detected, its identity is ascertained by multiple ion detection (MID). This is a technique whereby the mass spectrometer is monrtored to detect and measure the mtensrtres of more than one fragment ion in the mass spectrum of a compound. If the ratios of the ions produced by MID of the compound in the blologi-

K-MS

and AmIne Analysis

311

cal sample are comparable to those m the authentic standard and both have the same retention time, the possrbrhty that the two compounds are the same 1s strongly suggested. 2.7. Extraction Except for catecholammes, all other brogemc ammes and metabolites listed m Table 1 are extracted mto an organic solvent prior to derrvatrzation Whenever possible, extraction is carried out in 2 “Eppendorf’ tubes (Brinkmann Instrument mL polypropylene Inc., Westbury, NJ). Extraction IS carried out by vortex mixing the biological material with the organic solvent for 1 mm followed by centrrfugatron at 10,OOOg.A portion of the organic phase is separated and the extraction repeated once more after adding more organic solvent After centrifugation of the second extract, a portion of the organic phase is separated, combined with that of the first extract and evaporated to dryness under a gentle stream of NZ, The dried residue is then denvatrzed. 2.8. Assay of Catecholamines and Metabolites in Human Brain Tissues Brain trssues, 50-200 mg, are homogenized in 0.5 mL of a 1N HCl solutron contammg 100 ng/mL of the appropriate deuterated isomers. An alrquot, 10 pL, of the homogenate IS removed for protein assay (Lowry et al., 1951) The homogenate IS centrifuged at 12,OOOgfor 5 mm and the clear supernatant removed and stored at -16°C until analyzed For the assay of catecholammes, 50 PL of the above supernatant 1s transferred mto 1 mL mrcroflex tubes, evaporated to dryness under N2 and the PFP derrvatrve prepared. Acidic metabolites of catecholamines, 3,4-dihydroxyphenylacetic acid (DOPAC), homovamllrc acid (HVA), and vanilmandelrc acid (VMA), m the above supernatant (0.4 mL) are extracted mto ethyl acetate Two mL polypropylene tubes are used. The EE/ PFP derivative 1s prepared. 3-Methoxy-4-hydroxyphenylglycol (MHPG; MOPEG) IS assayed as follows: 0.2 mL portrons of the above supernatant m 2 mL polypropylene tubes are mixed with 15 PL of 10N NaOH, followed by the addition of 500 FL of 1M acetate buffer, pH 6 2. To this mixture, 10 ng of deuterated MHPG (2H3-MHPG) IS added, followed by the addmon of 100 LJ of crude aryl-sulfatase from Hellx pomatza, type H-l (Sigma Chemical Co., St. LOUIS, MO). The mixture is incubated at 40°C for 1 h and then extracted wrth ethyl acetate. The PFP derivative IS then prepared.

312

Karoum

Gas chromatographic separation is carried out on a 10 ft 3% SE-54-packed column The m/z of the fragments selected for MF are listed m Table 1.

2.9. Assay of Catecholamine Metabolites in Plasma and Cerebrospinal Fluid (CSF) Protem m plasma is precipitated by mixing 0 5 mL of plasma with 0.5 1M ZnSO+ After centrifugation, the clear supernatant is removed and used for analysis It should be pointed out that if deuterated solutions in 1N HCl are to be added before protein precipitatron, the HCl must be neutralized with NaOH before ZnS04 is added Failure to neutralize the HCl will lead to poor protein precipitation. For the assay of the acidic metabolites, 0 4 mL of the above plasma clear supernatant or CSF is mixed with 0 2 mL 2N HCl and 10 ng of the appropriate deuterated isomers are added. The mixture is extracted with ethyl acetate Two mL polypropylene tubes are employed. The EEPFP derivatives are prepared. For the assay of MHPG, 0.4 mL of the above plasma supernatant or CSF m 2 mL polypropylene tubes is mixed with acetate buffer; deuterated MHPG and sulfatase are then added exactly as described for brain tissues Mass fragmentography IS carried out on a lo-ft 3% SE 54-packed column The m/z values of fragments selected are listed m Table 1.

2.10. Assay of Catechofamines and Their Me tabolites in Urine Noradrenaline (NA), dopamme (DA), and normetanephrme (NMN) are measured by mixing 25 PL of urine with 25 PL HCI solution of the deuterated isomers, 1 kg/mL The mixture is then evaporated to dryness under a gentle stream of N2. The PFP derivative is prepared The acidic metabolites/DOl?AC, HVA, and VMA as well as p-hydroxyphenylacetic acid are measured by mixing 50 PL of urine with 50 PL of a 1N HCl solution contammg the deuterated isomers at a concentration of 1 pg/mL and adding 0.2 mL of 1N HCl. The mixture is extracted with ethyl acetate and the EE/PFP derivative is prepared MHPG is assayed by mixmg 50 ~.J,Lof urine with 0 4 mL 1M acetate buffer, 100 U of sulfatase, and 50 ng of 2H,-MHPG The mixture is incubated at 40°C for 1 h. The sulfatase employed also contains glucurorudase. After mcubation, the mixture is extracted

GC-MS and Amine Analysrs

313

with ethyl acetate as described for brain tissues. The PFP derivative is prepared MF 1s carrred out on a lo-ft 3% SE 54-packed column. Table 1 lists the nziz values of the fragments selected.

2.11. Assay of lndole Amines and Metabolites in Urine Indoleacetic acid and 5-hydroxymdoleacetic acid and their precursor amines, tryptamme and serotonm, are assayed as described for the catecholammes and their metabolites except for the followmg modifications* (1) a 6-ft 3% SE 54 column is used, and (2) the PFP derivative 1s prepared by heatmg for 15 min at 100°C

2.12. Assay of Phenylethylamine (PEA) in Urine To 1 mL of urine m a 10 mL round-bottom glass tube, 5 mL of n-heptane and 50 ng of deuterated PEA (2Hg-PEA) are added, followed by the addition of 1 mL phosphate buffer (prepared by mixing one part 0 5M Na2HP024 with three parts 0.5M NalP04, pH 13). The mixture is then vortex-mixed for 1 mm and centrifuged at SOOOgfor 3 min. A 4-mL portion of the heptane phase is separated mto a 15 mL conical bottomed glass centrifuge tube and the mixture IS extracted with an additional 5 mL of heptane. After centnfugation, another aliquot is removed and mixed with the previous 4 mL extract The combmed heptane extract is then mixed vigorously with 0 3 mL 1N HCL and the heptane evaporated under vacuum (Buchler Instrument, Fort Lee, NJ), taking care not to evaporate the aqueous phase. The aqueous phase IS transferred mto a “Micro-Flex” vial and dried under N2 The PFP derivative is prepared Mass fragmentography IS carried out on a 30-m fused-silrca capillary column bonded wall-coated with 15% SP 2250, 60% SE 54, and 25% SP 2401. Alternatively, a 12-ft stainless-steel column, i/Rm od, packed with a mixture of 0 5% OV22 + 2% SE 54 + 1% OV210 coated on W(HP) 80/100 mesh support (Karoum et al., 1979) may be used. The m/z values of the fragments employed are listed m Table 1 The method described here may be used to assay amphetamine, methamphetamine and other noncatecholic ammes m urine (Karoum, 1983)

2.13. Assay of Phenylalanine and Tyrosine in Urine Protein intake may be evaluated from the rate of phenylalanme and tyrosme excretion. These ammo acids are measured by mixmg 10 PL of urine with 50 FL of a solution of

Karoum

314

*H8-phenylalanme and *H7-tyrosme (5 mg/mL in HCl) and evaporating the mixture to dryness under N2 The PFP esters are prepared and a lo-ft 3% SE 54-packed column 1s used.

2.14. Assay of Phenylacetic Acid (PM)

in Urine

Free and conjugated PAA are measured as follows 50 PL of urine IS mixed with 50 PL of a 1 mg/mL solution of PAA in 1 N HCl Aliquots (20 pL) are transferred mto two “Micro-Flex” vials To one vial, 10% trlethylamine in methanol (50 FL) IS added, and the mixture IS evaporated to dryness under N2. The residue is used to assay free PAA To the other vial, 6N HCI (50 FL) is added and the vial is heated at 100°C for 45 min After cooling and adding 5 PL 10% triethylamme in methanol the mixture 1s evaporated to dryness under N *. The residue 1s used for the assay of total PAA (Martin et al., 1979). The free or total PAA m the above residue is converted to its pentafluoropropanol ester. The denvatizatlon proceeds as follows: C2H5CH2COOH

+ C2F5CH20H PAA

(C2F,CO)20 b

pen tafluoropropanol

ChH5CH2COOCHZC2F5

+ H20

PAA pen tafluoroproplonate A packed fragments

3% SE 54 column is used for MF. The selected are listed in Table 1.

m/z values of the

2.15. Assay of PAA in Plasma and CSF Because of the relatively low concentration of PAA m plasma and CSF as compared to urine, a different approach to its assay has to be taken. This procedure includes extracting PAA mto ethyl acetate and using a 30 m fused slhca column bonded wall-coated with SPB-5 As previously reported (Karoum et al , 1983) phenylacetyl glutamme (PAG), the conjugated form of PAA found m humans, exhibits the followmg important properties (1) it IS not hydrolyzed in 1 or 6N HCl at room temperature, (2) it IS hydrolyzed completely m 6N HCl, when heated at 100°C for 45 mm , (3) It is extracted quantitatively into ethyl acetate at acid pH; (4) exposmg PAG to the derlvatlzatlon reagent produces no hydrolysis nor does It yield products that interfere with the derlvatlves corresponding to unconlugated PAA

GC-MS and Amine Analysis

315

For the assay of free PAA n-rplasma, equal volumes of plasma containmg 500 ng of *H7-PAA (taken from a solutron of 100 &mL m water) and acetone, 0.5 mL, are thoroughly mrxed in a 2 mL polypropylene tube and then centrifuged The clear supernatant IS transferred mto a 10 mL glass tube, acidified by adding 0.5 mL of 2N HCI and extracted twrce with 5 mL ethyl acetate. The ethyl acetate extract IS evaporated uz wcuo and the pentafluoropropanol ester is prepared For the assay of total plasma PAA, plasma 1s treated as described for the assay of free PAA except that 2H7-PAG IS added instead of the free form of deuterated PAA. The residue obtained after evaporating the ethyl acetate extract to dryness 1s reconstituted m 0 7 mL 6N HCl, transferred to a 10 mL roundbottom glass tube, capped, and heated at 100°C for 45 min After heating, 0.4 mL of 10N NaOH IS added to neutralize most of the acid and the solutron extracted and derrvatrzed as described for free PAA Free and total PAA m CSF are assayed as described for plasma but the protein precrprtatron step by acetone IS omitted.

3. Conclusions The concentration of a number of biogemc ammes and then metabolites m the urine, plasma, CSF, and brain tissues obtained from normal sublects are summarrzed m Table 2 Typical MFs of a number of brogemc amines and their metabolites m urine are shown m Figures 1 through 5. The feature that distmgurshes MF from other analytical procedures IS the combinatron of two powerful techniques: gas chromatography and mass spectrometry. The former offers selechvity while the latter mcorporates structural mformatron. These two characteristics of MF can be explorted to render high speclficitres m the detection of compounds, but they cannot on their own assure good reproducrbrlrty because reproducrbrlity 1s largely dependent on the procedure employed to prepare the brologrcal sample for MF and on the day-to-day conditron of the mass spectrometer. Thus, although the peaks that are measured on a given day may correspond to the pure compound (high specificity), the absolute concentratrons to which these peaks correspond may vary from day to day (sometrmes as much as 20%). Such a variation is tolerable m studies that involve one experiment, because the differences between the tested materials and the controls will

02

(PEA) Phenylethanol-

300-l 50-200 (7-13) x IO’

14bk74

377 t 56

85 2 14

(9 5 _f 0 9) X IO’

(19 7 i

3-Methoxytyramme

p-Tyramme nl-Tyramme

Phenylalanme

II-Tyrosme

1 6) x 10’

144

735 t

Dopamme

50-500

(l&27)

620

0 1

0103rO01Y

n,g’mL

Plasma

002-01

Range

concen tra tlon

0061 01

2001

ngimL

-

o-o

Range

tra twn

Blologlcal

CSF concen

TABLE 2 and Metabolltes m Various TABLE 2 (co&nucd)

x 10’

500

200-2000

150-300

116 2 27

-

3-12

Range

175 2 41

excretion

Ammes

Normetanephrme

?I03

h

Urme

of Blogemc

Noradrenalme

amme

71

&g/24

Concentrations

Phenylethylamme

Name

Normal”

100

Bram

10 81 ? 13

Y3-+68

ndmg protem

59 8 t 34 5

0 82 2 0 60

-

Wmg protein

nucleus,

Caudate

Humans

concentratmn

from

Hypothalamus,

Materials

phenylacetlc

acid

t

19) x 10’

‘The data were obtained

WVA)

St3

100tL3000

from at least SIX sublects

(4 0 ? 0 6) x lo3

+ 144

2350

acid

coqugate Total

Homovamlhc

30&900

20-50

400-1000

74

(0 f&2 1) x 10’

10’

x 10’

X 10’

x 10’

(3-9 0) x

(14)

(15-50)

(40-63)

845 L 74

49 i

(1 77 f 0 2) x 10’

(4 1 2 0 5) x 10’

(1 9 2 0 4) x lo?

(29 8 t 3 8) x 10’

(162

979 t 84

conjugate

acid

Glucuromde

Sulfate

acid

acid

oxyphenylglycol (MHPG) Free

(DOPAC) 3-Methoxy-Chydr-

phenylacetlc

(VMA) 3,4-Dlhvdroxy-

Vamlmandek

acid

@ydroxymandek

acetlc

(PAA) ;?-Hydroxyphenyl-

Total

13

+ 06

117219

10 + 10

79205

83

58’06

1 3 + 0 19

12 4 I

16525

11 3 5 0 9

459 i- 77

j-10

0 5-2 0

8-15

S-15

Z-14

7 30-620

t 003

51 -t 8

10 2 2

020

17205

11 o+

12

f 0 18

k 0-l

30516

046

13

99t85

416228

-

30

30-80

36 3 t- 21 4

-

8-12

so5

1 13 + 1 04

1 57 + 1 69

0520

8-15

1-l

0 2-O 8

05

30-50

+ 600

905

2 175

0 71 r 0 21

03-rOlh

1037

318

Karoum

m/z

592 -A

DA

NMN

NA

,

m/z

458

ii (1

I

m/z

590

/(

m/z

431 -1)

d

INJ I

A 0

0

1

2 MIN

Fig 1 Typical mass fragmentograms adrenaline (NA), normethanephrme (NMN), human urine.

of PFP derlvatlves of norand dopamme (DA) from

not be greatly affected if the results are 10 or 20% higher or lower than the accepted values. However, this situation 1shighly undesirable in clinical mvestlgations where large number of samples are analyzed. In our laboratory we have adopted a number of steps that assure high reproducibility. The most important of these 1s the approach by which we normalize all our results against predetermined values corresponding to reference biologlcal samples (see methods se&on). Normahzatlon of results has enabled us to study the metabolism and turnover of catecholamines m depressed patients receiving a variety of treatments (Linnoila et al , 1982a, b, c, d, 1983a, b) As concluded from various assessments, the reproduclblllties of the methods described here showed intraclass correlations (ICCs) that are better than 0.95. The MF methods described m this chapter are modlflcatlons of previously reported methods for the assay of biogemc ammes m both man and experimental animals (Karoum and Neff, 1982, Karoum, 1983). Their appllcatlon to the routme assay of biogenic ammes and their metabolites has been tested extensively in depression (Liebowitz et al., 1983), schlzophrema (Potkm et al ,

K-MS

319

and Amine Analysis HVA

WA

1

I

0

1

2

I

,

I

0

1

2

Fig 2 Typrcal mass fragmentograms of EE/PFP derlvatrves of vamlmandelic acid (VMA), 3,4-dlhydroxyphenylacetrc acrd (DOPAC), and 3-methoxy-4-hydroxyphenylacetrc acid (HVA) Isolated from human urine.

3

5

6

7

MIN

Fig. 3 Separation of PFP derlvatrves of phenylethylamme (l), phenylethanolamme (2), o- (3), m- (4), and p-tyramme (5) on a 30 m fused silica column bonded wall-coated with SP 2250, SE 54, and SP 2401 (see Materials sectron)

Karoum

320

of PFP derlvatlves Fig 4 Typlcal mass fragmentogram p-tyramme Isolated from human urine Fused slllca capillary mass spectrometry was employed

of m- and column/E1

,, INJ

D-TYR

I’I ,

I -._----_-----_-,^I 0

5 1

1 2

-uL

1 3

4

5

MIN

of PFP derlvatlves of M- and Fig 5 Typical mass fragmentogram p-tyramme Isolated from human urine Fused sAca capillary column/C1 mass spectrometry was employed

GC-MS and Amine Analysis

321

1982), hyperactivity m children (Zametkin et al., 1984), Parkmsomsm (Karoum et al., 1982), idiopathic apnea of premature birth (Bhat et al., 1983a,b) and anorexia nervosa (unpublished) Specificity m MF is controlled by focusing the mass spectrometer on specific fragments and by employing selective and efficient GC columns. Selection of appropriate fragments for MF IS made after careful studies of the mass spectra of the compounds of interest and after exhaustive MID evaluation of these compounds in biological materials. Having selected the best fragment for MF, the next important decision that has to be made is the selection of the most convenient mode of ionization. Of the various types of ionization that are available, electron bombardment (EI) and chemical ionization (CI) are the two most frequently employed For routine analysis of large numbers of samples, EI is more convenient than CI Chemical ronization should be used when all possibilities mvolving the use of EI have failed. In this context, of the two types of GC columns that can be used, packed columns are more convenient and easier to use than capillary columns For this reason, capillary columns are recommended only when packed column fail to offer good separation of the compounds of interest. A good example of this latter situation is encountered in the assays of PEA and PAA. Although the fragment selected for the MF of PEA (m/z 104) employing EI is not an ideal fragment and therefore CI, which gives a prominant molecular ion, is more desirable, we have overcome this drawback by employing highly selective packed or capillary columns (Karoum et al., 1979). Using these columns with CI did not improve the specificity nor the precision of our assay In the case of PAA, on the other hand, packed columns are best suited for the assay of PAA m urine, but for the assay of PAA m plasma and CSF, capillary columns are recommended (Karoum et al., 1983) In conclusion, a number of MF methods are described for the assay of a variety of important biogenic amines m human biological media. These methods were specifically developed to enable the accurate and reproducible measurements of these compounds in biological materials obtained from humans and therefore are expected to prove useful m clmical investigations.

References Bhat A M , Scanlon J. W , Lavenstem B., Chuang L -W., and Karoum F (1983a) Effect of theophyllme on neurotransmltters m preterm mfants with apnea. CIUZKQ~ Neuropharmacol. 6, 71-74.

322

Karoum

Bhat A. M , Scanlon J W , Lavenstem B , Chuange, L. -W., and Karoum F. (1983b) Cerebrospmal fluid concentration of biogemc amme metabohtes m idiopathic apnea of prematurity Blol Neonate 43, 16-22. Karoum, F and Neff, N. H (1982) Quantitative gas chromatography mass spectrometry (GC-MS) of biogemc amines* Theory and practice, m Modern Methods In Pharmacology, Spector, S and Back, N. eds) pp. 39-54, Alan R. Liss Inc , New York Karoum F (1983) Mass fragmentography m the analysis of biogemc ammes a clmical, physiological and pharmacological evaluation, m Methods ITI Blogenrc AmnzeResearch, (Parve2 S., Nagatsu T , Nagatsu I , and Parvez H , eds.) pp 237-255 Elsevier Science Publications, New York Karoum F , G&n J C , Wyatt R J , and Costa E (175) Massfragmentography of nanogram quantities of biogemc amme metabelites m human cerebrospmal fluid and whole rat bram Biomed Mass Spectrom., 25, 653-658

Karoum F , Nasrallah H., Potkm S., Chuang L , Moyer-Schmmg J, Phillips I , and Wyatt, R. J (1979) Mass fragmentography of phenylethylamme, m- and p-tyramme and related amines m plasma, cerebrospmal fluid, urme and brain ] Neurochem 33, 201-212 Karoum F., Potkm S., Murphy D. and Wyatt R. J (1980) Quantrtation and metabolism of phenylethylamme and tyramine’s three isomers m human, m Non-Catechokc Phenylethylamwes, Part II, (Mosnaim, A D and Wolf, M. E , eds), pp 177-191. Marcel Dekker Inc , New York Karoum F , Lmnoila M,, Potter W. Z , Chuan L -W., Goodwin, F K. and Wyatt, R J (1982) Fluctuatmg high urinary phenylethylamme excretion rates m bipolar affective disorder patients. Psychuzf. Res. 2, 215-222 Karoum F., Chuang L. -W., Mosnaim A D., Staub R. A and Wyatt R. J (1983) Plasma and cerebrospmal fluid concentrations of phenylacetic acid m humans and monkeys ] Chromatog Su 21, 546-550. Liebowitz M R., Karoum F , Quitkm F M , Davies S. 0 , Stewart J W., McGrath I’. J,, Harrison W , Schwartz D , Levitt M., Lmnoila M , Wyatt R J., and Klem, M. (1984) L-deprenyl m atypical depression II. Biochemical effects. Arch Gen. Psychzatry, (m press). Linnoila M , Karoum F , and Potter W Z (1982a) Hugh posmve correlation between urinary free tyramme excretion rate and “whole body” norepmephrme turnover m depressed patients Blol Psychratry 17, 1031-1036. Lmnolla M , Karoum F and Potter W Z. (1982b) Effects of low dose clorgylme on 24-hour monoamme excretion m rapidly cycling bipolar disorder patients Arch Gen Psychratry 39, 513-516 Lmnoila M , Karoum F , and Potter W. Z (1982~) High correlation of norepmephrme and its malor metabolite excretion rates Arch Gen Psychuby 39, 521-523

K-MS

and Amine Analysis

323

Lmnoila M , Karoum F , Call1 H M , Kopin 1.J and Potter W Z (1982d) Alteration m norepmephrme metabolism with desipramme and zimelldme m depressed patients. Arch Gen Psychmy 39, 102.51028 Lmnoila M , Karoum F , Cutler N R , and Potter W Z. (1983a) Temporal association between depression dependent dyskmesias and high urmary phenylethylamme output. Brol Psycllrnfry 18, 513-516 Lmnorla M , Karoum F and Potter W Z (198313) Effects of antidepressant treatments on dopamme turnover m depressed patients Arch GUI Psyckuatry 40, 1015-1017 Lowry 0 H , Rosebrough N J , Farr A L., and Randall, R J (1951) Protein measurement with the folm phenol reagent ] B~ol Ckem , 193, 265-275 Martin M E , Karoum F , and Wyatt R. J. (1979) Phenylacetic acid excretion m man Anal Bzockm 99, 283-287 Potkm S G , Jeste D V , Karoum F , Doongali D R., Apte J S , Sheth A. S , Chuang L. -W , and Wyatt R J (1982) A cross-cultural design to test a biological hypothesis of schizophrema m Blolug~cal Mnrkeus 171Psycklatry and Neurology, (Hanm I and Usdm E , eds ) pp. 49-59 Pergamon Press, New York Sandler M and Reynolds G P (1976) Does phenylethylamme cause schizophrema? lancet, I, 70-71 Zametkm A. J , Karoum F , Lmnoila M , Rapoport J L , Brown G L , Chuang, L. -W , and Wyatt R. J (1984) Stimulants, urinary catecholammes and mdoleammes m hyperactivity. A comparison of methylphemdate and dextroamphetamme Arch Gen. Psyckzatry (In press)

Chapter 8

High Resolution and Met&able Mass Spectrometry of Biogenic Amines and Metabolites DAVID A. DURDEN 1. Introduction Mass spectrometry (MS) is now widely used in the neuroscrences to identify and quantify a variety of brogemc compounds. When used m combinatron with chromatography, i.e., packed column or high resolutron capillary column gas chromatography (CC or HRGC) or thm layer chromatography (TLC), in either its low resolutron (LRMS) or high resolutron (HRMS) modes, rt IS capable of great precrsion and has been used to quantrtate, at subnanomolar levels, the putative and associated neurotransmitters, their precursor ammo acrds, and then acidrc and alcoholrc metabolites. Mass spectrometrrc methods have been developed, m this and m other laboratones for the trace ammes, phenylethylamme (PE) (Durden et al., 1973, Wrllner et al., 1974; Anderson and Braestrup, 1977; Reynolds et al., 1978, Edwards et al., 1979a, Karoum et al., 1979; Suzuki and Hattorr, 1983), phenylethanolamine (PEOH) (Willner et al , 1974; Durden, 1978, Edwards et al , 1979b), IT&IIand para-tyramme (m-TA and p-TA) (Phrlrps et al , 1974a, Phrlrps et al., 1975; Edwards et al , 1979a, Karoum et al , 1979), &ho-, meta- and pnra-octopamine (o-OA, m-OA, p-OA) (Buck et al., 1977; Williams and Couch, 1978, Durden et al , 1980, Duffield et al., 1981), meta- and paua-synephrme (m-SYN and +p-SYN) (Durden et al., 1978, Midgley et al , 1980) and tryptamine (T) (Phrllps et al , 197413, Warsh et al., 1977b; Artigas and Gelpr, 1979), the catecholammes, dopamme (DA) (Curtrus et al., 1974, Ko et al., 325

326

Durden

1974; Miyazaki et al., 1974, Wang et al , 1975, Wiesel, 1976; Freed et al., 1977; Kilts et al, 1977; Hashimoto and Myazaki, 1979, Mizuno and Ariga, 1979, Holdiness et al , 1980; Lhuguenot and Maume, 1980, Warsh et al., 1980), noradrenalme (NA) (Miyazaki et al., 1974; Erhardt and Schwartz, 1978; Jacob et al., 1978, Hashimoto and Miyazaki, 1979, Lhuguenot and Maume, 1980; Yoshida et al., 1980), adrenaline (epinephrine,, A) (Koslow and Schumpf, 1974, Juorio and Durden, 1977, Jacob et al., 1978) the mdolyl amme, 5-hydroxytryptamine (5-HT, serotonm) (Markey et al , 1981); the lmidazolyl ammes, histamme (HA) and t-methyl histamme (t-MHA) (Mita et al , 1980a,b), the polyammes (Smith and Daves, 1977, Shipe et al , 1979); the cycloalkylamme, piperidme (PIP) (Miyata et al., 1979) and the quaternary ammes, choline (Ch) and acetylcholine (ACh) (Jenden et al., 1973, 1978) The precursor ammo acids phenylalanme (Phe) and p-tyrosme (p-Tyr) (Zagalak et al., 1977; Sloquist, 1979; Trefz et al., 1979) and tryptophan (Trp) (Wegmann et al , 1978, Martinez and Gelpi, 1978) have been determined primarily by low resolution GC-MS The acid metabolites of the trace ammes have been determined by GC-MS methods, using low and high resolution MS packed or capillary column GC and electron impact (EI) or chemical ionization (CI) Thus, methods have been reported for phenylacetic acid (PAA) (Fellows et al., 1978, Martin et al., 1979, Durden and Boulton, 1982a, Karoum et al., 1983), ~zeta-and parahydroxyphenylacetic acid (nz-HPAA, p-HPAA) (Karoum et al , 1975a,b, Narasimhachari et al., 1978; Durden and Boulton, 1981) and the o&o-, meta- and ynvn- isomers of hydroxymandelic acid (o-HMA, un-HMA and p-HMA) (Midgley et al , 1979, Davis and Boulton, 1981). Methods for the mdolyl acids have been reported for mdole-3-acetic acid (IAA) (Bertilsson and Palmer, 1972, Warsh et al , 1977a, Artlgas and Gelpi, 1979) and 5-hydroxymdole-3acetic acid (5-HIAA) (Fri et al , 1974b; Beck et al , 1977, Godse et al , 1977, Artigas and Gelpi, 1979; Faull et al., 1979). The acid metabolites of the catecholammes, 3,4-dihydroxyphenylacetic (DOPAC), homovanilhc (HVA) and vanlllylmandehc (VMA) have been the sublect of many GC-MS analyses Notable among these are procedures by Sloquist et al , 1973, Fri et al , 1974a,b, Gordon et al , 1974; Wiesel et al., 1974; Sloquist, 1975, Karoum et al , 1975a,b; Gordon et al , 1976, Takahashi et al , 1977; Takahashi et al., 197813, Muskiet et al , 1978b,d, Vogt et al,, 1980 Finally, methods for the alcoholic metabolites of the catecholammes predominate m the literature over methods for those of the trace acid metabolites Many of the procedures m-

Metastabfe

Mass Spectrometry

327

volve GC-MS detection For the metabohtes of PAA and p-HPAA, phenylethyl glycol (PEG), p-hydroxyphenylethanol (p-HPE) and p-hydroxyphenylethylene glycol (p-HPG) Edwards et al. (1979b) used CJ-GC-MS The mdolyl alcohol 5-hydroxytryptophol (5HTOL) has been determined by methods developed by Takahashi et al. (1978a), Diggory et al (1979) and Beck et al. (1980). The catecholamme metabohte that has received most attention is 4-hydroxy-3-methoxyphenylethylene glycol (HMPG or MHPG) (Bertilsson, 1973; Gordon et al., 1974, Sloqmst et. al., 1975, Karoum et al., 1975b, Swahn et al., 1976; Takahashi et al , 1977, Muskiet et al., 1978b,d,1979,1980); it IS frequently determmed at the same time as DOPAC, HVA and VMA. The less concentrated metabolites 4-hydroxy-3-methoxyphenylethanol (HMPE), 3,4-dihydroxyphenylethylene glycol (DHPG) and 3,4-dihydroxyphenylethanol (DHPE) have also been determined by GC-MS (Karoum et al., 1975a; Muskiet et al , 1978a, b,c,d; Edwards et al , 197913) A critical assessment of the various mass spectrometric methods for the neurotransmitters, their precursors and metabolites has recently appeared (Durden and Boulton, 198213) In the present review, the methods used in this laboratory only will be presented, i.e., the thm layer chromatographic high resolution mass spectrometric (TLC-HRMS) procedures for amines and the GC-MS procedures high resolution capillary column gas-chromatographic-high resolution mass spectrometric (HRGC-HRMS) for amino acids, acids and alcohols. Other mass spectrometric techmques that are becoming of mcreasmg importance to the analysis of neurotransmitters and metabohtes will be discussed briefly.

2. Mass Spectrometry The organic mass spectrometer is a chemical analytical tool that provides qualitative data that can be used to identify compounds and determine their structures. The instrument consists principally of three parts-an ion source in which the sample is turned into gaseous ions, an analyzer m which the ions are separated according to their mass to charge ratio (m/z), and an ion detectorall of which are contained m a high-vacuum envelope The ions may be charged positively or negatively, and may consist of the charged molecule, the molecular ion, and fragments thereof, and perhaps ions of greater m/z due to formation through chemical reaction m the ion source via chemical ionization (CI) The mass

Durden spectrometer provides two pieces of mformation: the m/z ratios of the ions and their relative mtensities Because relative mtensities only are measured, the mass spectrometer is not normally considered to be a quantitative instrument, but can be used to obtain qualitative results by use of appropriate internal standards The mass analysis may occur due to the effect on the ions of electric and magnetic fields (magnetic sector mass spectrometers), electric and radiofrequency fields (quadrupole mass spectrometers), electric, radiofrequency and magnetic fields (Fourier transform mass spectrometers) or electric fields alone (time of flight) By use of these electromagnetic fields m various combmations, it is possible to observe not only the conventional ions formed m the ion source, but also ions formed outside the ion source due to spontaneous or collision-induced dissociation using metastable or multiple analyzer techniques Mass spectrometers may be classified by the resolvmg of their analyzers mto low resolution instruments or high resolution instruments. They are normally operated m one of two modes the scan mode in which all ions are detected and the mass spectrum obtained for qualitative analysis, or m a selected ionmomtormg (SIM) mode, m which only a few ions m the spectrum are detected for quantitative analyses. In the SIM mode, sensmvity is increased by a large factor (100 to 1000) due to the increase m time spent detecting each ion and the correspondmg increase m the signal-to-noise ratio produced by the time-averaging process.

2.1. Low Resolution Mass Spectrometers Low resolution mass spectrometers separate ions of different m/z values by unit mass with the possible structures of the ions being determined by assuming integer mass numbers of the elements. Thus, although m most instances each ion observed may be due to one particular structure, it is possible that the ion is due to the combined signals of ions of two or more different elemental compositions In MS analysis of low concentrations of neurotransmitter compounds from tissue or biological fluids, there may be a contribution to the ion intensity from other ions from compounds present m the MS high vacuum (MS background signal), from column bleed if a CC column is attached to the mass spectrometer, or from compounds, present m the tissue, that have not been completely removed by purification procedures. This problem becomes exacerbated as detection of smaller and smaller quantities is attempted. Low resolution mass spectrometers used for biomedical anal-

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329

ysrs are the quadropole type and the smgle or double focusmg magnetic sector type The quadrupole instruments have the advantage of providing a linear mass scale and raprd scanning of the mass spectrum and are easier to interface to gas and lrqurd chromatographs since only low ion source potentials are used The mstruments tend, however, to have a limited mass scale (less than 1000) and to have reduced sensitivity to high-mass ions (see Durden and Boulton, 1982b for an example) and are not capable of detectmg metastable Ions. The latest generation of mstruments have ameliorated some of these problems by use of longer analyzers and improved electronic controls that permit mass ranges up to 1,500 to 2,000 and have lower mass-Intensity drscrrminatron. The magnetic sector instruments appear to have relatrvely constant sensrtrvrty with mcreasmg mass and are capable of detecting both normal and metastable Ions at very high-mass values. The mam drsadvantages are that the mass scale IS not lmear, rt 1s more difficult to interface to gas and lrquld chromatographs due to the high ionization potentials (1,000 to lO,OOO),and the magnetic fields cannot be changed as rapidly as electric fields. The latest generatron of magnetic instruments have overcome these difficulties because the magnetic field now can be changed rapidly (0.1 set per decade in mass) and new electronic circuits provrde direct mass readings. Use of modern let separator mterfaces or silica capillary column inlets and low capacitance power supphes have reduced the GC interface problems. The double-focusing instrument has the additional advantage of enhancing metastable ion signals by suitable control of the electric and magnetrc fields.

2.2. High Resolution Mass Spectrometers By mcreasmg the resolutron of the mass spectrometer, rt becomes possrble to separate ions of the same nominal mass into different elemental cornpositrons due to different exact mass values (based upon the scale using 12C = 12 00000 U) During a quantrtative analysis, by focusmg on a precise mass value at high resolution, rt is possrble to verrfy the instrument IS detecting an ion due to the compound of interest, and to reduce the background signal by excluding the other ions from detection. Increasmg the resolutron of a conventronal double focusing magnetic sector mass spectrometer causes the absolute srgnal mtensrty to be reduced The signal intensity due to the background and other interfering ions, however, decreases at a greater rate, especially when the ion of interest and the interfering ions are completely resolved. Thus se-

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lectlvity and sensitivity of analysis may be improved (Mlllmgton, 1975; Vogt et al., 1980; Durden, 1984). One type of high resolution mass spectrometer suitable for biomedical research 1s the double-focusing magnetic instrument. This mass spectrometer, which can operate at high or low resolution, uses three electromagnetic fields to achieve high resolution (Fig 1). the acceleratmg field (V) which gives the ions their mltlal velocity, the electric-sector field (E), which provides energy resolution of the ions and increases the selectlvlty of the magnetic field (B), which disperses the ions according to their m/z ratios. In the conventional geometry, the ions pass first through the electric sector and then are mass analyzed m the magnetic field In the reverse geometry instrument, the magnetic field B IS m front of the electric sector E. Both geometries are capable of providing resolution up to l:lOO,OOO (10 parts per mllhon), and by suitable

2V=RE Ion Trajectory

DETECTOR

Fig 1. Schematic representation trometer with conventional geometry

of a double-focusmg

mass spec-

Metastable Mass Spectrometry

331

linking, V, E and B can be used to detect metastable transitions of ions useful for quantitation (Gaskell and Millington, 1978; Gaskell et al., 1980; Durden, 1982). A recently developed instrument, the Fourier transform mass spectrometer (Wilkms and Gross, 1981) in which the ions are mass analyzed by combmed radio frequency and magnetic fields, can be used m both the high and low resolution mode depending upon the number of cycles in the combined fields the ions are permitted to take. One limitation, that of mamtammg low pressure in the instrument, which had limited its usefulness for combined GC-MS analysis, appears to have been overcome by use of a pulsed valve interface to limit the inflow of GC carrier gas (Sack and Gross, 1983). Because of its ability to scan the mass spectrum very rapidly and also to be able to switch from low to high resolution under computer control, without requiring mechanical adlustment, as is required with the magnetic sector instrument, the Fourier transform mass spectrometer appears to be very suitable for integration with high-resolution capillary GC for analysis of low concentrations of blogenic compounds.

2.2.1. Operating Modes of the Double Focusing Mass Spectrometer 2.2.1.1. DETECTION OF NORMALIONS In the conventional double focusmg mass spectrometer, normal ions formed in the ion source are detected when the electric sector field (E) is linked to the accelerating field (V) at a constant ratio determined by the physical dimensions of the mass spectrometer Ions at different r~/z values can be detected either by changing B at constant E and V, and thus ~&z becomes proportional to B2, or by changing E and V at constant B, m which case m/z is proportional to l/V (or l/E) To obtain complete mass spectra, the first method is usually used For quantitative analysis using selected ion momtormg (SIM), either procedure may be used Changing B permits monitormg over a wide mass range, but is slower due to the necessity of changing the magnetic field in a precise manner Thus it is usually used for low resolution SIM analysis. The second procedure is used for high resolution SIM, as the voltages can be more rapidly and precisely controlled For this type of analysis, B is set to focus the lowest mass ion, usually a mass reference ion, at the maximum value for V and E, and V and E are reduced to detect the higher mass ions. For maximum precision of mass measurement and greatest sensitivity, the ratio of higher mass to reference is kept below 1.1, but ratios up to 2 may be used on modern mstruments at the cost of reduced sensi-

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tivity for the higher mass ions. If a sample contains a mixture of compounds with characteristic ions with a wider range of m/z values, the sample may have to be reinlected for each compound or group of compounds at different magnetic field settings using new reference ions. The main advantage of this type of SIM analysis is that the mass spectrometer may be operated at an optimum resolution, i.e., up to 10,000 or 15,000, and the ion masses may be verified to a few parts per million. 2.2.1.2. DETECTION OF METASTABLE PEAKS In the conventional double focusing mass spectrometer (Fig 1) there are two field free regions (FFR) that are useful for analytical applications; the first between the fields V and E and the second between E and B Ions that leave the ion source, but decompose mto smaller fragments as they pass through the first FFR, may be detected as metastable peaks by appropriate marupulation of V, E and B. In these “metastable” modes of operation, the normal ion signal is suppressed. The mass spectrometer is operated at low resolution to give maximum signal and the specificity, equivalent to that of a high resolution analysis, is obtained through choice of the metastable fragmentation transition (Gaskell and Millmgton, 1978; Durden, 1982) Metastable transitions of ions decomposmg m the first FFR can be detected by linking E and B There are two possible modes of operation. If V is set to the normal operating potential of the mass spectrometer, the ratio E/B may be set under conditions the parent ion is detected as a normal ion. B and E can be reduced at constant ratio E/B until the fragment ion is detected as a metastable peak. This method gives constant sensitivity as V is constant, but when used in the SIM mode, i.e., for selected peak monitoring (SMPM), the switching rate is slow since the magnetic field is changed. The alternate method is to adlust B and E at a reduced value of V to focus on the fragment ion; V is then increased so that the metastable peak from the parent ion is detected. This method permits a faster switchmg rate for SMPM, but the ion source sensitivity for each mass changes somewhat as V IS changed (Boyd and Beynon, 1977).

2.3. Other Mass Spectrometric

Techniques

The interest m using metastable peaks to increase specificity of analysis leads to the use of multiple mass spectrometer analyzers m a technique labeled mass spectrometry-mass spectrometry (MS-MS). An ion characteristic of the compound is selected by the first mass spectrometer; it is then excited by collision with a neutral gas m an intermediately located cell and then a second mass spectrometer records the spectrum of the decomposmg ions By

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Mass Spectrometry

careful selection of ions by each MS it was hoped to increase the speclhclty so that crude mixtures could be analyzed directly Speclflclty appears msufflcient for unambiguous ldentlflcatlon but, similar to HRMS or metastable MS, this instrument can provide very specific analysis when used with a chromatographic purification step such as TLC or GC

2.4. Sample Introduction and Associated Chromatography 2.4.1. Direct

Probe

and Thin Layer

Chromatography

The direct probe mlet 1s used for mtroducmg compounds that are solids at room temperature. The sample must be purlfled by a chromatographic procedure such as thm layer chromatography After denvitizatlon and separation, the samples are eluted from the chromatogram with a very small volume of an orgamc solvent and placed in the direct probe tip. The solvent is removed by heat from a small electric hot plate before the probe 1s inserted mto the direct msertlon lock. The probe tips are usually made from a melting-point capillary and constructed so that the sample m a small cup at the tip (volume, 5-7 pL) will be as close to the electron beam as possible. This maximizes sensitivity We have found that quartz tips that are cleaned by heating m a flame are not suitable as they appear to cause decomposltlon of the sample (Durden et al , 1974) The man-t advantage of use of the direct probe is the speed of analysis, use of the MS 1s maximized as all chromatography takes place independently of the MS operation and many samples can be run each day Because the samples are well purified, the sample load 1s small and the mass spectrometer Ion source remains clean. 2.4.2. Gas Chromatograph

inlets

2.4.2.1. PACKED COLUMNS In all gas chromatograph

inlets the sample is transported from atmospheric pressure mto the high vacuum of the MS ion source. In the case of packed column GC, the carrier gas must be removed preferentially. Most modern mass spectrometers use a single-stage glass Jet separator which has a separation factor proportional to the square root of the molecular weight. Thus, if helium 1s used as the carrier gas (optimum flow 25-35 cm3 atmimm) it will be removed preferentially The yield of organic molecules is usually of the order of 2040% The glass let 1s attractive for biological samples that are usually quite labile and would tend to decompose on metal surfaces (see McFadden, 1973, for a review of GC-MS interface types).

334

Durden

Packed columns have been used for many analyses of blogemc amines m the past, due to their ease of construction and high sample load They are, however, being supplanted by caplllary columns for ultrasensltlve analysis as their low resolution and tendency for high column bleed severely limit the speclflcity and sensitivity of GC-MS procedures 2.4.2.2. CAPILLARY COLUMNS Most modern mass spectrometers now use large capacity pumping systems on the Ion source region These pumps are capable of accepting flows of up to 5 cm3 atmimm of He without degrading MS performance and consequently capillary columns may be used wlthout a separator. It has also been shown that operating the capillary column with its outlet at vacuum increases speed of analysis without a slgnlflcant decrease m GC separation (Cramers et al., 1981, LeClerq et al., 1982). Several types of capillary columns have been used for HRGC-MS. The support-coated open-tube (SCOT) column permlts a relatively high sample load (300 ng to 700 ng per component, with an intermediate resolution (600 to 1200 effective theoretical plates, N&m) These columns can be used with on column mIectlon (0 1-O 5 PL solvent) and requu-e carrier gas flows of 1 to 4 cm3 atm/mm) Wall coated open tube (WCOT) columns have lower permlsslble sample loadings (20-200 ng, depending upon size), but much greater efflclencles (N,Jm = l,OOO-5,000) The smaller diameters must be used with inlet splitters on the GC. The most significant step m HRGC has been the development of vitreous slllca columns and bonded liquid phases The silica columns are ideal for GC-MS as they are very robust and flexible. The column may be inserted through the GC inlet right up to the ion source for maximum sample yield This mmlmlzes loss of chromatographic resolution due to dead volumes The bonded phases are very stable and exhibit very low column bleed. They have the added advantage that they can be rinsed with organic solvents when they become contaminated (Blomberg et al., 1981). Capillary columns increase both the selectlvlty and sensltivlty of GC-MS analysis. Because of this high resolving power, It 1s possible to readily separate structural Isomers and because the peaks are narrowed, and column bleed is reduced, the ion signals are increased. Finally, wide bore, thick film bonded phase columns may be used In two modes. with high carrier gas flows (20 cm3 atm/mm) as low resolution columns m which case they perform m a manner similar to packed columns, or with low carrier

Metastable

Mass Spectrometry

gas flows (1-3 cm3 atm/mm) capillary columns. 2.4.3. Llquld

Chromatograph

335 for increased

resolution,

as regular

Inlets

The combmation of hquid chromatography with mass spectrometry (LC-MS) is a newly developing method with possible applications m the study of neurochemistry. LC has the advantage of not requiring derivatization of the sample (as is the case for GC) and the capability of handling labile compounds. Removal of the carrier liquid is much more difficult than removal of carrier gas and at present compromises m both LC and MS are required. Two types of interfaces are presently m use direct liquid introduction (DLI) and the movmg belt (MB) interface. In the DLI interface, a small portion of the solvent and sample is inlected into the MS, usually via an expanding let that removes some of the solvent. In the MB interface, the column effluent flows onto a movmg belt on which the solvent is evaporated The belt circulates through the ion source at which point a flash heater evaporates the sample. At present, the method is limited to use of easily evaporated organic solvents, although recently success has been obtained using reverse phase columns and aqueous buffers with a contmuous solvent extraction procedure (Karger et al., 1979) and is limited by sensitivity, since nanogram or microgram quantities are required. As with GC-MS, the development of narrower columns, m this case microbore LC columns, may increase the sensitivity of LC-MS to make it a useful procedure for analysis or neurotransmitter compounds. LC-MS has been reviewed recently by McFadden (1980) and several examples of its utility are presented.

2.5. Ion Formation 2.5.1. Electron

Impact

Electron impact (EI) ionization is the conventional method for forming ions m the MS ion source. A beam of electrons (energy. 50-70 eV) passes through the gas molecules (pressure less than 10P4 mb) and forms mainly excited positive ions by strippmg an electron from the molecule. The molecule ion may then dissociate into a series of fragment ions and neutrals or may leave the ion source intact It is possible to determine the structure and identity of the compound from the m/z ratios and relative mtensities of the molecular ion and its fragments. The molecular ion is most useful for quantitation but many compounds, especially biogemc ammes, exhibit very low molecular ion intensities under EI En-

336 hanced molecular ion intensities able derivative or less energetic

Durden may be obtained by use of a suitforms of iomzation

2.5.2 Chemical lonlzation Chemical ionization (CI) is a technique m which the sample is ionized by means of ion molecule reactions from a reagent gas Both positively and negatively charged ions are produced. A reagent gas is admitted to the ion source, along with the sample, at a greater pressure (up to 1 mb) and mtereacts preferentially with the electron beam. Ions from the reagent gas then react with and ionize the sample molecules by a variety of mechanisms (Harrison, 1983). 2.5.2.1. POSITIVE ION CHEMICAL IONIZATION Reagent gases for positive ion CI suitable for analysis of biogenic ammes and metabolites include methane, isobutane and ammoma. These compounds react with themselves to form protonated, molecular and adduct ions For methane, the malor ions m the CI spectrum are CHs+, C2H5+ and C3 H5+ (&z 15,29, and 41) Such ions may react with the sample molecule M either as Bronsted acids to produce the pseudomolecular ion MH’ by proton transfer or as Lewis acids to produce the (M-H)+ ion by hydride ion abstraction Adduct ions may also be produced, for example, isobutane may complex to produce (M + C,H,) ’ ions as well as MH’ or (M-H) ’ ions. Molecular ions are not usually produced in positive ion CI Using CI, the amount of energy transferred is much less than with EI and fragmentation IS reduced, giving much simpler spectra. The amount of fragmentation is very dependent upon the choice of reagent gas and the chemical structure of the sample molecules. By careful choice of reagent gas, it is frequently possible to mmlmize fragmentation so that the ionization is concentrated m pseudomolecular or adduct ions and to increase the specificity and sensitivity of analysis. Methane, the origmal reagent gas for CI, is most popularly and easily used. It is relatively simple to use but because of reaction energetics causes the greatest amount of fragmentation, such that some compounds may not exhibit MH’ or (M-H) ’ ions On the other hand, it does react with all types of compounds Isobutane is a milder reagent gas, consequently MH’ and (M + C4H9)+ ions are observed with many compounds Many researchers have found, however, that it tends to contaminate the ion source much more rapidly than does methane Ammonia is an even less reactive reagent gas and since it cannot protonate carboxy1 or ether oxygens, mtroduces a mode of selectivity. Its rea-

Metastable

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Mass Spectrometry

gent ion NH4-’ readily reacts with acid labile compounds, such as primary, secondary, and tertiary amme functional groups, alcohols and ammo acids; the factors affecting its reactivity have been studied by Keough and DeStefano (1981) 2.5.2.2. NEGATIVE ION CHEMICAL IONIZATION Negative ion CI is a most selective ionization method that is ideal for use with compounds contammg halide or other atoms with high electron affu-uties Three malor processes may occur m the ion sourceresonant capture ionization, dissociative electron capture or negative ion molecule reactions If methane or mtrogen is used as the reagent gas, the electrons are rapidly deenergized to thermal energies. These electrons are captured and compounds with electron affinities greater than 0 5 eV may be detected as M- ions In the drssociative mechanism, hydrogen atoms or larger groups may be lost with the charge reman-ring on the fragment with highest electron affinity When reagent gas mixtures such as methane-nitrous oxide or methane-acetylene are used, Bronsted base reagent ions (e g , 02-, OHP,C2H-) may deprotonize the compound to (M-H)ions, or form adduct ions [(M + OH)), (M + NO - H))] (Hunt et al., 1976). By derivatizing the compounds with electron-capturmg groups (especially those containing large numbers of fluorme atoms), it is possible to increase the sensitivity of analysis of neurotransmitter compounds by several orders of magnitude when compared to EI or positive ion CI procedures (Hunt et al , 1978; Lewy and Markey, 1978; Markey et al., 1981; Wood, 1982) Faull and Barchas have recently revrewed the use of negative-ion mass spectrometry for the analysis of neurotransmitters and related compounds (Faull and Barchas, 1983). 2.5.3. Fast Atom Bombardment

lonlzation

The above methods of iomzation require that the sample be presented into the ion source in the gaseous phase and thus many samples require heatmg. This precludes analysis of compounds that are thermally labile, or have low volatilities due to large molecular weights or polar nature. Fast atom bombardment (FAB) uses a beam of atoms of about 8 keV energy that impinges on the sample, that has been dissolved m a low-volatility solvent, such as glycerol, and then coated onto a target. The sample IS sputtered from the surface as neutral molecules and as both posrtrvely and negatively charged ions with equal facility Most compounds do not give molecular ions, but the even electron species encountered in CI, 1 e., (M + H)+ or (M - H)). Their mtensities may be

338

Durden

enhanced by the addition of protonatmg agents, such as p-toluenes u If onic acid. If the solvent is “doped” with a salt such as NaCl or KCl, (M + Na)+ or (M + K)+ ions may be observed Because of the sputtering technique, FAB produces ions at room temperature from thermally labile or involatile samples from such large molecules as peptides up to molecular weights of 7000. FAB ionization does not, at present, produce the sensltlvity that EI or CI do, but with improvements m technology could be applied to analysis of neurotransmitters, especially usmg LC-MS, for which rt is perhaps the most appropriate method of lomzation The technique has been the subject of a recent review (Barber et al , 1982).

2.6. Ghan titative Mass Spectrome tty Mass spectrometry is not inherently a quantitative procedure since the ion signal 1s dependent upon such adlustments as resolution, mstrument focusing and the molecule’s fragmentation, which is influenced somewhat by the electron beam energy. Use of an internal standard overcomes these variations as well as loss of sample through variations m yields of derivatization, extraction or chromatographic separation and adsorption on the glassware or mass spectrometer inlet system. The ideal internal standard should have identical chemical and physical properties to those of the compound to be quantitated. Stable isotope labelled analogues (isotopomers) are thus the most suitable internal standards. Atoms such as r3C, 15N, or 180 have been used to label mternal standards (e.g., 15N is used for histamine, Mita et al., 1980) but deuterium (2H) is used primarily due to the ease of synthesis of the appropriate isotopomer It is preferable that more than one atom of deuterium be used to avoid the contribution of the natural abundance of 13C to the ion to be observed It appears that labeling with three or four deutermm atoms is preferable, as most spectra usually contain very low abundances of M ” or M ’ ’ ions Greater numbers of deutermm atoms may produce an isotopomer with physical and chemical characteristics different from the compound of Interest For example, we have observed that nonadeutero-phenylethylamme (PE-d9) is partially separated from phenylethylamme using thin-layer chromatography of the l-dimethyl-ammonaphthalene-5-sulfonyl derivatrve (Durden and Dyck, unpublished observations). Even tetradeutero or pentadeutero hydroxyphenylacetic acids may be partially separated when using capillary column GC-MS (Durden and Boulton, 1981). Other limitations of use of deutermm labeling is that the

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Mass Spectrometry

339

deutermm atoms must be located in non-exchangeable (or very slowly exchangeable) positions m the molecule so that they are not lost during extraction or derivatization and in parts of the molecule that are not lost m the neutral fragment if the molecular ion is not to be monitored. In addition the deutermm-labelled compound should be a single isotopomer, rather than a mixture of several and should, of course, have a very low concentration of the proteo compound. A wide variety of stable isotope-labeled neurotransmrtters, precursors, and metabolites are now available commercially (e.g., Merck, Sharpe and Dohme Canada, Pt. Claire, Quebec, Can., KOR Isotopes, Cambridge, MA). Synthetic procedures are described for the compounds as follows: PE-d2 (Durden et al., 1973), PE-d4 (Philips and Boulton, 1979; Davis and Boulton, 1980), m-TA-dz (Philips et al , 1975), p-TA-d2 (Philips et al., 1974a), p-TA-d4 (Phil ups and Boulton, 1979; Saraswat et al., 1981), T-d;! (Philips et al., 1974b), T-d5 (Raisanen and Karkkainen, 1979), A-d3 (Juorio and Durden, 1977; Muskiet et al., 1978e; Miyazaki and Hashimoto, 1982), NA-da (Murphy, 1975; Muskiet et al., 1978e, Saraswat et al., 1981; Mryazaki and Hashimoto, 1982), NA-dh and d7 (Kalir et al , 1977), DA-d2 (Perel et al., 1972, Miyazaki and Hashimoto, 1982), DA-da (Lindstrom et al., 1974; Ko et al., 1974, Saraswat et al., 1981), 5-HT-d4 (Beck and Sedvall, 1975, Shaw et al., 1976, Raisanen and Karkkainen, 1979), o-, m-, p-OA-d3 (Durden et al , 1980, Couch et al., 1983), PAA-d:! and d5 (Davis et al., 1982), m-HPAA-d2 (Davis et al , 1982), m-HPAA-d5 (Durden and Boulton, 1981), p-HPAA-d2 (Davis et al., 1982), p-HPAA-d4 (Karoum et al., 197513; Durden and Boulton, 1981), IAA-d6 (Musklet et al , 1978e), IAA-d4 and d5 (Magnus et al., 1980), DOPAC-da (Muskiet et al., 1978e), DOPAC-dS (Karoum et al., 197513;Gordon et al., 1976; Saraswat et al., 1981), HVA-d2 and d5 (Lindstrom et al., 1974), HVA-ds (Sjoquist and Anggard, 1972; Fri et al., 1974a, Gordon et al., 1974, Muskiet et al., 1978e; Markey et al., 1980; Saraswat et al., 1981), VMA-d2 (Lindstrom et al., 1974), VMA-d3 (Gordon et al., 1974; Karoum et al., 1975b; Faull et al., 1981); o-, m-, p-HMA-d3 (Midgley et al., 1979; Davis and Boulton, 1981; Couch et al., 1983), p-HMA-dz (Karoum et al., 1975b), 5-HIAA-d2 (Beck and Sedvall, 1975), 5-HIAA-d5 (Muskiet et al., 1978e); MHPG-d3 (Lmdstrom et al , 1974; Gordon et al., 1974; Karoum et al , 1975b) and MHPG-d6 (Murray et al., 1981) A second choice of internal standard would be a homolog or structural isomer, although these tend to have significantly different physical and chemical properties. Fmally a few researchers

340

Durden

have used radiolabeled isotopomers (e.g., Lhuguenot Maume, 1980), but this is not recommended as a general dure as the mass spectrometer becomes contaminated radioactivity.

and procewith

3. Derivatives of Amines and Metabolites The analysis of neurotransmitter ammes and their metabolites by MS procedures requires that derivatives be made since most of the parent compounds are only slightly volatile and have unsuitable spectra. Derivatization improves the speclficlty and sensmvity of the method by making the compounds more amenable to isolation via extraction and chromatography, by changing their volatilities and by forming compounds with spectra suitable for MS analysis. Suitable spectra are those that preferably exhibit an intense molecular ion or alternatively form fragments which contam all of the structure of the origmal compounds. If fragments are to be monitored, more precise results are obtained if the site of fragmentation is not adlacent to the site of deuterium atom labelling so that deutermm atoms are not lost through rearrangement. Ions of mass greater than m/z about 200 are preferred as the background spectrum of the mass spectrometer due to the high vacuum diffusion pump 011s and due to GC column bleed is lower. Finally for HRMS it is preferable that the derivative contam mass deficient elements, such as fluorme (m/z 18 998402), silicon (m/z 27.97693), phosphorus (m/z 30 973704) or sulfur (m/z 31.972073) instead of carbon, hydrogen, nitrogen and oxygen so that the resultant ions to be momtored have exact m/z values lower than those of hydrocarbons and are thus easily resolved. On the other hand, since fluormated compounds, perfluoro kerosene or perfluoro tri-n-butylamme, which are very mass deficient, are used as mass reference compounds, the derivative should not contain a large number of fluorine atoms Derivatives which contam chlorme or bromine, which have two abundant isotopes (35C1 and 37C1 or 7yBr and ‘*Br), are not normally used as the higher mass isotope may interfere with the use of the stable isotopelabeled mternal standard Other factors mfluencmg the choice of a derivative are that it should have good chromatographic properties and perhaps be specific for certain functional groups. GC chromatographic properties would include producing well-separated, narrow, nontailmg peaks on the low bleed liquid phases used for GC-MS (1.e , primarily the polysiloxanes, such as SE-30, OV-1, OV-17, SE-54),

Metastable

Mass Spectrometly

341

and sufficient volatlllty so that high GC temperatures are not requu-ed For TLC, a physlochemlcal property that enables the compound to be visualized without use of a spray reagent, i e , fluorescence under UV or color, 1sa necessity Most of the denvatives chosen fit these requirements

3.1. Derivatives for Amines 3.1.1. Derivatwes

for GC-MS

Many derlvatlves have been proposed for the analysis of blogenlc ammes by GC-MS These include the trimethylsllyls (TMS) (Abramson et al., 1974; Hattox and Murphy, 1978), the fluonnated acyls, trifluoroacetic (TFA), pentafluoroproplomc (PFP) and heptafluorobutyrlc (HFB) (Angaard and Sedvall, 1969, Karoum et al , 1972); dmltrophenyl (DNP) (Edwards et al., 1979a); lsothlocyanate (NCS) (Naraslmhachari and Vouros, 1972, Suzuki and Hatton, 1983); pentafluorobenzylimmo (PFBI) (Moffat and Hornmg, 1970, Lhuguenot and Maume, 1974) and the flophemsyl derivatives (Francis et al , 1978) Spectra of some of these derivatives of phenylethylamme are shown m Fig 2 The PFP (or HFB) derlvatlves are popular, pnmanly for their ease of preparation, but none of the ammes exhibit any slgmflcantly intense molecular ions Catecholammes, such as DA, exhibit fragment ions with reasonable W/Z values (428 for DA), but the trace amines, such as PE, fragment to give low mass and relatively nonspeclflc ions (e.g , TX/Z 91 1s C7H7’ and may come from many aromatics m tissues and physlologlcal fluids). The TMS derivatives again do not exhibit slgmflcant M ’ ion mtensltles and do not appear to give the required sensltlvlty. Under EI condltlons, the DNP-derivative does not give a large M ’ ion (Fig 2), but under CI gives intense pseudomolecular ions that have been used for analysis of PE, PEOH, nz-TA and p-TA (Edwards et al , 1979) The lsothlocyanate derivative produces compounds of relatively low mass, but with more intense Mm’ ions (Naraslmhachan and Vouros, 1972) and has been used for PE analysis (Suzuki and Hatton, 1983). Due to the presence of the sulfur atom, it 1s mass deficient when compared to hydrocarbons and would be a good candidate for HRMS analysis. Finally, the PFBI derivative of the amme m conlunctlon with the TMS of the phenollc group appears to be suitable for analysis of the catecholamines and tyramines (Lhuguenot and Maume, 1974, 1980) Again the molecular ion intensity 1s small, and since the major fragmentation IS via loss of CH2N=CHChF5, only low mass,

342

Durden

F% 2 Mass spectra of some GC denvatlves of PE b) 2,kdmrtrophenyl, c) pentafluoroproplonyl, isothrocyanate, pentafluorobenzylrmme. nonspecrfrc as PE.

ions are observed

3.1.2. Denvatwes

m the spectra of trace ammes

a) d)

such

for TLC-MS

Vu-tually all of the very sensmve derwatrves that have been proposed for analysis of ammes using TLC are fluorescent (see re-

view by Seiler, 1977), as they are visualized to much lower levels (l-10 been

ng) than are colored derrvatrves The reagents that have used for MS quantrtation of biogenic ammes include

Metastable

Mass Spectrometry

343

4-chloro-7-nitro-benzo-[cl-1,2,5-oxadrazole (NBD) (Relsch et al , 1970) and the fluorescent sulfonyl chlorides, l-drmethylammonaphthalene-5-sulfonyl chloride (dansyl chloride) (Seiler, 1971, Durden et al , 1973), 1-drbutylammonaphthalene-5-sulfonyl chlorrde (bansyl chlorrde) (Seiler et al., 1973, Lehman et al , 1976), and 1-dipropylaminonaphthalene-5-sulfonyl chloride (propansyl chlorrde) (Davrs, 1979). Some spectra of fluorescent derivatives of PE are shown m Fig. 3 Although fluorescamine 1s used extensively for lrqurd chromatography of ammes, we found rt fragmented extensively m the mass spectrometer and was not suita-

Fig. 3

Mass spectra of some fluorescent denvatlves of PE. a)

nltro-benzo-oxadloazole

(NBD),

b) fluorescamme,

c)dansyl,

d) bansyl

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Durden

ble for analysis of trace ammes The NBD derivative exhibits a more intense molecular ion and this is more suitable We have found the dansyl derivatives of the monofunctional trace ammes such as PE or tryptamme very useful as they exhibit very intense molecular ions while the ammes with several functional groups also exhibit sigmficantly intense molecular ions (Durden et al , 1974) The reagent (Dansyl-Cl) is readily available and the denvatives are easily prepared under mild alkali conditions The bansyl and propansyl derivatives exhibit, m their spectra, intense fragment ions, (M-43 and M-29 respectively) which may be used to improve the sensitivity of analysis (Davis, 1979). Unfortunately the reagents, bansyl chloride and propansyl chloride, are less stable than dansyl chloride and are not commercially available. Thus, u-r this laboratory we routmely use the dansyl derivative of the amines. In order to increase the sensitivity for analysis of multifunctional ammes, the dansyl group may be used m a mixed derivative, i e., with alkylation (methyl, ethyl or propyl) of phenolic groups (Davis, 1979) or with acetylation of B-hydroxylphenylalkylamines (Durden et al , 1980). Besides providing an mtense molecular ion for selected ion momtormg, the dansyl derivatives contam sulfur atoms that make them somewhat mass deficient and easily resolvable from background material at MS resolution settmgs of 5,000 and 10,000 (Durden et al , 1974). Finally, the use of TLC to isolate and purify the ammes increases the efficiency of use of the time of the HRMS.

3.2. Derivatives of Acids The analysis of the acid metabolites of the trace and catecholamines have been accomplished primarily through use of volatile fluorinated derivatives, 1 e., trifluoroethyl (TFE), pentafluoro-n-propyl (PFnP) hexafluoroisopropyl (HFIP) or pentafluorobenzoyl (PFB) esters of the carboxylic acid, with the phenolic group acylated as the pentafluoropropionyl (PFP) or heptafluorobutyryl (HFB) derivative. These derivatives are easily prepared as the fluorinated acyl anhydride acts as a catalyst m the esterfication of the carboxyl group with the appropriate alcohol Methyl (Me) esters, with PFP or HFB acyls have also proven to be useful for the catecholic and indolyl acids, but require a two step derivatization, 1.e , methylation with diazomethane or methanolic HCl followed by acylation with the anhydnde. The TMS derivative has been used, but appears to be much less sensitive. A homolog, the t-butyl dimethylsilyl (TBDMS), derivative appears to have more suitable fragmentation

Me&stab/e

Mass Spectrometry

345

The TFE ester (with the PFP acyl) usually produces compounds whose spectra exhibit intense molecular ions (e.g., PAA, Durden and Boulton, 1982a) as is shown m Fig. 4. The derrvatives are somewhat mass defrcrent due to the presence of fluorme, and their ions are conveniently separated from other background ions at about 5000 resolutron. The next homolog m the series, the PFnP ester, has had wider use and also produces derivatives that exhibit intense molecular ions, as was shown in the spectra of the

Fig

tnmethylsllyl, pentafluoro

4

spectra of some GC-MS derlvatlves b) tnfluoroethylester, c) pentafluoro-n-propyl benzyl ester Mass

of PAA: a) ester, d)

346

Durden

following acids: DOPAC and HVA (Wiesel et al , 1974, Fri et al., 1974a, Gordon et al., 1976, Girault et al., 1980), PAA (Martin et al., 1979, Durden and Boulton, 1982a) and 5-HIAA (Fri et al., 1974b, Beck et al , 1977, Faull et al., 1979) In our use of this derivative for analysis of PAA, we experienced problems m momtormg the molecular ion at nz/z 268 due to bleed from the OV-1 coated capillary column, and we presently use the TFE derivative as described below. The HFIP-TFA derivatives of VMA, HVA and isoHVA also exhibit high yields of molecular ions (Takahashi et al , 197813) The PFB derivative has proven useful for the analysis of PAA (Fellows et al , 1978), but when combined with PFP or HFB for the multifunctional acids, produces less volatile derivatives, that require considerably higher GC temperatures for elution. The methyl esters (Me-TFA, Me-PFP and Me-HFB derivatives) have been most widely used for the acid metabohtes of the catecholammes. Although the molecular ion is reasonably intense with these derivatives [as has been reported for the followmg acids* m-HPAA and P-HPAA (Durden and Boulton, 1981), DOPAC (Karoum et al., 1975b), HVA (Sloquist et al., 1973; Fri et al., 1974a), IAA (Warsh et al., 1977a, Artigas and Gelpi, 1979), 5-HIAA (Artigas and Gelpi, 1979)], fragment ions are frequently used for increased sensitivity. The Me ester is not suitable for PAA and hence cannot be used if PAA is to be determined with the other metabohtes m a smgle GC-MS analysis Although the TMS derivatives of acids are easily prepared, they appear to fragment extensively under EI with low densities being recorded for the Mf or (M-15)+ ions (Hattox and Murphy, 1978). The TBDMS derivatives have much more useful spectra as the t-butyl group is readily lost to produce intense (M-57)+ ions (deJong et al , 1980), but they do not appear to have been used for neurochemical analyses.

3.3. Derivatives

of Alcohols

The alcohol metabolites of the biogemc ammes are most conveniently determined by GC-MS m conluncuon with the acids since the perfluoroacyl derivatives are formed by the same reaction mixtures The PFP and TFA derivatives have been used most for the catecholamme and trace acid alcohol metabohtes (Gordon et al , 1974; Karoum et al , 1975a,b, Takahashi et al , 1977) The molecular ion is relatively intense, but fragment ions due to alkyl chain cleavage are usually momtored so that the ions detected are m the same mass range as the ions of the acids Unfortunately, the fragment ion of the internal standard deutermm isotopomer

Metastable

Mass Spectrometry

347

may lose deuterlum atoms if it is labeled in the alkyl carbon chain. The TMS derivative has been used (Muskiet et al., 197&z), but does not appear to give the sensltlvlty of the PFP derlvatlve due to the low yield of molecular or (M-15) ions (Hattox and Murphy, 1978).

4. Protocols 4.1. Amines by TLC-HRMS-SIM The followmg procedures are based upon methods previously reported by this laboratory for the analysis of trace ammes in tissues (Durden et al , 1973; Boulton et al., 1973; Philips et al , 1974a, 1974b, 1975; Durden, 1978; Durden and Boulton, 1979; Durden and Phlhps, 1980) and m urine (Slingsby and Boulton, 1976; Huebert and Boulton, 1979), for P-hydroxy amines m tissue and physiological fluids (Durden et al., 1978; Durden et al , 1980) and for catecholamines (Juono and Durden, 1977). 4.1.1. Direct

Dansylatlon

of Trace Amlnes

For the analysis of PE, m-TA, p-TA and T m small amounts of tlssue (up to 200 mg), or m physiological fluids, 1 mL of CSF or plasma, 0.5 mL urine, the fluid or homogenization medium is dansylated without preseparatlon of the amines. The tissue homogenate (m 2 mL 0.05N HC104) or aliquot of fluid IS transferred to 16 x 100 mm glass culture test tubes, and ahquots of stock solutions containmg 25 ng each of the tetradeutero-PE, m-TA, p-TA and T internal standards are added. A mixture of Na2C03 and NaHC03 (1:l w/w), approximately 300 mg, is added to the homogenate. This usually dissolves completely Two mL of acetone containing 20 mg dansyl chloride are then added with mixing (Vortex mixer). Some of the carbonate will precipitate m the presence of acetone. The tubes are sealed and left overnight m the dark at room temperature to permit the reaction to go to completion and excess dansyl chloride to hydrolyze. Next day, the tubes are centrifuged at low speed (1600 rpm) m a clinical centrifuge and the supernatants transferred to a second set of 16 x 100 mm test tubes. The supernatant 1s extracted twice with 2 mL hexane and the organic layer saved m test tubes. The tubes are then placed in a water bath at 4045°C and the organic layer evaporated m a stream of nitrogen. The dansyl derivatives are dissolved m a few drops (100-200 pL) of the mixture acetone-toluene (1:l v/v) and applied to the silica gel layers (Merck silica gel 60, 20 x 20

348

Durden

cm, 0.25 mm thick, No. 5763, Brmkmann Instruments, Rexdale, Ontario, Can.) m a 2 cm streak Six samples plus a concentrated dansyl standard may be accommodated on one plate. After development m the first solvent system (toluene-ethyl acetate, 5.2), the zones are located under long-wave UV light, and scraped into test tubes and extracted twice with acetone-methanol (1.1 v/v). The samples are transferred to new tubes in which the organic layer is again removed under a nitrogen stream and the dansyl derivative redissolved in acetone-toluene. The samples are then spotted on the second TL plate and developed with a second solvent system [I’E, toluene-triethylamme, 5.1 (v/v); IPTA, p-TA, toluene-triethylamine-methanol, 50.5 1 (v/v/v), T, toluene-triethyl ammemethanol, 50:5~7 (v/v/v)] The zones are again visualized under UV light and circumscribed with a metal stylus. The silica gel is carefully powdered m situ and then sucked mto one-half of a melting point capillary constricted midlength to hold a small piece (1 mm X 5 mm) of glass fiber filter paper (Whatman GFA) The dansyl amme is then eluted from the silica by drawing 25 FL of ethyl acetate through the silica and the capillary is sealed with hematocrit clay so that there is an air gap adlacent to the ethyl acetate (Philips et al., 1974a) Samples are stored m a freezer (-20°C) until analyzed by mass spectrometry Minor variations on this procedure are used by different researchers m this laboratory, tissue may be homogenized m 0 1M-ICl containing 5 ng/mL ascorbic acid and 1 mg/mL EDTA, the reaction catalyzed by Na2C03 (150mg) alone and the dansyl ammes extracted from the reaction mixture with toluene-ethyl acetate (9 1 v/v). For safety reasons, benzene is no longer used for this step, but is still used for chromatography, which is now done m a fume hood. In all cases a set of blanks containing the deutero internal standards m the homogemzmg medium, and a set of “checks,” that contam known amounts of proteoammes along with the internal standards (in the homogenlzmg medium) are taken through the entire procedure with each batch of samples The checks from several batches of samples are used to prepare a standard curve from which the isotopic purity factors of the mternal standards are calculated. All solvents for the procedure are of highest chemical purity, usually HPLC grade. Water is prepurlfied by reverse osmosis (Barnstead RO pure 40, SybroniBarnstead, Boston, MA) and then either distilled from glass or purified using a cartridge system (Barnstead NAN0 pure) capable of providing “HPLC” grade water.

Metastable 4.12.

Mass Spectrometry

Preseparation

349

of Trace Amlnes

Larger amounts of tissue or urine may be used for determinations of trace amines if it IS desired to work with lower MS sensitivity, 1.e , larger MS ion currents to obtain greater precision of analysis The sample must be partially purified by ion exchange chromatography before preparation of the dansyl derlvatlves Blo-Rad AG 50 W-X2 ion exchange resin (about 500 g) 1s cleansed of amines by the procedure of Kaklmoto and Armstrong (1962) by washmg with dilute HCI, acetone, dilute ammonia, water, dilute HCl and water until blanks, run through the TLC-HRMS procedure, are less than 0 5 ngig resin. For urine analysis, 10 mL m the case of PE or 1 mL for MPTA, p-TA and T, allquots are diluted to 15 mL with water that contains the internal standards (200 ng PE-d4, or 200 ng m-TA-d4, 1,000 ng p-TA-d4 and 500 ng T-de) and the pH 1s adjusted to between 6.8 and 7.2 with 2iV NaOH (urine samples are acidic as they are usually collected in polyethylene bottles contaming 10 mL concentrated HCI and frozen until analyzed) The sample 1s then percolated through a chromatography column contammg a 6 x 1 cm bed of the AG 50 W-X2 resin and washed with 10 mL distilled water, 10 mL O.lM sodium acetate and 10 mL distilled water. M.I-TA, p-TA and T are eluted with 10 mL 1M NH40H in 65% ethanol. PE, in a separate analysis, IS eluted with 10 mL methanol-hydrochloric acid (73.27). The extracts are dried under reduced pressure at 4O”C, redissolved m 1 mL 10% Na2C03, reacted with dansyl chloride in acetone, and separated as described m the above procedure The zones containing the dansyl ammes are scraped from the TLC plate into the capillary tubes, as described above. After extraction with ethyl acetate, the extract is transferred to a small vial and the volume increased to 100-200 PL before MS analysis Only 5 PL of the extract 1s used for the MS analysis to prevent overloading of the instrument. Extraction of the dansyl ammes into the capillary tubes and then transferring to the vials is preferable to extracting the silica gel with a large volume of ethyl acetate (and then removmg the slhca by centrlfugatlon), as the latter procedure permits large amounts of silica to remam m the sample and this causes the sample to decompose during MS analysis (Durden et al , 1973) If it 1s desired to determine the trace ammes m large quantities of tissue (e g., whole rat brain, heart, kidney, liver and spleen), the ion exchange step again must be used. The tissues are homogemzed m 30 mL 0.4&I HC104 to which is added 25 ng of the internal standard The suspension is well mixed and then cen-

350

Durden

trrfuged at 12,OOOg for 10 min The supernatant 1s decanted, and Trrton X-100 detergent (J.T Baker Chemical Co , Phllhpsburg, N.J.) is added to a concentratron of 0 05% (v/v) to prevent bmdmg of the lrpophrlrc ammes I’E or T (Durden et al , 1973; Phllrps et al , 1974b). The pH IS then adlusted to 7 and the solution percolated through a bed (3 cm x 1 cm) of resin washed with water, 0 1M sodium acetate and water, as described above The ammes are eluted, dansylated, and isolated as described m 4 1 1 Since only 25 ng of internal standard 1s used, the final extracts m the caprllary tube are not diluted before MS analysis 4.1.3. Analysis of p-Hydroxy Amlnes The B-hydroxy trace ammes, PEOH, VZ-OA, r?-OA, wSYN and p-SYN, are d e t ermmed as then dansyl-acetyl derrvatrves since the spectra of the dansyl derrvatrves contam srgmfrcant mtensmes of (M-H,)’ ions that cause problems of quantrtatlon using internal standard labelled with deutermm m the alkyl chain For the analysis of PEOH or nz-OA and p-OA together or nz-SYN and y-SYN together, i.e , m- and p-isomers not separated, the followmg procedure may be followed. After drssectron and weighing, tissues (up to 200 mg) are homogenized m 1 mL O.lM HCl contammg ascorbic acid (5 mg/mL) and EDTA (1 mg/mL) to which IS added 25 ng of the trrdeutero internal standard (PEOH-d3, p-OA-d3, or p-SYN-d3) The homogenate IS dansylated overnight as above with 16-20 mg dansyl chloride in 2 mL acetone and the solutron 1s saturated wrth sodium carbonate. Next day, the excess sodmm carbonate is precrpstated by the addition of 2 mL toluene-ethyl acetate (9 1, v/v) and the organic layer IS transferred to a new test tube and evaporated to dryness under a stream of nitrogen m a water bath at about 40°C. The extract 1s redrssolved m 2 mL ethyl acetate-pyndine-acetic anhydrrde (redrstrlled before use) (10 1.1, v/v) and the acylatron reaction allowed to proceed for one h at room temperature, again m the dark The mixture IS then evaporated to dryness under a stream of nitrogen, redrssolved m 200 PL toluene and applied to the first srhca gel TLC plate SIX samples plus a chromatogram reference can be accommodated on one 20 X 20 cm plate The plate IS developed m the solvent system chloroform-ethyl acetate 6:l (v/v) The zones contammg the dansyl acetyl derrvatrves are scraped from the plate and drssolved m ethyl acetate (0.2-O 5 mL). After centrrfugatlon (1600 rpm m a clmlcal centrifuge, 5 mm) the organic layer 1s transferred to a new test tube, dried under nitrogen and the amme redrssolved m 02

Metastable Mass Spectrometry

351

mL toluene and applied to a second plate. This plate IS developed in one of three solvent systems depending upon the amme under analysis; PEOH, carbon tetrachlonde-triethylamine 10.1 (v/v); octopamines, chloroform-n-butyl acetate 5:2 (v/v) or synephrmes, carbon tetrachlonde-triethylamine-methanol 10:2:1 (v/v). After development, the zones are located under UV at 254 mm, and the amines are eluted in the capillary tubes, as described above. Blank values, and hence sensitivity of analysis, of synephrmes may be improved by use of a third chromatogram using the solvent system carbon tetrachlonde-triethylamine 5.1 (v/v). As usual, blanks and standards are carried throughout the procedure. This method may be used to measure “total” octopamme or “total” synephrme usmg the pava lsotopomer internal standard and is appropriately used m tissues in which the mefa concentration may be expected to be extremely low. Since it was found possible to separate the m- and p-Isomers of OA and SYN only as their bls-dansyl derivatives, the above procedure may be modified as follows. The method 1s followed to the point prior to acetylahon. The toluene-ethyl acetate solution is evaporated to dryness, the amines are redissolved m toluene and applied to a thin layer plate developed m the solvent system chloroform-butyl acetate (5:3 v/v). The two zones, corresponding to OA and SYN, are scraped into separate test tubes, eluted (toluene-ethyl acetate 9:l v/v), evaporated, redissolved m toluene and applied to separate thin layer plates. The plates are developed in the system benzene-triethylamine (5:2 v/v) that is capable of separating the m- and p-isomers. The zones, now totaling four, are scraped into separate test tubes, eluted, and acylated with 1 mL of ethyl acetate-pyndine-acetic anhydride as above. The bisdansyl-acetyl octopamines and synephrines are separated from other reaction products by a third TLC run using the system chloroform-ethyl acetate (6:1, v/v). It 1s important to verify the resolving power of the TLC methods for m-OA vs p-OA and m-SYN vs p-SYN by using mixtures of p-OA with m-OA-d3, m-OA with p-OA-d3, p-SYN with m-SYN-d3 and m-SYN with p-SYN-d3 (25 ng of each amine). Cross-contamination is usually less than 5%. 4.1.4. Catecholamines

The catecholammes A (Juorio and Durden, 1977), NA and DA (Dewar, Durden and Dyck, unpublished) may also be determined mass spectrometrlcally as their tns-dansyl derivatives. The procedure is especially useful for followmg metabolic pathways using

Durden

352

deuterlum labeled precursors since, unlike the GC MS methods in which fragment ions are monitored, it 1s used to monitor molecular ions which contain all the labeled posltlons Tissues, after freezing and weighing, are homogenized in O.lM HCl containing EDTA (1 mg/mL) and ascorbic acid (5 mg/mL) and appropriate amounts (100-1000 ng) of the Internal standards (A-d3, NA-d6 or DA-d4) are added and well mixed. The homogenate is saturated with mtrogen gas to remove dissolved oxygen and NaHC03-Na2C03 (1.1 w/w) 1sadded. The homogenate may be frozen and thawed twice to fracture any nonhomogenized cells. Dansyl chloride (16-20 mg) m two volumes of acetone 1s then added and the reaction allowed to go to completion overmght. The ammes are extracted and chromatographed as above using the followmg solvent systems. A, NA and DA, solvent I, chloroform-butyl acetate (5.2, v/v), solvent II, benzene-triethylamme (12:1, v/v) (Juorlo and Durden, 1977); DA, solvent I, benzene-cyclohexane-methanol (17.2:1, v/v/v), solvent II, cyclohexane-ethyl acetate (2.3, v/v) The tns-dansyl ammes are eluted from the silica gel in capillary tubes, as above. The addition of EDTA and ascorbic acid and saturation of the solution with nitrogen gas 1s essential to reduce oxldatlon of the catecholamines under the alkali reaction condltlons. Addition of more dansyl chloride (4-5 mg) after one or two h may be used to promote the formation of the tns-dansyl derivatives. Although NA may be determined as the tns-dansyl, the tns-dansyl-acetyl derlvatlve gives a somewhat better signal (Durden et al., 1980). Thin-layer chromatographlc solvent systems and Rf values for the amme dansyl derivatives are given m Table 1. 4. I .5. HRMS-SIM

Procedure

The ammes extracted from the TLC are quantltated using SIM by integrating the signals, usually from the molecular ions, due to the proteo and deutero compounds as they evaporate from the direct probe during a period of 30-60 s The signal usually vanes m an approximately Gausslan manner and the areas are proportional to the amounts evaporated (Jenkins and Maler, 1967). The mass spectrometer 1s adjusted at a relatively high resolution, 7,000-10,000, (Durden et al., 1974) to detect ions of specific elemental compositions (e g., C20H22N202S m/z 354 1402 for dansyl-PE and C20H18N202SD4 m/z 358 for dansyl-PE- d4) using a mass reference ion from a compound such as perfluorokerosme (PFK) or perfluoro-tn-n-butylamme (PFTBA) which IS admitted at constant partial pressure through a second inlet. See Table 2 for elemental composltlon and precise mass values. The high resolu-

carbon tetrachlonde. 10 1 carbon tetrachlonde 10 1

#DNS- I-Dlmethylammonapthalene-5-sulfonyl ‘AC Acetyl denvatlve of /3-hydroxy

Tns-DNS-AC-A

Tns-DNS-Ac-NA

Tns-DNS-NA Tns-DNS-A DNS-AC”-PEOH Bls-DNS-AC-~, p-OA Bls-DNS-AC-m,p-SYN

toluene tnethylamme, 5I1 toluene tnethylamme methanol, toluene tnethylamme methanol, toluene. tnethylamme methanol, benzene tnethylamme, 5 2 benzene tnethylamme, 5 2 benzene tnethylamme, 5 2 benzene tnethylamme, 5 2 cyclohexane ethyl acetate, 2 3

2

of Ammes

derivative of amme or phenol

carbon tetrachlonde . tnethylamme methanol, 10 2 1

0 03

67 49 55 55 20 22 35 39 45

Solvent

Derwatwes

tnethylamme,

0 0 0 0 0

0 0 0 0 0 0 0 0 0

X,

Dansyl

tnethylamme,

TABLE 1 for Isolatmg

67 benzene. tnethylamme, 12 : 1 53 benzene trlethylamme, 12 1 30 carbon tetrachlonde tnethylamme, 20 chloroform butyl acetate, 5.2 37 carbon tetrachlonde tnethylamme methanol, 10 2 1 0 04 chloroform ethyl acetate, 2 1

toluene ethyl acetate, 5 2 toluene ethyl acetate, 5 2 toluene ethyl acetate, 5 2 toluene ethyl acetate, 5 2 chloroform butyl acetate, 5 3 chloroform butyl acetate, 5 3 chloroform butyl acetate, 5 3 chloroform butyl acetate, 5 3 benzene cyclohexane methanol 17 2 1 chloroform. butyl acetate, 5 2 chloroform butyl acetate, 5-2 chloroform.ethyl acetate, 6 1 chloroform ethyl acetate, 6 1 chloroform ethyl acetate, 6 1

DNS”-PE DNS-T Bls-DNS-m-TA Bls-DNS-p-TA Bls-DNS-m-OA Bls-DNS-p-OA Bls-DNS-m-SYN Bls-DNS-p-SYN Tns-DNS-DA

1

and Rf Values

Solvent

Systems

Amme

Solvent

10 1

50 5 7 50 5 1 50 5 1

53 38 42 38 18 22 44 48 68

0 48

0 58

0.03 0 24 0 02 0 24 0 61

0 0 0 0 0 0 0 0 0

Rf

‘nm. not measured

Tns-DNS-AC-A

Tns-DNS-Ac-NA

Bls-DNS-AC-m,p-SYN

Bls-DNS-AC-m,p-OA

DNS-AC-PEOH

Tns-DNS-A

Tns-DNS-NA

Tns-DNS-DA

Bls-DNS-m,p-TA

DNS-T

DNS-PE

Compound

Mass

h de h 4 h 4 h & h 4 h 4 h 4 h d3 h d3 h d6 h 4

Isotopomer

Spectrometrlc

TABLE 2 for the Analysis

G&3sW%W6 C&&G& C&&b%bD3 CdW’J204S CA-~IN~O.W~ GA&O,% CMH&‘J~O&D~ G&N&S2 GdW%W2D~ C4&4&010% &&oN4010%& C&4&101oS3 ~&42N4O,oW3

G&J%OYS~

C32H@@& G&GJ3OsS2D~ G&&J4Q& G~H~oN~Q&D~

G~HIYWWD~

CzoJLN2W CZOHIRN~WD~ CzzHd’J@zS

Composition

Condltlons

354 358 393 397 603 607 852 856 868 874 882 885 412 415 661 664 675 678 910 916 924 927

1402 1653 1511 1762 1861 2113 2321 2572 2270 2647 2427 2614 1457 1645 1916 2104 2073 2261 2376 2753 2532 2720

Mass

of Ammes

nm”

nm”

02

02

02

nm”

02

01

01

01

Mm practical detectable amount (ng)

by HRMS-SIM

300

300

270

250

300

300

300

280

260

250

Evaporation temperature

Metastable

Mass Spectrometry

355

tion mode of the VG digital multiple ion detector (DIGMID) is set to the ratios of the three masses (to ppm accuracy), with the lowest mass (e.g , m/z 354.1402) at ratio 1.000000 and the higher masses at ratios greater than 1.0 (e.g., 358.1967, ratio 1 011366 and m/z 363.9807 ratio 1.027787), and controls the accelerating and electric sector potentials. The magnetic field is adjusted so that the mass reference ion IS centered in the oscilloscope display and the peak width span adlusted so that the peak fills the screen (i.e , span = 100 ppm for 10,000 resolution, 140 ppm for 7000 resolution) The DIGMID is thus operating as a multiple channel peak matching device For metabolic studies m which deuteriumlabeled precursors are used, the ratios correspondmg to the m/z values of the expected elemental compositions of the amme metabolites may be placed in additional channels (up to eight total) that may be monitored sequentially. For simple quantitation, with three channel operation, the peak scan time is set to 0 5 s so that 60-120 peaks are recorded as the sample evaporates from the probe. If more than three channels are used, the scan time is reduced to 0.2 s. The sample probe for this work is a modified version of the MS9 design. It is of fixed length and constructed to hold a short length of 1 8 mm capillary tubing so that the tip will be m close proximity to the electron beam to provide maximum ionization of the sample and maximum sensitivity (Durden et al., 1974). The capillary tube is constricted about 5 mm from the tip to form a cup with a volume of 7-10 PL and is held mto the probe tip high-voltage msulator by a short spring, which permits some flexibility and prevents the tip from lammmg inside the ion source. These probe tips are discarded after each batch of 20 or 30 samples and whenever the evaporation profiles become nonas the latter indicates that silica gel has been Gaussian, transferred to the probe tip An aliquot of the sample, usually 5 pL, is transferred to the probe tip and the solvent evaporated on a a small heater. The direct probe is then passed mto the MS vacuum housing and the mass reference Ion recentered The probe IS pushed fully mto the ion source and the signals due to the proteo and deutero compound are recorded on the UV oscillographic recorder and by the VG data system (PDP8E based) controlled by one of several programs written either m machme code (Durden, unpublished) or m BASIC (Durden, 1978) The program mtegrates the two signals, subtracts the baseline signal, corrects for contributions due to the presence of 13C, 180, 5N, 04S isotopes and calculates the number of ng of proteo amme m the sample (Durden, 1978)

356

Durden

Because the samples are well purified and the MS ion source remains quite clean, the background signal at high resolution is very small and remains constant as a large number of samples are processed. In this laboratory, two mass spectrometers are used for these analyses* an AEI (Kratos) MS-902s with a VG ZAB electronics console update, and a VG 70-70F (VG Analytical Ltd.). The MS-9 gives better results for the TLC-HRMS-SIM analyses as It 1s easier to obtain the necessary resolution and the ion source appears to give the more Gaussian shaped evaporation profiles. The 70-70F on the other hand 1s more suitable for GC-MS analyses by GCHRMS-SIM or GC-MS-SMPM as the magnetic field can be stepped more rapidly and precisely.

4.2. Acids and Alcohols by GC-MS 4.2.1 Extraction and Derwatization After dissection, freezing on dry ice and weighing, tissues are homogemzed m 2 5 mL ZnS04 (O.lM). Known quantities of the mternal standard (20-500 ng, approximately equal to the amount expected) are added and thoroughly mixed These are usually PAA-d2, m-HPAA-d2 or d4 (Durden and Boulton, 1981, 1982), but may also include DOPAC-d5, HVA-d3, VMA-d3, m-HMA-d3, 2 or MHPG-d3 Protein 1s precipip-HMA-d4, IAA-d2, 5-HIAA-d tated by addltlon of 2 5 mL Ba(OH)2 (0 1M) solution and followed by centrlfugatlon at 10,000~ for 15 mm at 4°C The supernatant 1s transferred into 15 x 100 mm glass test tubes, acidified with HCl to obtain a pH between 1 and 2 and extracted twice with 3 mL of ethyl acetate For analysis of body fluid samples (0 5 mL plasma, 0.5 mL csf or 0.2 mL urine, the ZnS04/Ba(OH)2 step IS omitted. The samples are acidified with HCl and extracted mto ethyl acetate. The ethyl acetate solution 1s reduced to about 0.1 mL under a stream of nitrogen and 1s transferred with two 0 1 mL washings to 1.0 mL Reactlvlals (Pierce Chemical Co , Rockford, IL) After addition of 25 PL trlethylamme (pnmanly to reduce losses of PAA or IAA), the solution 1s evaporated to dryness Traces of water are removed azeotroplcally by addition of 100 FL benzene or methanol followed by further evaporation to dryness under a nitrogen stream. These operations are performed m an efficient fume hood. The sample 1s redissolved m 50-100 PL pentafluoroproplomc anhydride (PFPA) and 100 PL 2,2,2-tnfluoroethanol (TFEOH) 1s added. 3,3,3,2,2-Pentafluoro-n-propanol (PFPOH) may be used instead if the PFnP-PFP derivatives are desired. The vials are then heated for 1 h at 80°C m a dry block heater (Pierce,

Met&able

Mass Spectrometry

357

Reactiblock heater). The vials are cooled to about 30°C and the excess reagent 1s removed by reducing the volume to about 20 FL under a stream of nitrogen. A second amount (100 FL) of PFPA is added and the mixture allowed to react for another hour at 80°C. The vials are again cooled and the volume reduced to about 20 PL The mixture is diluted by addition of 100 PL hexane and is shaken with 100 PL phosphate buffer (pH 6) for about 30 s. The hexane layer 1s transferred to a 100 PL Reactivial and the volume reduced to 20 FL Samples of 0.5 FL are then inlected mto the GC-MS for analysis Blanks are prepared by adding the internal standards to ZnS04 solutions, and calibration curves by adding various amounts of the proteo acids to the internal standards in ZnSOd solutions, which are then processed as described above When very small concentrations of nz-HPAA and p-HPAA in small tissue samples are to be determined, the procedure is modified slightly The volumes of the ZnS04 and Ba(OH)2 solution are reduced to 1 mL and only 1.0 or 2.5 ng of the internal standards m-HI’AA-d2 are added. 4.2.2. HRGC-HRMS-S/M

Analysis

GC-MS analysis of the acidic and alcoholic metabohtes is performed using the VG 70-70F or AEI MS902S double focusmg mass spectrometers. Both are equipped with HP 5700 gas chromatographs and capillary columns connected directly to the mass spectrometer ion source with a length of 0.15 mm id fused silica capillary. Two types of columns are normally used, a 50 m SGE-OV-101 SCOT capillary (Mandel Scientific, Rockwood, Ont., Can.) operated with a helium flow of 29 cm/s and a J&W 60 m DB-1 bonded thick film (1 km thickness) wide bore (0.35 mm id) fused silica capillary (Chromatographic Speciahtres, Brockvrlle, Ont., Can.) operated with a helium flow of about 40 cm/s. Since the capillaries are connected directly to the ion source they operate with part of their lengths under partial vacuum. Both columns permit high sample loads and are used with direct mlection inlets. The MS9 system has a Valco YK, m high-temperature valve (Valco Instruments Co., Houston, TX) in line which can be used to direct the flow and close off the MS inlet to reduce column bleed contammatlon of the ion source when the column is not m use. The acid and alcohol metabolites are usually analyzed usmg two GC-MS runs, the trace acids PAA, m-HPAA and ,PHPAA m a short isothermal run with the reference mass ion of PFTBA cho-

358

Durden

sen so that the ratio to PAA M+ Ion is close to 1.0, and the remammg acids and alcohol in a temperature-programmed run using a higher mass reference ion. Typical conditions and the precise masses of the ions are shown m Table 3. The mass chromatograms are recorded using pen charts attached to the eight integrated outputs of the DIGMID and by the data system Mass

Compound Ruil 2” PAA

and Retention Alcohols

Rt (mm s) 2 48

wHPAA

4 50

/Y-HPAA

5 20

Run 2’ m-HMA p-HMA DOPAC

3 50

TABLE 3 Times for Analysis by HRGC-HRMS-SIM”

Isotopomer

218 220 223 253 257 253 257

0555 0680 0868 0288 0539 0288 0539

M+ M’ M+ F’ F+ F+ F+

h 4

415 0028 418 0216 415.0028 419 0279 415 0028 420 0342 445 0134 448 0322 410 0400 413 0589 445 0134 446 0197 403 0454 405 0580 438 0188 440 0314

F+ F+ F’ F’ F’ F’ F’ F+ M’ M’ F’ Fi M’ Mf F+

h

5 53

4 h 4

6 55

HVA

7 37

MHPG

7 49

IAA

11.30

5-HIAA

13 24

“Column

Mol or frag ion

h d2 ds h 4 h 4

4 50

VMA

Mass

of Acids

h 4 h d? h dl h 4 h 6

J and W DBI-30 W, 30 m ska,

F+

0 35 mm id, hellurn

lution 5000 at rniz 219 “Run 1, 150°CIsothermal ‘Run 2, 8 mm at 16O”C,lO”/mm to 2OO”C,4 mm at 200°C

and

Reference ion mass 213 9903 213 9903

Mass ratio 1018995

1028402 213 9903 1 042509 213 9903 1 182431 213 9903 1 201241 213 9903 1182431 213 9903 1201241 1 103788 375 9807 1 111817 375.9807 1 103788 375 9807 1 114493 375.9807

375 375 375 375 375 375 375 375 375 375 375 375

9807 9807 9807 9807 9807 9807 9807 9807 9807 9807 9807 9807

1103788

1 117170 1183607 1191636 1090588 1098617 1183607 1186283

1071984 1077337 1.165003

1170356

flow 40 cm/s MS reso-

Met&able

using

359

Mass Spectrometry

a program

4.2.3. HRGC-SMPM

written Analysis

in OS/8 BASIC

(Durden,

of m-HPAA

and p-HPAA

unpublished).

Subnanogram quantities of nz-HPAA and ,PHPAA may be determined usmg the same derivatives and GC condmons with the mass spectrometer adlusted to detect metastable transitions of m/z 380 to nLz 253 and mlz 382 to mlz 255 of nl-HPAA and p-HPAA, and m-HPAA-d2, and p-HPAA-d2, respectively The VG 70-70F instrument is more suitable for this analysis as the magnetic field can be switched rapidly and precisely. The mass spectrometer resolution is set to 1000 and adlusted in conventional mode of operation to detect U~/Z 380 with the DIGMID controllmg the magnetic field The instrument IS then adlusted to linked B/E metastable mode and the DIGMID controlling voltage reduced so that the metastable transitron m/z 380 to m/z 253 is detected. By switchmg another circuit, the metastable ratro amplifier (Durden, 1982), mto operation the DIGMID can be programmed to detect the second metastable ratio m/z 382 to N/Z 255. The sample is then inlected mto the GC and the signals due to the two transrtions are recorded by SMPM m a manner analogous to conventional SMPM Because of the selectivity obtained by use of the metastable transitions, the mass chromatograms are very clean and the sensitivity is increased by about a factor of 10 over conventional SIM operation (Durden, 1983). The procedure is limited to compounds that provide hrgh yields of metastable ions and at present we have used it only for determmations of IwHPAA and p-HPAA as th en- TFE-PFP derivatives (Durden 1982, 1983)

5. The Case for HRMS and HRGC The use of high resolution mass spectrometry provides two mam advantages over the use of low resolution mass spectrometry. Firstly, the precise mass and hence a specific elemental composition provide an increase m specificity in the analysis of trace amounts of biogemc compounds by excluding the possibility that the recorded signal is due to ions of other elemental compositions. Secondly, because the HRMS can separate ions of the same nommal, but different precise, mass values, the blank values are reduced, enhancing the sensitivity of analysis This was demonstrated recently by examples of analysis of the dansyl derivatives of nz-TA and ,PTA from rat bran-t tissues (Durden, 1984) The blank values for wTA and P-TA at low resolution were 4.2 and 2 6 ng, respectively, whereas at high resolution they were reduced to

360

Durden

140 low tities sue

and 250 pg, respectively. Because of the high blank values, resolution analysis was not capable of determmmg the quanof ammes (with amounts between 1 5 and 4 6 ng) m the tissamples A comparison of low resolution GC-MS to HRGC-HRMS for the analysis of PAA and p-HPAA are also given In both cases, the low resolution analysis gives significantly higher values for PAA and p-HPAA m a CSF sample For PAA it appeared that with a packed column other compounds that produced ions with VI/Z 218.0555 (M+ of TFE-PAA) were not resolved from the PAA derivative by the GC column and thus a capillary column IS required In the analysis of ,u-HPAA, the malor factor affecting the result was the mass spectrometer resolution Thus, to obtain reliable results m the analysis of the acids listed above, it appears that the combmation of capillary column HRGC with HRMS provides the most suitable conditions for all of the compounds

Acknowledgments I would like Division for here and Dr. uscript The Health and

to thank my colleagues m the Psychiatric Research their assistance m discussmg the methods presented A A Boulton for helpful suggestions with this manresearch program is supported by Saskatchewan the Medical Research Council of Canada.

References Abramson F I’ , McCaman M. W , and McCaman R E (1974) Femtomole level of analysis of blogemc ammes and ammo acids using function group mass spectrometry Anal Bmche~rz 51, 482-499 Andersen H and Braestrup C (1977) Mass fragmentographlc demonstration of low amounts of beta-phenylethylamme in human urme Stand 1 cl111 Lab invest 37, 3>37 Anggard E and Sedvall G (1969) Gas chromatography of catecholamme metabolltes using electron capture and mass spectrometry Am1 Chem 41, 1250-1256 Artlgas F and Gelpl E (1979) A new mass fragmentographlc method for the simultaneous analysis of tryptophan, tryptamme, mdole-3acetlc acid, serotonm, and 5-hydroxymdole-3-acetic acid m the same sample of rat brain. Allal Bmhen~. 92, 233-242. Barber M , Bordoll R S , Elliott G J , Sedgwlck R D , and Tyler A N (1982) Fast atom bombardment mass spectrometry Aual Chew 54, 645A-657A Beck 0 and Sedvall G (1975) The synthesis of 5-hydroxymdole-3-(-2-

Metastable

Mass Spectrometly

d,-acetic acrd) and Compd 11, 57-61 Beck 0 , Wiesel, F -A determmation of acetic acid m brain Chromatogr

361

CY,cy’, J3, J3’-d4-5-hydroxytryptamme.

J. Labelled

, and Sedvall G (1977) Mass fragmentographic 5-hydroxytryptamme and 5-hydroxymdole-3tissue using deuterated internal standards. 1

134, 407414

Beck 0 , Borg S , Holmstedt B , and Stabler H (1980) Levels of 5-hydroxy-tryptophol in cerebrospmal fluid from alcoholics determined by gas chromatography-mass spectrometry Blochem Pharmacol

29, 693-696.

Bertilsson L (1973) Quantita&e determmation of 4-hydroxy-3methoxyphenyl-glycol and its conjugates u-t cerebrospinal fluid by mass fragmentography \ Chromatqr 87, 147-153 Bertilsson L and Palmer L (1972) Indole3-acetic acid m human cerebrospinal fluid. Identification and quantification by mass fragmentography Scrence 177, 74-76. Blomberg L., Builten J , Markides K , and Waunman T. (1981) Evaluation of bonded methylsilicone rubber as a stationary phase for glass capillary columns ] Chromatogr 208, 231-238 BouIton A A , Philips S R , and Durden D A (1973) The analysis of certain ammes m tissues and bodily fluids as their dansyl derrvatives J Chromatugr 82, 137-142 Boyd R K and Beynon J H (1977) Scanning of sector mass spectrometers to observe the fragmentations of metastable ions Or8 Mass Spectrom

12, 163-165

Buck S H., Murphy R C , and Molmoff P B. (1977) The normal occurrence of octopamme m the central nervous system of the rat Braw Res 122, 281-297

Couch M W , Gabrielsen B M , and Midgley of deuterated hydroxyphenylethanolammes ] Labelled Camp

Radzopharm

J. M (1983) The synthesis and their metabohtes

20, 93%949

Cramers C A., Scherpenzeel G J., and LeClerq I’ A. (1981) Increased speed of analysis m directly coupled gas chromatographtc-mass spectrometry systems Capillary columns at sub-atmospheric outlet pressures ] Chromatogr 203, 207-216. Curtms H -Ch , Wolfensberger M , Stemmann M , Redwerk U., and Siegfried J (1974) Mass fragmentography of dopamme and Applrcations to the determmation of 6-hydroxydopamme dopamme m human brain biopsies from the caudate nucleus J Chromatogr

99, 529-540

Davis B A. (1979) Some new fluorescent derivatives for the mass spectrometric quantitation of biogemc ammes Blamed. Mass Spectrom. 6, 149-156

Davis B A and Boulton A A (1980) The metabolism of ingested deuterated @phenylethylamme m a human male Eur 1 Mass Spec. Blochem Med Envwon Res. 1, 149-153 Davis B A and Boulton A A (1981) Excretion of m-hydroxymandehc acid m human urine J Chromatugr 222, 271-275

362

Durden

Davis B A , Durden D A , and Boulton A A (1982) Plasma concentrations of p- and m-hydroxyphenylacetlc acid and phenylacetlc acid m humans Gas chromatographlc-high resolution mass spectrometrlc analysis 1 Chromatogr 230, 219-230 deJong A P J M , Elema J , and van de Berg B J T (1980) Gas chromatography-mass spectrometry of t-butyldlmethylsllyl denvatlves of organic acids Blamed Mass Spectrum 7, 3591364 Dlggory G L , Ceasar I’ M , Hazelby D., and Taylor K T (1979) Endogenous 5-hydroxytryptophol m mouse brain 1 Ne~ro&m 32, 1223-1325. Duffleld I’ H , Dougan D F. H , Wade D N , and Duffleld A M (1981) A chemical lonlzatlon gas chromatographlc-mass spectrometnc assay for octopamme and tyramme m rat brain Blamed Mass Spectrom 8, 170-173. Durden D A. (1978) Analysis of ammes by mass spectrometry Identlflcatlon and quantltatlon of trace ammes at the plcomole level, m Research Methods III Neurochemrstry (Marks N and Rodnight, R eds ), pp 205250 Plenum Press, New York Durden D A (1982) Selected metastable peak morutormg m mass spectrometry with stable-isotope-labelled Internal standards and linked magnetic and electric sector fields. Anal Chem 54, 66G670 Durden D A (1983) Determination of meta- and paYa-hydroxyphenylacetlc acid levels m single caudate nuclei by selected metastable peak morutormg. a new sensitive gas chromatographlc-mass spectrometrlc procedure 1 Neuroscl Methods 7, 61-66 Durden D A (1984) Quantlflcatlon of trace ammes and their metabolutes by high resolution or metastable analysis usmg double focussmg mass spectrometry, m Necl~oDlolo~~/ of the Tvacc Aml~les Physlolopcal, Pharmacolopcal, Behavloural and Cllmcal AS~JCC~S (Boulton A A , Baker G B , Dewhurst W G and Sandler, M eds ), pp 2740 Humana Press, Clifton, New Jersey Durden D A. and Boulton A A (1979) Mass spectrometnc analysis of metabolltes with specific reference to blogemc ammes, m Tcch~uq~es m the Life Sciences Techmques m Metabolx Research B224 (Kornberg H L , Metcalfe J C , Northcote D H , Pogson C I and Tlpton K F , eds.) pp l-25, Elsevler/North-Holland, Amsterdam Durden D. A and Boulton A. A (1981) Identlflcatlon and dlstrlbutlon of m- and p-hydroxyphenylacetlc acids m the brain of the rat 1 Neurochem 36, 129-135 Durden D A and Boulton A A (1982a) Identlflcatlon and dlstrlbutlon of phenylacetlc acid m the brain of the rat 1 Necrrohem 38, 1532-1536 Durden D A. and Boulton A A. (1982b) Mass spectrometnc analysis of some neurotransmltters and their precursors and metabohtes, m Handbook of Neuvochemlstry Vol 2 (Laltha A , ed ), pp 397428. Plenum Pubhshmg, New York Durden D A and Philips S R (1980) Kinetic measurements of the turn-

Metastable

Mass Spectrometry

363

over rates of phenylethylamme and tryptamme 111zuuo m the rat brain. ] Neurochenr 34, 1725-1732. Durden D. A., Phlhps S R., and Boulton A A (1973) Identlflcatlon and dlstrlbutlon of P-phenylethylamme m the rat Can ] Bfochenz 51, 995-1002 Durden D. A., Davis B. A , and Boulton A A (1974) Qualitative and quantitative mass spectrometry of some non-catechohc blogemc ammes and related compounds as their dansyl derlvatlves Blomell Mass Spectrom 1, 83-95 Durden D. A , Juorlo A V , and Davis B A (1978) Analysis of p-synephrme and related beta-hydroxyphenylethylammes by direct probe high resolution mass spectrometry Qunrlf Mass Spectrorr~Life SC{ 2, 389-397 Durden D A, Juorlo A V , and Davis B A (1980) Thin-layer chromatographlc and high resolution mass spectrometrlc determlnation of beta-hydroxyphenylethylammes m tissues as dansyl-acetyl derivatives Anal Chew 52, 1815-20 Edwards D J , Doshl P S., and Hanm I (1979a) Analysis of phenylethylammes by gas chromatography-chemical lomzatlon mass spectrometry Anal Bmckern 96, 308-316 Edwards D J., Rlzk M , and Nell J (1979b) Simultaneous analysis of phenylglycols and phenylethanols m human urine by gas chromatography-mass spectrometry ] Ck?onzatqr 164, 407-416 Ehrhardt J D and Schwartz J (1978) A gas chromatographlc-mass spectrometrlc assay of human plasma catecholammes CIlrl Clzl~z Acfa 88, 71-79. Faull K F and Barchas J D (1983) Negative-ion mass spectrometry fused-silica capillary gas chromatography of neurotransmltters and related compounds, m Methods of Bmkermcal Aualysls Vu1 29 (Gllck, D ed ), pp 325-383, John Wiley & Sons Inc , New York Faull K F , Anderson P J , Barchas J D , and Berger P A (1979) Selected ion momtormg assay for blogemc amme metabolltes and probenecld m human lumbar cerebrospmal fluid ] Chromatugr 163, 337-349 Faull K F., Anderson P J , and Barchas J D. (1981) Deuterated blogemc amme metabolltes Preparation of ring-deuterated 4-hydroxy-3methoxymandellc acid J LabelledComp Radmpkam 18, 1075-1079 Fellows L E , Kmg G S , Pettlt B R , Goodwin B L , Ruthven C R J , and Sandler M (1978) Phenylacetlc acid m human cerebrospmal fluid and plasma selected ion monltormg assay B~o~wl Mass Spectrom 5, 508-511 Francis A. J , Morgan E D , and Poole C F (1978) Flophemsyl denvatlves of alcohols, phenols, ammes and carboxyl acids Derlvatlves for mass spectrometrlc ion momtormg and structure determination Or8 Mass Spectrom 13, 671-674 Freed C R., Wemkam R. J , Melmon K L , and Castagnoll N (1977) Chemical ionization mass spectrometric measurement of

364

Durden cx-methyldopa Blochem

and s-Dopa metabolltes

m rat bran-t regions

Anul

78, 319-332.

Fri C. -G., Wlesel F -A , and Sedvall G (19974a) Mass fragmentographlc analysrs of homovanillic acid and its homo-iso analogue m cerebrospinal fluid using the cx-dideutero acrd as internal standard Psychopharmacologla 35, 295-305. Fri C -G., Wiesel F -A., and Sedvall G (197413) Simultaneous quantlfrcatlon of homovanlllic acid and 5-hydroxymdoleacetic acid m cerebrospmal fluid by mass fragmentography. Llfr Scr 14, 2469-2480 Gaskell S J and Millmgton D S (1978) Selected metastable peak momtormg a new specific techmque m quantitative gas chromatography mass spectrometry Blamed Mass Spectrorn 5, 557-558 Gaskell S J., Fmlay E M H , and Millmgton D S (1980) The determination of testosterone m human blood plasma using multiple metastable peak monitormg Adz) Mnss Specfrom 8B, 1908-10. Girault J., Lefebvre M A , Fourtillan J B , Courtois Ph , and Gombert J (1980) Dosage simultane de l’acide hydroxy-4-methoxy-3phenylacetlque et de l’acide hydroxy-4-methoxy-3 mandelrque plasmatiques par fragmentographie de masse en utilrsant des standards mternes deuteries Annales Pharrrrnceutqucs Francnlses 38, 439-446, Godse D D , Warsh J J,, and Stancer H C (1977) Analysis of acidrc monoamine metabolites by gas chromatography-mass spectrometry Amd Chcm 49, 915-918 Gordon E K , Oliver J , Black K , and Kopm I J (1974) Simultaneous assay by mass fragmentography of vamllyl mandellc acid, homovanilhc acid, and 3-methoxy-4-hydroxyphenethylene glycol m cerebrospmal fluid and urme Blochem Med 11, 32-40 Gordon E. K., Markey S. P , Sherman R L , and Kopm I J. (1976) Conlugated 3,4-dihydroxy phenyl acetic acid (DOPAC) m human and monkey cerebrospmal fluid and rat brain and the effects of probenecld treatment L$e Scl 18, 1285-1292 Harrison, A. G (1983) Chemxal lonlzaflon Mass Spectrometuy, CRC Press Inc , Boca Raton, Fla , U S A Hashlmoto Y and Miyazakr H (1979) Simultaneous determmation of endogenous norepmephrme and dopamme-beta-hydroxylase activmaterials by chemical ionization mass 1tY in biological fragmentography 1 Chromntogr 168, 59-68 Hattox, S E. and Murphy R C (1978) Mass spectrometry and gas chromatography of trimethylsilyl derivatives of catecholamme related molecules Blamed Mass Specfrom 5, 338-345 Holdmess M R , Rosen M T , Justice J B. and Neil1 D. 8. (1980) Gas chromatographic-mass spectrometric determmtation of dopamme m subregions of rat brain. ] Chromatogr 198, 329-336. Huebert N D and Boulton A A (1979) Longitudmal urinary trace amme excretion m a human male ] Clrromatogr 162, 169-176

Metastable Hunt

Mass Spectrometry

365

D F and Crow F W (1978) Electron capture negatrve Ion chemical romzatron mass spectrometry Anal Chem 50, 1781-1784. Hunt D F , Stafford G C Jr , Crow F W , and Russell J. W (1976) Pulsed posmve negative ran chemical romzatlon mass spectrometry Anal Chem 48, 209%2105. Jacob K., Vogt W , Knedel M , and Schwertfeger G. (1978) Quantrtatron of adrenaline and noradrenalme from human plasma by combined gas chromatography-high resolution mass fragmentography J ChromafoXr 146, 221-226 Jenden D J , Roth M , and Booth R. A (1973) Simultaneous measurement of endogenous and deutermm-labelled tracer varrants of choline and acetylcholine m subplcomole quantmes by gas chromatography/mass spectrometry Anal Blochenr 55, 43-48 Jenden D. J,, Roth M., and Famman F. (1978) Estlmatlon of dean01 and choline by gas chromatography/mass spectrometry Lrfe Scr 23, 291-300 Juorlo A V and Durden D A (1977) The effect of some phenylethanolamme N-methyltransferase mhlbltors on the adrenaline content m the domestic fowl dlencephalon Carl ] B~~chm 55, 761-765 Kakrmoto Y and Armstrong D (1962) The phenolrc ammes of human urine J Blol Clzelrl 237, 208-214 Kallr A., Freed C , Melmon K. L., and Castagnoll N , Jr (1977) The synthesis of deutermm enriched erythro-a-methylnorepmephrme and norepmephrme 1 Labelled Camp Radzopharm 13, 41-58 Karger B L , Krrby D I’, Vouros P , Foltz R L , and Hrdy B (1979) Online reversed phase llqmd chromatography-mass spectrometry Anal Chem 51, 2324-2328 Karoum F , Cattabem F , Costa E., Ruthven C R J , and Sandler M (1972) Gas chromatographrc assay of ptcomole concentratrons of brogemc ammes Aual B~oclwm 47, 550-561 Karoum F , Glllm J C , and Wyatt R J (1975a) Mass fragmentographrc determmatlon of some acrdlc and alcoholic metabohtes of brogemc ammes in the rat brain J Neuroclrerrl 25, 653-658 Karoum F , Grllm J C , Wyatt R. J., and Costa E (1975b) Mass fragmentography of nanogram quantmes of brogemc amine metabohtes m human cerebrospmal flutd and whole rat brain Boomed Mass Specfrom 2, 183-189. Karoum F , Nasrallah H , Potkm S , Chuang L , Moyer-Schwmg J , Phrllrps I and Wyatt R J (1979) Mass fragmentography of phenylethylamme, UT- and /I-tyramme and related ammes m plasma, cerebrospmal fluid, urine and brain ] Nel4roclzenz 33, 201-212 Karoum F , Chuang L W , Mosnalm A D , Staub R A , and Wyatt R J (1983) Plasma and cerebrospmal fluid concentrations of phenylacetlc actd m humans and monkeys 1 Chrorrmtogr Scr 21, 546550

366

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Keough T and DeStefano A J (1981) Factors affecting reactivity m ammoma chemical lonlzatlon mass spectrometry Or8 Mass Spectronr 16, 527-533 Kilts C. D., Vrbanac J J , Rlckert D E , and Rech R H (1977) Mass fragmentographlc determination of 3,4-dlhydroxyphenylethylamme and 4-hydroxy-3-methoxyphenylethylamme m the caudate nucleus 1 Neurochem 28, 465467 Ko H , Lahtl R A , Duchamp D J , and Royer M E (1974) A GC-MS procedure for the measurement of dopamme m mouse strlatal tlssue Anal. Left 7, 243-255 Koslow S and Schlumpf M (1974) Quantltatlon of adrenaline m rat brain nuclei and areas by mass fragmentography N&we (Lond ) 251, 530-531 LeClerq I’ A , Scherpenzeel G J , Vermeer E A A , and Cramers C A (1982) Increased speed of analysis m directly coupled gas chromatography-mass spectrometry systems. II Advantages of vacuum outlet operation of thick-film capillary columns I Chromatogr 241, 61-71 Lehmann W D., Beckey H D., and Schulten H. -R (1976) Qualitative and quantitative analysis of bansyl derlvatlves of dopamme and some of its metabolltes m urine Samples by electron impact and field desorptlon mass spectrometry. Anal Chem 48, 1572-1575 Lewy A J and Markey S I’. (1978) Analysis of melatonm m human plasma by gas chromatography negative chemical lomzatlon mass spectrometry Sclelzce201, 741-743 Lhuguenot J -C and Maume B F. (1974) Improvements m quantitative gas phase analysis of catecholammes m the plcomole range by electron capture detection and mass fragmentography of their pentafluoro-benzyllmme-tnmethylsllyl derlvatlves J Ckromafpyr Scl. 12, 411-418 Lhuguenot J, -C and Maume B F (1980) A method for the analysis of catecholammes by selected ion momtormg and ‘*C isotope dllutlon m adrenal medullary cell culture Blotned Mass Spectrom 7, 529-532. Lmdstrom B , Sloqulst B , and Anggard E (1974) Preparation of deuterIurn labelled catecholammes, catecholamme precursors and metabohtes for use as internal standards m mass fragmentographlc determmatlon and for turnover studies / Lab Corny Radzopkarm 10, 187-195 Magnus V., Bandurskl R S , and Schulze A. (1980) Synthesis of 4,5,6,7 and 2,4,5,6,7 deuterlum -labeled mdole-3-acetic acid for use m mass spectrometrlc assays PIant Pkys~ol 66, 775781 Markey S I’ , Powers K , Dubmsky D , and Kopm I J (1980) General methods for the synthesis of methyl isotope labelled catecholamme metabohtes, preparation of 4-hydroxy-3-methoxy-d3-(mandellc aad, phenylacetlc acid and phenylethylene glycol) J Lab Camp Radzopkarm 17, 103-114

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367

Markey S. I’ , Colburn R W., and Johannessen J N (1981) Efficient extraction and mass spectrometnc assay of serotonm u-t brologrcal fluids. Bromed Mass Spectrom. 8, 301-304 Martin M E , Karoum F , and Wyatt R. J (1979) Phenylacetic acid excretion m man Anal Blochem 99, 283-287. Martinez E. and Gelpr E (1978) Mixed pentafluoropropionyltrrmethylsilyl derrvatrves of 5-hydroxytryptophan for mass fragmentographic detection Development of a retention index model for substituted mdoles. I Chromafo~r 167, 77-90. McFadden W H (1973) Techniques of Combwed Gas Ch~omatppraphyiMass Spectrometry, John Wiley & Sons Inc , New York McFadden W H (1980) Liquid chromatography/mass spectrometry systems and applications 1 Chromatogr Scr 18, 97-115 Midgley J M , Couch M W , Crowley J, R , and Wrllrams C M (1979) Identification and quantitative determmation of o- and m-hydroxymandelic acid m human urme Blamed Mass Syectrom 6, 485-490 Midgley J M , Couch M W , Crowley J R , and Williams C M (1980) m-Synephrme Normal occurrence m adrenal gland 1 Neurochem 34, 1225-1230 Mlllmgton D S (1975) Determmation of hormonal steroid concentrations m biologrcal extracts by high resolutron mass fragmentography ] Steroid Blochem 6, 239-245 Mrta H., Yasueda H , and Shrda T (1980a) Quantitative analysrs of histamine m biological samples by gas chromatography-mass spectrometry 1 Chromatogr 181, 153-159. Mita H , Yasueda H , and Shlda T (1980b) Srmultaneous determmation of hrstamme and Nt-methyl-histamine m human plasma and urine by gas chromatography-mass spectrometry ] Chyomatogr 221, l-7. Mlyata T., Okano Y , Murao K , Fukunaga K , Takahama K , and Kase Y (1979) Analysis of physiological variations of piperldme levels m trssues by mass fragmentography Lzfe Scl 25, 1731-1738. Miyazaki H and Hashimoto Y (1982) Determmation of catechol-omethyltransferase (COMT) activity by gas chromatography-mass spectrometry usmg a mrxture of deuterated catecholamme as multisubstrate system, m SfaDk Isotopes (Schmidt H. -L , Forstel, H , and Hemzmger, K , eds ), pp 247-252 Elsevier, Amsterdam., Mryazakr H , Hashlmoto Y , Iwanaga M., and Kubodera T. (1974) Analys1s of brogenic ammes and their metabolites by gas chromatography-chemical ionization mass spectrometry j Chromatogr 99, 575-586

Mrzuno Y. and Arrga T (1979) Gas chromatographic chemical ronrzation-mass fragmentometric assay of catecholammes m the brain Clan Churn Acta 98, 217-224 Moffat A C and Hornmg E C. (1970) A new derivative for the gasliqurd chromatography of prcogram quantities of primary ammes of the catecholamme series. Blochem Bzophys Acta 222, 248-250

Durden

368

Murphy R C (1975) Synthesis of stable isotope labelled norepmephrme. J Lab Conlp Radlopharnz 11, 341-347 Murray S , Bailhe T A , and Davies D S (1981) Synthesis of l-(3-trideutero-methoxy-4-hydroxyphenyl)-[1,2,2 -2Hi] ethylene glycol ([2H6] MHPG) ] Lab Camp Radzopham 18, 1135-1140 Muskiet F A J , Fremouw-Ottevangers D C , Nagel G T , Wolthers B G , and de Vries J A (1978a) Determmation of 3-methoxy-4hydroxyphenylpyruvic acid, 3,4-drhydroxyphenylethylene glycol and 3,4-dihydroxyphenylmandelic acid m urine by mass fragmentography, with use of deutermm labelled internal standards Clllr Chem 24, 2001-S Musklet F A J , Fremouw-Ottevangers D C , van der Meulen J , Wolthers B G , and de Vries J A (197813)Determmation of some L-3,4-dihydroxyphenylalanme and dopamme metabolites m urine by means of mass fragmentography CII~Z Clzcl~ 24, 122-127 Musklet F A J , Jeurmg H J , Adler J I’, and Wolthers J P (1978~) Identification and quantification of 3-methoxy-4-hydroxyphenylethanol (MOPET) m human cerebrospmal fluid and rat brain by means of gas chromatography-mass spectrometry 1 Neu~oc/ze~r~ 30, 1495-9 Muskiet F A J., Jeurmg H J , Nagel G T , de Bruyn H W A , and Wolthers B G. (1978d) Mass-fragmentographrc determmahon of catecholamme metabolites m ammotic fluid and its possible clmlcal usefulness C/in Chem. 24, 1899-1902 Musklet F A J , Jeurmg H J , Thomasson C G , van der Meulen J , and Wolthers B G (1978e) Deuteration of catecholammes, catecholamme metabohtes and tryptophan metabohtes 1 Lnb Comp Radtopharm 19, 497-505 Muskiet F. A J., Jeurmg H J , Korf J , Sedvall G , Westermk B H , Teelken A W , and Wolthers B G (1979) Correlations between a fluorimetrlc and mass fragmentographlc method for the determmanon of 3-methoxy-4-hydroxyphenylacehc acid and two mass fragmentographic methods for the determmation of 3-methoxv-4hydroxyphenylethylene glycol m cerebrospmal fluid 1 Neurochenz 32, 191-194 Muskiet F A J , Nagel G T , and Wolthers B G (1980) Simultaneous determmahon of unconmgated homovamllic acid, vamlmandehc acid, and 3-methoxy-4-hydroxyphenylethylene glycol, m serum by mass fragmentography and deuterated internal standards Arm/ Bmchw

109,

130-136

Narasimhachari N and Vouros I’ (1972) Gas-liquid chromatography and mass spectrometry of biogenlc ammes and amphetamines as their isothiocyanate derivatives Anal. Bml7em 45, 154-163 Narasimhachari N , Prakash U , Helgeson E., and Davis J M (1978) Simultaneous determmahon by CC-MS-SIM of o-, !u-, p-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid and homo-

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Mass Spectrometry

369

vamlhc acid m blologlcal samples using a common selected ion 1, Chromatogr Scz 16, 263-267. Perel J M , Dawson D K., Dayton D G., and Goldberg L. T (1972) (Y, c-u’-and p, p’-deutermm-labelled dopamme synthesis and pharmacologlcal actlons 1 Med. Chem. 15, 714-716. Phlhps S R and Boulton A. A. (1979) The effect of monoamme oxldase mhlbltors on some arylalkylammes m rat strlatum J Neurochem. 33, 159-167. Phlhps S R , Durden D A , and Boulton A. A (1974a) Identlflcatlon and dlstrlbutlon of r?-tyramme m the rat. Can. I Blochem 52,

366-373. Phlllps S. R , Durden and dlstrlbutlon

D. A , and Boulton A A (2974b) Identlflcatlon of tryptamme m the rat. Can J Blochem 52,

447-451 Phlllps S R , Davis B A , Durden D. A , and Boulton A. A (1975) Identlflcatlon and dlstrlbutlon of m-tyramme m the rat Can. J Brochem 53, 65-69 Ralsanen M and Karkkamen J (1979) Deutenum labellmg of tryptamme, serotonm and their N-methylated metabolltes usmg solvent exchange reactions Acfa Chemlca ScatId 33, 11-14 Relsch J., Alfes H , Kommert H -J , Jantos N , Mollman H , and Clasmg D. (1970) Die Massenspektrometrle der NBD-denvate von emlgen phenylathylamm-abkommlmgen. Pharmazx 25, 331-334 Reynolds G I’ , Rlederer I’ , Sandler M , Jellmger K., and Seemann D (1978) Amphetamine and 2-phenylethylamme m post-mortem Parkrnsoman brain after ( - ) deprenyl admmlstratlon J Neural Trans 43, 271-277 Sack T. M. and Gross M. L (1983) Pulsed valve interface for gas chromatography/Fourier transform mass spectrometry Anal Chem 55, 2419-2421 Saraswat L D , Kenny J. M , Davis S K., and Justice J. B (1981) Preparation of deutenum labelled catecholammes J Lab Comp Radlopharm 18, 1507-1516 Seller N (1971) Identlflcatlon and quantltatlon of ammes by thin-layer chromatography I Ch?omatogr. 63, 97-112 Seller N (1977) Chromatography of blogemc ammes I. Generally apphcable separation and detection methods J Chromatogr 143, 221-246. Seller N , Schmidt-Glenewmkel T., and Schneider H H (1973) 1-Dl-nbutyl-ammonapthalene-5-sulphonyl chloride A new reagent for fluorescence labellmg of ammes, ammo acids and peptldes 1, Chromatogr 84, 95 Shaw G J , Wright G J , and Mllne G W A (1976) The synthesis of (Y, (Y -p, P-d4-serotonm Blamed Mass Spectrom 3, 14&148 Shape J R Jr , Hunt D F., and Savory J (1979) Plasma polyammes determined by negative-ion chemical lomzatlon/mass spectrometry Clln Chem 25, 15641571.

370

Durden

Sloqulst B (1975) Mass fragmentographlc determmatlon of 4-hydroxy-3methoxy-mandehc acid m human urine, cerebrospmal fluld, bram and serum usmg a deutenum-labelled internal standard \ Neurochem 24, 199-201 Sloqulst B (1979) Analysis of tyrosme and deutenum-labelled tyrosme m tissues and body fluids Blamed Mass Spectronz 6, 392-395. Sloqulst B. and Anggard E (1972) Gas chromatographlc determination of homovamlhc acid m human cerebrospmal fluid by electroncapture detection and by mass fragmentography with a deuterated internal standard Anal Chem 44, 2297-2301 SJoqulst B Lmdstrom B , and Anggard E (1973) Mass fragmentographlc determination of homovamlllc acid m tissues and body fluids usmg the deuterlum labelled species as internal standard. L$e Scr 13, 1655-1664 Sloqulst B , Lmdstrom B., and Anggard E (1975) Mass fragmentographic determination of 4-hydroxy-3-methoxyphenyl glycol (HMPG) m urine, cerebrospmal fluid, plasma and tissues usmg a deutenum-labelled internal standard 1 C/~~~nntc~~~r 105, 309-316 Slmgsby J M and Boulton A A (1976) Separation and quantltatlon of some urinary arylalkylammes 1 Chiomfqy~ 123, 51-56 Smith R G and Daves G D , Jr (1977) Gas chromatography-mass spectrometry analysis of polyammes usmg deuterated analogs as internal standards Blowed Mass S~JCC~IYJHI4, 146-151 Suzuki, 0 and Hattorl H (1983) Determmatlon of l&ophenylethylamme as its lsothlocyanate derivative m blologlcal samples by gas chromatography-mass spectrometry Bm~cd Moss Spectrom 10, 430-433 Swahn C -G , Sandgarde B , Wlesel F -A , and Sedvall G (1976) Slmultaneous determmatlon of the three malor monoamine metabolites m bram tissue and body fluids by a mass fragmentographlc method Psycl~oyhnnnacolu~~y 48, 147-152 Takahashl S., Godse D. D , Warsh J J , and Stancer H C (1977) A gas chromatographlc-mass spectrometrlc (GC-MS) assay for 3-methoxy4-hydroxyphenethyleneglycol and varulmandellc acid m human serum C/III Clzr~~ Acta 81, 18>192 Takahashl S , Godse D D , Naqvl A, Warsh J J , and Stancer H C (1978a) 5-Hydroxytryptophol m human cerebrospmal fluid quantltatlve determination by gas chromatography-mass spectrometry using a deuterated internal standard Cll~ C~IUI Acta 84, 55-62 Takahashl S , Yoshloka M , Yoshlue S and Tamura Z (1978b) Mass fragmentographlc determination of varulmandellc acid, homovan1111~acid and lsohomovanllllc acid m human body fluids ] Chronzatogr 145, l-9. Trefz F K , Erlenmaler T , Hunneman D H , Bartholome K , and Lutz I’. (1979) Sensitive uz z1~1uassay of the phenylalanme hydroxylatmg system with a small intravenous dose of heptadeutero

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Mass Spectrometry

371

L-phenylalanme usmg high pressure liquid chromatography and capillary gas chromatography/mass fragmentography Clan Chmz Acta 99, 211-220 Vogt W , Jacob K , Ohnesorge A -B., and Schwertferger G (1980) Highly sensitive method for the quantitation of homovamllic acid m cerebrospmal fluid. 1. Chromatogv. 199, 191-197. Wang M -T , Imai K., Yoshioka M and Tamura Z. (1975) Gas-liquid chromatographic and mass fragmentographic determmation of catecholammes m human plasma. Clan. Chum. Acta 63, 13-19. Warsh J J., Chan P W , Godse D D , Coscma D V , and Stancer H C (1977a) Gas chromatography-mass fragmentographic determination of mdole-3-acetic acid m rat bram ] Netwochem 29, 955-958 Warsh J. J , Godse D D , Stancer H. C., Chan P W., and Coscma D V (1977b) Brain tryptamme m rats by a new gas chromatographicmass fragmentographic method Bioclzem Med 18, 10-20 Warsh J. J , Chm A , Li I’ I’ , and Godse D D (1980) Comparison of liquid chromatography-electrochemical detection and gas chromatography-mass spectrometry method for brain dopamme and serotomn 1 Chromatogr 183, 483486 Wegmann H , Curtms H -Ch , and Redweik U (1978) Selective ion momtormg of tryptophan, N-acetyltryptophan and kynurenme m human serum. Application to the zn z~z~vo measurement of tryptophan pyrrolase activity 1, Chromafogr 158, 305-312 Wiesel F -A (1976) A mass fragmentographic method for the determination of 4-hydroxy-3-methoxyphenylethylamme and dopamme m brain tissue Advances zn Mass Spectromefry m Bzochemlstry and Medlczne Vol 1 (Frigerio, A. and Castagnoli N , eds). Spectrum Publishmg, New York. Wiesel F -A , Fri C -G , and Sedvall G (1974) Simultaneous mass fragmentographic determmation of 3,4-dihydroxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetic acid m brain tissue J Near Trans 35, 319-326 Wilkms C L. and Gross M L. (1981) Fourier transform mass spectrometry for analysis Anal Chem 53, 1661A-1676A. Williams C M and Couch M. W (1978) Identification of ortlzooctopamme and meta-octopamme m mammalian adrenal and salivary gland Lrfe Scr 22, 2113-2120. Willner J , LeFevre H F , and Costa E (1974) Assay by multiple ion detection of phenylethylamme and phenylethanolamme m rat bram J Neurochem

23, 857-859

Wood P L (1982) A selected ion momtormg metabolites using negative chemical Spectrom

assay for dopamme and its ionization Blamed. Mass

9, 302-306

Yoshida J -I., Yoshmo K , Matsunaga T., Higa S , Suzuki T., Hayashi A., and Yamamura Y (1980) An improved method for determmation of plasma norepmephrme Isolation by boric acid gel and assay by selected ion momtormg Blamed Mass Spectrom. 7, 396-398

372

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Zagalak M -J , Curtlus H -Ch , Lelmbacher W , and Redwelk U (1977) Quantltatlon of deuterated and non-deuterated phenylalanme and tyrosme m human plasma using the selective ion momtormg method with combined gas chromatography-mass spectrometry Appllcatlon to the VI uzztu measurement of phenylalanme-4monooxygenase activity 1 Chromato~r 142, 523-531

Chapter 9

Autoradiographic Methods for the Localization of Amine Receptor Sites in Neural Tissue R. A. LESLIE,C.SHAW,H. A. ROBERTSON, AND K. M.MURPHY 1. Introduction Many of the most srgmfrcant advances m neurobrology m the 1970s relate to the study of receptor functron Receptors are protemaceous membrane components that, when occupied by a specific lrgand (neurotransmitter, neuromodulator, or hormone), will mitrate a cellular response The most important techniques that have been developed recently to advance such studies involve ways of measurmg directly the mteractlons between a neurotransmrtter or drug and its receptor. With few exceptions, these procedures involve an m vitro technique m which animals are sacrificed, their brains removed and dissected into varrous specific regions according to some standardized procedure, and the dissected regions homogenized and centrifuged to yield a membrane preparation that mcludes the receptors of interest. Sometrmes crude synaptosomal (Pz) pellets are used m the final bmdmg assay that follows these procedures, but more often homogenates are the source of receptor material. Alrquots of the homogenate are then incubated with various concentratrons of a radroactrve &and, specific for the receptors of interest, m the presence or the absence of displacing concentratrons of a “cold” (nonradioactive) hgand, often called a displacer. The concentrations of displacer are some orders of magnitude higher than the radioligand concentrations m order that most bmdmg sites are 373

f esbe et al

occupied by the unlabeled &and After an appropriate period of time, the bound ligand is separated from the free ligand in the mixture by one of two techniques. Either the mixture goes through another centrifugation step, or is filtered rapidly on a vacuum manifold to trap the plasma membrane fragments together with their associated receptor-ligand complexes on the filtration disks while washing away unbound ligand. Bmdmg results obtained m experiments with displacer present in the incubation medium (which may be defined as nonspecific binding) are subtracted from results obtained with no displacer (which may be defined as total binding) The resultant values are a measure of specific b~~dzng of the radioligand with the tissue Generally experiments to determine binding values are run m triplicate, and mean values obtained from the experiments are calculated. These techniques have been described m detail by many authors (see, for example, Yamamura et al., 1978) and will not be treated at great length m this chapter. It is, however, important to understand how in vitro homogenate bmdmg assays work before one can fully understand the autoradiographic method of receptor localization, as the latter can be thought of as an elaboration or refinement of the m vitro homogenate binding technique Figure 1 illustrates some of the similarities and differences between the homogenate and autoradiographic techniques Basically, m place of the test tube used to manipulate a membrane preparation, for autoradiography one uses a slide-mounted histological section of the tissue in question. In place of a filtration manifold or centrifuge to separate free from bound ligand m the sample, one simply washes the free ligand away from the tissue section. Finally, instead of counting the bound radioactivity m a scintillation counter one determines the quantity of bound material by exammmg the reduced silver grains m a nuclear track emulsion, similar to a photographic emulsion, that has been exposed by the radioligand m the tissue sample.

1.1. Why the Autoradiographic

Method Is Used

Autoradiographic localization of receptors is often the only practicable way of determining receptor distribution anatomically, 1e , in histological sections It should be mentioned at this point that one determines the distribution of bzn&zg s&s for a specific radioligand m a tissue sample, one hopes that these binding sites represent sites of receptor-ligand complexes m which receptors specific for a compound of interest lie. There are various means one can use to determine with a reasonably good degree of accu-

Localization

of Amine

Receptor

Sites in Neural

Tissue

375

A

-

Sb

.Ib

B

\J

Rb

Fig. 1. This diagram illustrates the comparative stages employed in the in vitro homogenate binding technique (A) and the in vitro autoradiographic method (B). A vial of a brain membrane homogenate (wavy lines) suspended in buffer is shown in la. The homogenate is incubated with a radioligand (open circles) in 2a and a second vial (3a) is incubated with the radioligand plus an excess of a displacer compound (closed circles). These mixtures are washed and filtered so that the membrane fragments with their bound ligands are retained on the filter disks (4a and 5a). The disks are then placed into scintillation vials along with an appropriate scintillation cocktail, and the bound radioactivity is determined (6a). In B, a slide with attached tissue section (lb) is either incubated with a “bubble” of solution containing radioligand (2b) or radioligand plus an excess of displacer substance (3b). After an appropriate period of time and a wash, the slides are dried and apposed to a nuclear emulsion (4b and 5b) and exposed for a length of time. The emulsions are later developed, and the bound radioactivity in the tissue sections is visible as reduced silver in the emulsion (6b). racy whether this hope is realized in any given study (see Section 2.3). It is possible to dissect free very tiny regions of the brain and perform homogenate binding assays on them, but the structure of the brain is destroyed during the procedure and there is no effec-

Leshe et al

tlve way of checking exactly the boundaries of the excised tissue Thus, one cannot determine precisely whether a small brain nucleus 1s entu-ely within a sample or if a sample IS contaminated with adlacent tissue This 1s no longer a conslderatlon with the autoradlographlc technique as the tissue section 1s always available for comparison The actual location of the binding sites within the tissue can be determined (with a reasonably high degree of resolution at the light mlcroscoplc level) by apposmg the completed autoradlogram to the tissue section As we will see, this 1s not necessary with one version of the technique, as the autoradlogram 1s permanently apposed to the tissue sectlon on the completed slide The followmg example serves to illustrate how the autoradlographlc technique can be more powerful than the homogenate-bmdmg technique It was relatively easy to determine, with the homogenate-bmdmg technique that a large concentration of alpha-2 adrenerglc binding sites occurs m the medulla oblongata. It was not until the use of autoradlographlc locallzatlon of bmdmg sites became readily available, however, that the nuclear and subnuclear dlstnbutlon of these sites became known (Young and Kuhar, 197913, 1980a, Robertson et al , 1983, Unnerstall et al., 1984) It was only with the use of autoradlographic receptor bmdmg techniques that possible correlations between prolectlons from known target organs with then probable neurotransmitter complement became possible, as these prolectlons terminate m specific subnuclear regions of the nucleus tractus solitarius; m practice the resolution of homogenate bindmg 1s simply too poor to localize bmdmg sites accurately enough. The autoradlographlc procedure of studying receptor bindmg 1s not without its llmitatlons In cases when receptor preparations require some purification before optimal binding characteristics are attained, the autoradlographlc method 1s precluded Slmllarly, when llgands with low affmltles for the receptors are the washing procedure necessary for the being studied, autoradlographlc technique may eliminate any bound llgand (along with unbound hgand) from a tissue section before it can be processed. Finally, the autoradlographlc technique 1s much slower than the homogenate technique, which may be a serious llmitatlon for investigators requiring immediate answers regarding receptor locallzatlon. Exposure periods of several months are not uncommon for optimal vlsuallzatlon of the sites of some bound radloligands m autoradlographs Despite these drawbacks, the autoradiographic method 1s a very powerful way of locallzmg bmdmg sites of many llgands, the technique offers

LocahzatIon

of Amlne

Receptor

Sites m Neural

greater sensrtrvrty and resolutron vitro homogenate/scmtrllatron localization

377

Tissue

than its mam alternative, the m countmg method of receptor

2. Procedures 2.1. The Choice of Autoradiographic

Techniques

The first studies to use autoradrography to localize receptors to specific neurotransmitters were performed on cholinergic mcotmrc receptors (Waser and Luthi, 1962) with the use of r4C-labeled curare. The resolution of these studies was quite poor for several reasons. The high-energy beta particles emitted by ‘*C can travel much further through tissue than those of 3H, for example This means that reduced silver grains m a resulting autoradrogram can be found at a greater distance from the actual labeled membrane than would be the case with a tritium label Salpeter and Salpeter (1971) have shown that the distance 14C beta particles can travel through tissue can be five times as far as those of “H. Another problem associated with early autoradrographrc attempts at receptor localization was associated with the lack of methods to reduce the diffusion of lrgands through tissue that is being processed for autoradiography. For hgands that do not bmd particularly tenacrously to their associated receptor (1 e., have a low affinity constant), diffusion can result m a great loss of resolution or entire lack of success with locahzatron More recent studies have used 3H-labeled hgands specrfrc for cholinergic muscarmlc receptors, opiate receptors, and dopamme 1975; Pert et al , 1975, receptors (e g , Kuhar and Yamamura, Kuhar et al., 1978). These in vrvo studies made use of systemic inlectrons of the labeled hgands and subsequent autoradrographrc exammatron of brain tissue sections. Such studies have some severe hmrtatrons, however, including 1 The necessity of using a large amount of expensive radiolabeled lrgand to ensure that blood levels are high enough for effective labeling of receptors, 2. The hgands used must effectively pass the blood-brain barrier m order to label brain sites, 3. The lrgands must not be degraded by metabolism before they label then specific receptors, 4. There IS little opportunrty of controllmg precisely the amount of time the ligands or drsplacers are m contact, m appropriate concentratrons, with the receptors of interest, and 5. Studies cannot be performed on human tissue A most important advance for autoradrographic receptor studies

occurred

when

Young

and

Kuhar

(1979a)

introduced

a

378

Leslie et al.

method of m vrtro labeling of hrstological bram sections with specific radiohgands and coupled such treatment with autoradiography These authors adapted an apposrtron technique that made use of dry emulsrons, described by Roth and his colleagues (1966, 1974) They also performed the mcubations with radioligands in vrtro, I e., on slide-mounted tissue sectrons such as had been done by Polz-Telera et al. (1975). The Young and Kuhar method eliminated m one stroke all the problems consrdered above for the in vivo labeling studies. It relies upon apposing labeled tissue sections to a cool, dry layer of nuclear emulsron, previously deposited on a large flexible glass coverslrp. Thus procedure ehmmates any need to dip the labeled tissue section m a warm, lrqurd emulsion bath to coat the sectrons, a method very often used m older brological applicatrons of autoradrography (and revised u-r a recent study by Herkenham and Pert, 1982; see below). The elrmmatron of the drppmg step reduced srgrufrcantly the process of hgand diffusion throughout the brain tissue, so that ligands that bind to receptors with less than optrmum affinity could be considered for autoradrographrc studies Smce the mtroductron of the Young and Kuhar method, a film sensrtrve to beta particles has become available commercially LKB Ultrofilm-3H has been m use since 1981 and 1s currently being used instead of emulsron-coated coverslrps m many m vitro-labeled autoradrographic studies (e.g , Palacios et al., 1981a). Most of the advantages of the covershp method are retained with the film technique Together, these two techniques are, u-rgeneral, so superror to earlier methods of receptor locahzatron by autoradiography that the choice of techniques now really seems to be between only these two, although a modrfrcatron of older methods recently has been introduced and should be considered. This method makes use of formaldehyde vapor to fix slide-mounted tissue sectrons hrstologically after they have been incubated with the radroligand The trssues up to this point are completely unfixed. The sectrons are then dipped m warm, molten nuclear emulsron to coat them with an even film, and then are allowed to expose in the normal way. Development of the emulsron and staining of the underlying tissue sectrons are carried out u-r the normal manner This technique IS described m detail by Herkenham and Pert (1982) who also compared results from the method with those obtamed with the LKB film technique This paper drscusses the relative merits of the two techniques and IS a useful reference for more general mformatron about autoradrographrc receptor locahzation methods. In the rest of this chapter, the coverslip and film techniques will be discussed m detail

Locahzatlon

of Amlne

Receptor

Sites In Neural

Thsue

379

When one compares the recent techniques, the film method clearly has some very attractive advantages over the covershp method; still, it is necessary to examine the advantages of both before making a final choice. The LKB film 1s very much more eas11y handled during all stages of the autoradrographic process. Many slides with their attached labeled sections can be quickly apposed to a single sheet of film taken straight off the shelf The film will not break, as will a thin coverslip, and so the development of the photographic emulsion is a much less tedious process than that needed for the coverslip method Probably the most rmportant reason for choosmg LKB film rather than emulsion-coated covershps, however, is that the tissue sections are completely removed from the film at the development stage, so that densitometric quantlflcation of the distribution of reduced silver grains is possible on the developed film. With the coverslip and liquid emulsion techniques, the apposition of tissue section and photographic emulsion is permanent, so that the underlying tlssue section interferes with densitometry A tedious process of manual countmg of mdlvidual silver grams is necessary to quantify the amount of bmdmg observed m these methods. On the other hand, the coverslip and hqurd emulsion techmques have at least two dlstmct advantages over the film method. In the first place, the permanent apposition of tissue section and autoradiographic image can be very advantageous when the pattern of labeling does not give a clear indication of the cytoarchitecture of the matching brain slice This can happen when very few brain regions are labeled. This limitation of the film method can be overcome somewhat by examinmg a temporary sandwich of the autoradlographlc film and matching hlstological section on a microscope stage or by superimposing photographs of the tissue section and matching autoradlogram Exact regrstration of the two images is extremely difficult to achieve, however The second advantage of these two techniques 1s that the resolution of the resulting autoradiogram is somewhat better than that of the film method as the silver grains m the nuclear track emulsion are approximately one-tenth the size of the grams m the film emulsion. These two considerations may be enough m some cases to make the more tedious coverslip or liquid emulsion method the one of choice.

2.2. Preparation of Tissues for Autoradiography One of the first questions to be addressed, after choosing which autoradiographic technique to use, is how to prepare the tissues of the experimental animal for autoradiography Since the

Leslie et al

380

autoradrographic technique IS an anatomical one, it is logical to consider performing a histological fixation step to preserve tissue mtegrity before cutting sections The problem with most histological fixatives, however, is that they alter the conformation of membrane proteins, including those of receptors, and thus compromise the binding of hgands to receptors. As a result, either no fixative at all may be tolerated, or very weak solutions of routine fixatives may sometimes be used to help preserve tissue mtegrity, yet allow the receptors to mamtam most of their normal binding characteristics. It has been found, for example, with some ligands used for neurotransmitter receptor bmdmg studies, that fixative solution consistmg of 0.1% formaldehyde (0.27% formalm) can be used without seriously affecting autoradiographic results (Young and Kuhar, 1979a; Wamsley et al , 1981) This small amount of fixation not only helps to preserve histological details in tissue sections, but also makes it easier to cut frozen sections, so It is worth investigating m any specific case if a fixative can be used. This can be done either by referrmg to any relevant reports m the literature if data are available, or by mcludmg some experiments dealing with different fixation regimes in a series of “wipmg experiments” that can be done to establish optimum bmdmg parameters (see section 2.3 below) If it is determmed that no fixation can be tolerated by the technique, a perfusion of the experimental animal with a simple solution consistmg of an ice cold isotonic buffer or saline solution should be considered. This may help to preserve tissue integrity somewhat, and ~111 at least wash out any blood remaining m the vessels of the experimental tissue; this should also help to remove endogenous ligand in the tissue that can interfere with binding of labeled ligand wrth receptors. If it is decided that a perfusion step will be performed, the method is as follows: 1. Prepare the perfusate and perfusion apparatus and position the assembled apparatus near a laboratory sink. The apparatus consists of a flask that will empty via a piece of tubing leading to a stainless steel or plastic cannula that may be inserted mto the aorta of the animal A 13-gage cannula is about the correct size for a rat. Approximately 500 mL of perfusate should

be available

to perfuse

2. Deeply anesthetize the intraperltoneal sodium amount for a rat would given mtraperitoneally,

an adult

rat

animal with intravenous or pentobarbital (an appropriate be approximately 60 mg/kg, see Skinner, 1971).

Localization

of Amlne

Receptor

Sites In Neural

Tissue

381

3. With the animal supme on a piece of wire mesh screening over the sink, open the thoracic cavity and quickly insert a perfusion cannula, already flushed through with the desired perfusate, through a slit in the wall of the left ventricle and into the ascending aorta. Mamtam the cannula in position with a clamp around the distal end of the ventricle. 4. Make a slit m the right auricle or atrium to allow efflux of blood and perfusate 5. Start the flow of perfusate, using a pressure head of approximately one meter of water, i.e., have the flask containing the perfusate on a shelf at least that high above the level of the heart of the animal Once the perfusion is complete, remove the brain of the amma1 and dissect free the block of tissue required Mount this block on a cryostat chuck This may be done with a commercial mounting medium (e g , Tissue-Tek II, Miles Laboratories Inc , Naperville, IL) although for very tiny pieces of tissue more support for the resulting sections may be gamed by mounting them m a paste of homogenized fresh brain tissue An alternative way of preparing experimental brains for sectionmg is to excise fresh (unperfused) brains from deeply anesthetized animals and mount blocks from these directly on the mlcrotome chuck A method of rapidly freezing such brains has been found useful by some laboratories (Mendelsohn et al., 1984), and consists of immersing the excised fresh brain m a bath of isopentane cooled to approximately -40°C over a slurry of acetone and dry ice. Once the brain block is frozen, it may be mounted in the normal way on a cryostat chuck At this stage the brain may be stored, before mounting, in liquid nitrogen or wrapped rn foil and placed in a -80°C freezer In any case, before sectioning begins, it is necessary to equilibrate the mounted tissue to the temperature u-r the cryostat chamber, which is usually mamtamed at about -20°C The choice of section thickness depends upon several factors. Beta particles emitted by tritmm only can penetrate a few micrometers of brain tissue, depending upon the u-utial energy of the tritium ( seeRogers, 1973) they may be able to penetrate perhaps five micrometers. If sections are cut thicker than this, no increase in labeling (or reduction m resolution) will occur for tritium-labeled binding sites. It is often expedient to cut sections of a greater thickness than this, however, as the actual cutting and handling of such frozen tissue sections (particularly unfixed ones) will be

382

Leslie et al.

easier. Thus, it is often useful to cut sections from about 10-20 km thick. With radiolabels other than tritium, however, the radiation may travel much further through tissue. Furthermore, within limits, the thicker a section is, the more label it will incorporate. The problem then becomes one of resolution, as the thicker a section is, the worse the resolution of the resulting autoradiograph becomes (see Fig. 2). An optimum thickness for tissue sectioning must therefore be determined by experimentation for each individual project. (For a more thorough discussion of factors affecting the resolution of the autoradiographic method, see Rogers, 1973.) Sections once cut are generally “thaw-mounted” onto precleaned, “subbed” glass microscope slides. Slides may be washed in a mixture of 1% glacial acetic acid in 70% ethanol, or ...........::........ :. .:..... ..... ..:.:.:.~:.~~~:.:.~:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.~:.:.:.~~~~~~~:.~:.:. .i, ,) I 7‘U’X---&** a+ A

/ ,,,,,,,,,,,,

B,,,,,RI!,

,,,,,,,,,,,,,

/

,,,

Fig. 2. The effect of section thickness on resolution of an autoradiogram, under certain conditions, is illustrated. A shows a section of thickness “a” mounted on a glass slide and containing two bound radioactive molecules separated by a distance x . The dotted lines represent possible trajectories of radioactive particles towards the surface of the section nearest the overlying emulsion (stippled). It can be seen that there is a very high probability that no overlap of any resultant silver grains at the bottom surface of the emulsion will occur. In B, however, two radioligand molecules occur at the same distance apart as in A, but one is deeper in the section since the section itself is thicker in B (thickness b). Now, the trajectories of radioactive particles have a higher probability of overlapping and the resultant autoradiogram could be more confusing. Another way of saying this is that the resolution of the autoradiogram in case B is poorer than that in A, even though nothing has changed except that the section thickness has increased.

Localization ofAmine Receptor Sites m Neural Tissue

383

one consisting of 100 g potassium dichromate in 850 mL distilled water with 100 mL of concentrated sulfuric acid added. Alternatively, slides may be washed m an aqueous solution of laboratory detergent. The washed slides must then be rinsed very thoroughly m running tap water, and finally rinsed several times m distilled water. A commonly used subbing solution contams 2 5 g of gelatm and 0.25 g of chrome-alum m a liter of distilled water. Cleaned slides are dipped m this solution and allowed to dry at room temperature before use. Sections are picked up on prepared slides that have been cooled down to the temperature of the cryostat chamber. Application of a finger to the face of the slide opposite to that contammg the section will cause the section to melt and flatten onto the surface of the slide. If the coverslip technique is going to be employed, it is very useful to mount the tissue section near one end of the slide rather than m the center. This leaves room for the long coverslip to be glued to the opposite end of the slide (see Section 2 5.). If the LKB film technique is to be used, one may consider collectmg the sections on coverslips rather than slides. If the sections are small (m area), correspondingly small coverslips may be used and more sections can then be positioned on a sheet of film during the exposure period. Mounted sections are then stored m a freezer overnight (-20°C or so) to allow them to adhere thoroughly to the slides

2.3. Determination of Appropriate Ligands and Binding Parameters One of the most important steps m preparmg a receptor bmdmg study using autoradiography or other means is to choose carefully an appropriate ligand for the receptor one wishes to examine. Ideally, one will be able to choose a ligand that differentiates receptors from other high affmity uptake sites, and further, that is specific for the type of receptor (or the subpopulation of receptor if appropriate) of interest. Additionally, the specific activity of the ligand, that is the activity per unit mass of the ligand, should be reasonably high. A useful rule of thumb is: use a specific activity of at least 10 Ciimmol for receptor binding studies. Before actually begmnmg the autoradiographic study for any given receptor, one should characterize the binding sites m question m such a way that they can be identified as true receptors. This step should not be omitted as many ligands used for receptor binding studies bmd not only to biological receptors, but to other binding sites of both biological origin and nonbiological origin (for example, glassware)

384

Lesbe et al.

To identify any binding site as a receptor, certain criteria must be met First, receptors, which are fume m number m any piece of tissue, exhibit saturable binding, i.e., a point IS reached after which no further addition of hgand will increase the amount of binding Normally, bmding to the total number of specific receptors (the maximum number of bmdmg sites for a tissue is known as the B,,,,) will b e achieved by quantities of ligand m the nanomolar range This characteristic is illustrated for gammaammobutyric acid (GABA) and acetylcholme (ACh) muscarmlc receptors m Figs 3C and 4C. Basically, the technique for determining an appropriate concentration of radiolabeled ligand to use mvolves cuttmg and mountmg sections of the tissue of mterest, then incubating them m vitro with various concentrations of the radiolabeled hgand The sections are then rinsed for the same length of time m each case and are then simply wiped off the slide with a filter disk such as is normally used m a filtration manifold as described earlier for homogenate binding experiments (Whatman GF/B, Whatman, UK, for example) These filter disks are then placed m scmtillation vials with appropriate amounts of scmtillation cocktail so that the amount of bound radiolabel on the tissue section can be determined m a scmtillanon counter The results of this experiment, when plotted, yield a curve such as that mdicated by the filled circles m Fig. 3C. The data can be plotted m another fashion and displayed as a Scatchard plot (Fig. 3C, see Yamamura et al., 1978) or an EadieHofstee plot (Fig 4C, see Zivm and Waud, 1982) These plots provide a convenient method of estimating values for B,,, and &. Secondly, receptor bmdmg should be reversible. Rmsmg the incubated sections m media without ligand or m the presence of appropriate displacers should decrease the amount of bmdmg, By performing “wiping experiments” m which the times of the postmcubation rinse are varied, and the amount of bmdmg remammg m the sections is determined m a scmtillation counter, one may plot a dissociation curve for the ligand as shown by the open circles in Figs 3A and 4A These curves will give an mdicanon of how long a rinse may be used before an unacceptable amount of radioligand bmdmg is lost through the rinsing procedure. It can be seen m Figs. 3A and 4A that a very short rmsmg time (less than 5 mm) should be used for the (3H)-muscimol experiments m cat visual cortex, whereas a rinse time of even 60 mm for (3H)-QNB bmdmg m the same tissue does not compromise binding. A measure of the time it takes to reach steady-state bmdmg at a given temperature and at one concentration of ligand is known as the association rate constant (K 1 ,), and the time it takes

385

Localization ofAmine Receptor Sites in Neural Tissue 0 picrotoxln l bwculllne A GABA

.

z yL7d

20. O.,' 015

10

20

013s

10

30

40

Time

Log

(minutes)

[displacer]

M

B

A

0

20

40

60

100

80

Free

Cone

120

j +i]

140

muscimol

160

180

(nM)

c

Fig. 3. Kinetic and saturation studies of (3H)-muscimol binding in sections of cat visual cortex. In A, the time course of association and dissociation of the ligand are shown. Sections were incubated in 6 nM (“H)-muscimol at 4”C, and either the duration of the incubation (0) or the rinse (0) was varied. B illustrates some displacement experiments of the same study. Sections were incubated with (“H)-muscimol (3-6 nM) and various concentrations of several displacers for muscimol: gammaaminobutyric acid (GABA), bicuculline, and picrotoxin. C illustrates a saturation experiment which examined different concentrations of (3H)-muscimol. All sections were incubated with the ligand for 30 min at 4°C. Nonspecific binding was estimated by coincubation with 10 -“M GABA. (0) = total binding; (0) = specific binding; (x) = nonspecific binding. Binding is expressed in counts per minute (cpm). Insert: Scatchard plot of these data. For further details, see Needler et al ., 1984. for the receptor-l&and sociation rate constant dissociation equilibrium

complex to dissociate is known as the dis(K-i). The ratio K-r/K+i is known as the constant or simply the dissociation con-

386

Leslie et al. I

. Atroplne o Carhachol

Log [displacer]

sulfate

M

B

Free

Cone

[‘H]

QNB

(nM)

C Kinetic and saturation studies of (3H)-qumuclldmyl benzllate (QNB) binding m sections of cat visual cortex A Time course of assoclatlon and dlssoclatlon The sections were incubated m 5 nM (‘H)-QNB at 20°C with varied incubation (0) or rmse (0) times B. Dlsplacement experiments Sections were incubated with various concentrations of atropme sulfate or carbachol, both displacers of QNB C Saturation experiment that used different concentrations of (3H)-QNB All sections were incubated with &and for 60 mm at 20°C Nonspeclflc bmdmg was estimated by incubation with lO-“M atropme sulfate (0) = total bmdmg, (0) = specific binding, (x) = nonspeclflc binding Binding 1s expressed m counts per minute (cpm) Insert Eadle-Hofstee plot of these data For further details, see Shaw et al , 1984a

Localizatron

of Amrne

Receptor

Sites in Neural

Tissue

387

stant or Kd, and its inverse IS a measure of how strongly the hgand is bmdmg to the receptor (i e , its affinity) Simply stated, the lower the Kd, the higher the affinity. Both the K, I and K-i are important for autoradiographic bmdmg studies; the first tells the experimenter the amount of time the incubation should proceed for the bmdmg to reach steady-state (equilibrium), and the second tells the experimenter the optimal wash time needed to remove unbound hgand and to diminish nonspecific bmdmg m order to achieve the highest possible specific binding Thirdly, the bmdmg site must have pharmacological properties consistent with its role as a specific receptor, the addition of unlabeled agonists or antagonists should displace the labeled hgand For example, GABA receptors, labeled with (“H)-muscimol, should be displaced by GABA and bicuculline, but not by nonGABAergic compounds. Similarly, muscarinic ACh receptors should be displaced by appropriate substances Figures 3B and 4B illustrate this type of “wipmg experiment.” For example, it can be seen m Fig 38 that, for any given concentration of displacer, “cold” GABA is more effective than bicucullme or picrotoxm m displacing (3H)-muscimol from cat visual cortex In addition to identrfymg the binding site as a receptor, performing the characterization of the receptors prior to attempting autoradiography allows one to determine optimal binding conditions Time course experiments provide optimal incubation and rinse times; the saturation binding studies provide a range over which one can work, and will show the concentration at which the highest specific to nonspecific binding ratio can be achieved. A very useful discussion of factors to consider when establishmg bmdmg parameters to use m receptor localization studies IS given by Burt (1978). This author details ways m which experiments can be designed to attempt to characterize bmdmg sites as true biological receptors.

2.4. Incubation Procedures It is generally easiest to consider the bmdmg stage of an experiment as three separate steps: premcubation, incubation, and final rinse. Each step may require its own special techmques. The chemical makeup and concentration of the medra for preincubation and mcubation, their pH, and their temperature are all important factors to consider in order to achieve optimal specific binding In principle, it is best to determine these variables each time when examining bmdmg in a new preparation For binding sites that have already been characterized, for which there IS already general agreement m the literature about optimal

388

Leshe et al.

bindmg conditions, it generally seems appropriate to follow the established protocols In Table 1, we refer the reader to some representative publications that describe the use of specific radiolabeled hgands for autoradiographic locahzation of different amme receptors 2.4.1

Premcubatlon

The slide-mounted sections are placed m slide racks and immersed in stammg dishes (Wheaton or Lipshaw, for example) that are filled with the appropriate premcubation medium at the chosen temperature For most of the receptors that we have examined, a time of 10 mm is suitable Since little or no fixative is used during the mitral perfusion step, a low concentration of formaldehyde (0 1-O 2%) IS sometimes added to the premcubation medium. This step has been found to preserve the histological quality of the sections through the subsequent stages of the mcubation and rmse without compromismg bmdmg characteristics for many receptors (see Young and Kuhar, 1979a; Needler et al., 1984; Shaw et al , 1984a) Two additional five minute washes m preincubation medium alone serve to remove the formaldehyde. Each of these three rinses should also help to reduce the endogenous hgand m the tissue sections 2.4.2. Incubation

The sections are now placed face up on a black plastic tray and dried under a stream of air (the cool setting on an ordinary hairdrier is useful for this) The black background aids m the visuahzation of the sections When the sections are dry, the mcubanon medium containing the labeled ligand is dripped onto each section with a disposable pipet, so that it forms a “bubble” of incubation medium over the section. Approximately 200 PL of solution will effectively cover a single section of rat bram While some evaporation can be expected during relatively long mcubations at room temperature or above, and must be taken mto account, the “bubble” technique keeps the amount of hgand and hence cost per experiment to a mmimum Evaporation can be kept to a mirumum by performing the mcubation m a covered tray, with the slides raised slightly off the bottom on glass rod or similar supports Between the supports are placed pieces of filter paper that have been moistened with distilled water to keep the humidity high within the tray. Samples of the bubble may be taken before and after the mcubation procedure to determine the concentration of “free” tracer within the bubble to see how this changes during the length of the procedure

nlcotmlc

adrenerglc adrenerglc

Beta adrenerglc Histamine H, Glyclne

Alpha-l Alpha-2

5-Hydroxytryptamme

Dopamme

ACh,

(5-HT)

muscarmlc

Acetylcholme

benzllate

‘H-dlhydroalprenolol ‘H-mepyramme ‘H-strychnine

(QNB) (PBC)

Use of Radlolabeled

Llgands,

Chan-Palay (1978) Penney et al (1981) Needler et al (1984) Wamsley et al (1980) Shaw et al (1984a) Rotter et al (1979a) Wamsley et al (1980) Polz-Telera et al (1975) Hunt and Schmidt (1978) Silver and Bllllar (1976) Kuhar et al (1978) Hollt and Schubert (1978) Palaclos et al (1981b) Melbach et al (1980) Young and Kuhar (1980) Young and Kuhar (1979b, 1980) Young and Kuhar (1979b, 1980) Unnerstall et al (1984) Palaclos and Kuhar (1980) Palaclos et al (1979) Zarbm et al (1981)

Reference

Studies Makmg Receptors

(LSD)

mustard

acid dlethylamlde

‘H-WB4101 “H-t>-ammoclomdme

‘H-lysergic

“H-propylbenzllylcholme ‘H-N-methyl scopolamrne ““I-alpha-bungarotoxm

3H-qumuclldmyl

“H-musamol

acid (GABA)

Gamma-ammobutync

(ACh),

Llgand

TABLE 1 Publlcatlons That Describe Autoradlographlc Specific for Several Types of Amme

Receptor

List of Some Representative

Lesbe et al. 2.4.3. Postmcubatron Rmse Followmg the mcubatron, the slides are again placed in slide racks They are then dipped m dishes containing ice-cold preincubatron medium for an appropriate length of time. The use of a cold rinse can be very important smce the drssocratron rate constant of specific bmdmg tends to be temperature dependent, whereas that for nonspecific bmdmg does not A cold rinse thus will allow nonspecrfrc dissociatron to proceed at a set rate while slowmg the drssocratron of the specific bmdmg The rmse time depends on the drssocratron rate constant (K-,, see Sectron 2.3 ) Rinses that are too long will cause drssoclatron and diffusion of the bound hgand from its receptors For receptors with very rapid Kls, a final dip m acetone/570 glutaraldehyde may promote rapid, even drying (Greenamyre et al., 1983). If the K-1 1s less rapid, the slides may be removed from the slide racks at this stage, placed sectron side up on a tray, and dried m a stream of cool dry au- as described above. When dry, the slides are placed overnight m slide boxes with some dessrcant (srllca gel, for example) to ensure complete drying

2.5. Exposure of the Labeled Sections to Nuclear Emulsion Once mounted tissue sections are labeled with the &and of interest under the condrtrons determined to be most appropriate, rt 1s necessary to appose them to a dry nuclear track emulsron m a darkroom and store them in the dark for a period of time so that the mcorporated radroactrvrty will expose the emulsron and reveal the location of bound hgand As outlined above, two recent techniques have been developed to accomplrsh this, each with its own advantages and hmrtatrons. Two control exposures should be performed near the beginning of any series of experiments to determine if spurious results may occur due to phenomena called “posrtrve” and “negative chemography.” These phenomena mvolve either an enhancement or interference with exposure of the nuclear emulsron by the presence of the tissue alone, thus confusing results due to exposure of the emulsron by bound radroactrvrty m the tissue These effects are generally not a problem with either of the techniques detailed below, but simple control experiments will confirm this in any given case These experiments mvolve m the first instance apposing a completely unlabeled tissue section to an emulsron to determine rf silver gram formation will result from posrtrve chemography Secondly, to see rf the tissue itself interferes with normal silver gram formation (1 e , to test for negative chemo-

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graphy) an unlabeled tissue section can be apposed to a layer of emulsion that has been previously exposed to light. These control emulsrons are developed m the normal way and any unlabeled tissue effects are noted. 2.51. The Coverslip Technique With this method, large, thin glass coverslips are coated with nuclear track emulsion m a liquid form that is allowed to dry before use. The coverslip then is glued at one end to the slide with its mounted tissue section in such a way that the section is covered with the emulsion A spring clip is placed around the sandwich of slide and coverslip to hold the tissue section and coated coverslip tightly together during the exposure period When the emulsion is to be developed, the clip IS removed, and the coverslrp IS gently sprung away from the section, making use of the great flexibility of the long, thm glass coverslip. The autoradiogram is then developed and fixed, and the tissue section is then histologically fixed and stained. Fmally, the coverslip is reapposed to the tissue section with a permanent mountmg medium to give the finished autoradiogram. It IS necessary to prepare a supply of emulsion-coated coverslips at least a day m advance of use This is done as follows In a darkroom under safelight conditions (Kodak Wratten Series 2, with a 25W bulb, for example; see the emulsion manufacturer’s recommendations), melt the emulsion rn a suitably shaped vessel that is standing in a waterbath kept at about 40°C Slide mailing tubes such as the Lab-Tek 4310 cytomailer, available from Canlab Laboratory Equipment, are suitably shaped containers that may be glued at their base to a weighted pedestal that will keep them upright and partially submerged m the waterbath. Kodak NTB2 or NTB3 Nuclear Track Emulsion, diluted 1.1 with distilled water IS commonly used for coating. Using a nonmetallic spatula, spoon out the required amount of emulsion mto the warmed distilled water in the small vessel. Stir gently, avoidmg air bubbles, until the emulsion is entirely melted When a homogeneous solution of emulsion is attained, dip the coverslips, one by one, into the emulsion for about two-thirds of their length. The coverslips should be at least 60-70 mm long and 22-25 mm wide. They may be thickness 0 or 1, but no thicker. They should be scrupulously clean before dipping, so it is a good idea to wash them m the same manner that the slides were washed, before sections were mounted on them (seeSection 2.2 , above). The dipped coverslips then must be allowed to dry m the dark. This may be accom-

Leslie et al.

phshed most easily by puttmg them m a drying rack prepared from small coil sprmgs that are screwed at their ends to a small board so that the uncoated part of the coverslip is gripped between the coils of the spring, and the coverslips are then held perpendicular to the board This board can then be placed m a lighttight cupboard or “papersafe” (light-tight box) with some drying agent, such as silica gel, so that the coverslips can dry overnight. The emulsion should not be forced to dry too quickly because cracks or stress marks may form m the coatmg, these will obscure the results of the autoradiography The slides with mounted sections then are brought mto the darkroom and laid out on a bench Under safelight conditions, the coated coverslips are glued to the slides with a small drop of cyanoacrylate glue (Krazy Glue, F I’. Feature Products, Inc , Mississauga, Ontario, for example). A small drop of glue is placed on the end of the slide opposite the section, but on the same side. The uncoated end of the coverslip is then placed carefully over the glue and a sandwich made of slide, section, and coverslip A small spring steel stationery clip is then placed over the slide to hold the coverslip m tight appposmon to the section throughout the exposure period. Clips about 2 cm long, for example, fold back clips No 1411, available from I. B F Canada, Willowdale, Ontario, are about right The slides are then placed m small, lighttight boxes along with a small package of desiccant. These are then sealed with tape and left m a refrigerator or cold room at about 4°C to expose. The exposure time will depend upon several factors, mcludmg the B,,,, of the area of interest, the concentration of the radioligand used, and the specific activity of the ligand. Once a few successful autoradiograms have been obtained, then it is possible to estimate with some degree of accuracy how long a reasonable exposure period will be m succeeding experiments. When it is desired to test a slide to determine if the exposure period has been long enough, one is removed from its box m the darkroom, the clip is removed, and a single-edged razor blade is used to pry the coated end of the coverslip very gently from the tissue section. The glue at the other end ensures that the coverslip can be reapposed with the identical orientation as before. A small plastic or wooden spacer is positioned between the coverslip and slide to hold them partially apart durmg the development procedure. The slide is then placed m freshly prepared and filtered photographic developer (such as Kodak D-19) m a stammg dish. It is a good idea to stack the slide m the stammg dish vertically, so

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that the glue will not become wet and soften The emulsion is developed for 2 mm with gentle agitation every 15 s, and then the slide is removed to a dish containmg distilled water for a rinse. It is then placed m a dish of freshly prepared and filtered photographic fixer and left for 2 mm with gentle agitation. Three 5 mm rinses in distilled water follow, and the darkroom lights then can be put on. The section then has to be dealt with to preserve it and stain it for further observation The slide, with its spacer still m place, is treated as an ordinary histological slide except that care is taken to avoid wetting the glue at one end. The section is fixed in 10% formalin for about 30 mm, and is washed m several rinses of distilled water. It is dehydrated through a graded ethanol series, and then is placed in xylene for 5 min to defat the tissue. The section is rehydrated in another ethanol series and stained m a solution of 0.2% aqueous cresyl violet. About 15 mm m the staining solution is generally sufficient. Excess stain can be removed by placmg the slide m 1% acetic acid m 95% ethanol for a few seconds. The section is dehydrated once again m an ethanol series, and placed m two changes of xylene to clear the tissue and render it miscible with a permanent coverslip mountmg medium (for example DPX, BDH Chemicals, Toronto). When the slide IS dry, a razor blade can be used to scrape the emulsion from the top surface of the coverslip and the subbing gelatin from the bottom surface of the slide The slide then can be examined m a microscope. By altering the plane of focus of the oblective in the microscope, the reduced silver grains can be seen m the plane of the emulsion, and the posltlons of these over the underlying tissue section can be determined. 2.5.2.

The LKB Ultrotilm-3H

Method

Slides with mounted trssue sections are taped to conveniently sized sheets of heavy cardboard (Bristol Board) with double-sided tape, with the sections facing away from the card. The card is usually cut to correspond with the size of a single sheet of LKB film. As noted previously, sections may be mounted on coverslips mstead of glass slrdes, and these are treated in exactly the same way sheets of as slides m the followmg description. In the darkroom, LKB Ultrofilm-3H are placed, emulsion side down, m apposition to the tissue sections. The sandwich thus formed can be placed m a standard X-ray cassette that will keep the film and cardmounted slides in close contact and prevent any reorientation of the two during the exposure period These cassettes often can be

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obtained from a hospital’s X-ray department at a nommal charge if they are slightly damaged and due to be replaced by the department. Should they be too expensive or otherwise unobtainable, another method of apposmon and storage may be used This mvolves making a stack of several film-slide sandwiches m the darkroom and wrapping the bundle m several layers of overlapping aluminum foil If tightly wrapped, the foil will prevent any light leaks mto the stack of film The wrapped stack then is placed on a shelf to be undisturbed during the exposure period, and some heavy weights are placed on top of the stack, pressing slides and film tightly together. After an appropriate exposure time, the film is removed from the sandwiches and developed m the darkroom m photographic developing trays m the conventional manner. The emulsion is very soft, so great care must be taken to avoid scratching the film during the followmg procedures. A standard protocol for development involves a 5 mm development m Kodak D-19 developer, a rinse m water or stop bath, and a fix m standard photographic fixer for 5 mm The films are then washed for 20 mm m running tap water before being allowed to dry They then are ready for exammatron. The slides (or coverslips) with attached tissues may be used again to perform a “coated coverslip” exposure If the autoradiograms that already have been prepared are sufficient, however, the sections may be fixed and stained m an identical fashion to that described above for the coverslip method, except one need not worry about immersmg the entire slide m any of the baths.

3. Assessment

of Autoradiograms

3.1. Qualitative Assessment Once autoradiograms have been produced, it becomes necessary to determine precisely the regions exhibitmg bmdmg above background levels. The assessment can be performed at various levels of resolution, for example at the regional, lammar, cellular, or subcellular levels within a section. To determine the identity of areas exhibitmg a high degree of bmdmg, the original sections must be compared with the autoradiogram. In the case of the coverslip technique, the comparison is straightforward, as the autoradiogram and tissue section are already apposed to one another When such a preparation is viewed with a microscope under medium or high power, the reduced silver grams m the emul-

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sion and the underlying cells of the tissue can be examined in turn by changing the field of focus. Low power dark-field microscopy is very useful when scanning autoradiograms prepared with the coverslip technique because the contrast between labeled regions of the section and unlabeled regions is generally much greater than that seen with normal bright-field microscopy. The film technique, on the other hand, produces autoradiograms that are prepared separately from the tissue sections in the latter stages of the procedure, and so these must be reapposed during the assessment. This precludes cellular or finer resolution with this method, but careful comparison of the autoradiogram with the stained sections still generates useful information about the regional (nuclear or subnuclear, for example) or laminar distribution of binding sites, depending upon the organization of the tissue. In Fig. 5, representative autoradiograms of cat visual cortex are shown for (“E-I)-muscimol binding sites (representing GABA receptors) and (“f-I)-quinuclidinyl benzilate (QNB) binding sites (representing muscarinic cholinergic receptors). Figure 6 illustrates an autoradiogram of (“H)-FJ-

Fig. 5. Representative LKB Ultrofilm-3H autoradiograms from sections of the visual cortex of a 95-day-old cat. A. (3H)-muscimol binding sites. Ligand concentration was 50 nM. B. (3H)-QNB binding sites. L&and concentration was 5 nM. The different laminar distributions for GABA (A) and muscarinic cholinergic (B) receptors is readily apparent. Dorsal is up, medial is left for both panels; calibration bars = 1 mm.

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Fig. 6. A is an autoradiogram prepared by the LKB Ultrofilm-3H method of a transverse section of dog medulla oblongata that had been incubated with (“H)-p-aminoclonidine to indicate the presence of alpha-2 adrenergic binding sites. The incubation was for 45 min in 1 nM ligand. Displacer experiments with 100 FM phentolamine indicated that the binding was specific. Heavy labeling of the nucleus tractus solitarius (nTS) and the dorsal motor nucleus of the vagus (dmnX) is evident, and less dense but specific binding in the area postrema is visible. The solitary tract (TS) is not labeled significantly above background levels on the tissue section. IV = fourth ventricle; calibration bar = 0.5 mm. B is a micrograph of the cresyl violet-stained tissue section used in the preparation of the autoradiogram in A; all labels are the same as in A. aminoclonidine binding sites in dog medulla oblongata (representing alpha-2 adrenergic receptors). Each population of receptors exhibits highly laminar or reqonal patterns of binding. Fig. 7 illustrates the laminar pattern of H-muscimol binding sites in the visual cortex of the macaque monkey. With appropriate staining of the original section and adjacent positioning of photographs of the section and the autoradiogram it is possible to identify the various laminae in relation to the binding pattern.

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Fig. 7. Representative LKB Ultrofilm-“H autoradiogram of a section from the visual cortex of a young adult female macaque monkey incubated with (3H)-muscimol. The visual cortex has been cut tangential to the cortical surface. Ligand concentration was 16 nM. After the autoradiogram was produced, the apposed section was stained with cresyl violet to reveal the cortical lamination. Photographs of both the autoradiogram and the stained section were then aligned to allow an identification of the laminar pattern of (“H)-muscimol binding. The laminae are labeled conventionally. D = dorsal; L = lateral; calibration bar = 1.25 mm.

3.2. Quantification of Autoradiographic Results 3.2.1. Grain Counts for the Coverslip Technique The density of binding sites can be estimated by manually counting the numbers of reduced silver grains lying over selected areas of the tissue. This is done by selecting a standard magnification in the light microscope (usually a 40-100~ objective lens is chosen for this), and a standard area (encompassing several square micrometers, for example) to examine for each count. Counts of the entire microscope field or use of a calibrated ocular grid fitted to the microscope are the easiest ways of providing standard areas of autoradiograms to count. An example of a silver

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gram count for simple quantification of a “coverslip autoradiogram” IS given m Fig. 8A. Other methods may mvolve prolectmg photographs of the autoradiograms onto a paper screen with a grid marked on it, using a standard magmfication each time. The prolected silver grams can be counted easily this way by marking with a pencil each gram on the paper screen as it is counted, thus avoidmg counting any grain twice If autoradiographic standards are employed, as described below, an mdication of how much bound l&and there IS in any counted area of the autoradiogram can be attained. 3.2 2. Densitometry of LKB Fj/m Autoradiograms Several recent publications have outlined methods of quantifymg the amount of bound ligand over selected regions of tissue sections by applying densitometry to the areas m correspondmg autoradiograms; some of these mvolve computer enhancement techniques. Examples of such papers that should be consulted for full details of the techniques include Palacios et al (1981a); Penney et al. (1981), Rainbow et al (1982), and Unnerstall et al. (1982) One method that is m use m our laboratories mvolves cuttmg out the individual autoradiograms from a sheet of film with scissors so that they will fit on the stage of a Zeiss Universal microscope that has been fitted with a Zeiss model MPM microdensitometer attachment This device allows very accurate measurements to be taken of the percentage transmittance of light through a small portion of the autoradiogram, correspondmg to a definite region of tissue within a tissue section The area of the sampled light varies with the aperture chosen m the densitometer and the size of the oblective lens chosen for the work A direct readout of percent transmittance is given on a meter, and the autoradiogram can be moved around the stage of the microscope to sample many different regions. The device is calibrated using a region of the autoradiogram containing background silver grains only The meter 1s set at 100% transmittance at this point, and m subsequent readings, transmittance readings of lesser values mdicate the density of receptor bmdmg The lower the reading, the denser the bmdmg One must take readings of the same regions of tissue in adlacent autoradlograms that have been prepared with an excess of displacer m the incubation medium. These two sets of readings can then be subtracted from one another to yield an mdrcation of specific binding. Another method m use makes use of a simpler densitometer

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Fig. 8. Laminar distribution of (“H)-muscimol binding sites in cat visual cortex. The ligand concentration used was 25 r&l. A. Silver grain counts from a Kodak NTB-2 emulsion-coated coverslip experiment were obtained at 1000x magnification from layers I-VI through the medial bank, and are plotted as a function of depth from the cortical surface. The highest grain concentration is shown to be in a position corresponding to layer IV. B. With a section adjacent to that used to produce the autoradiogram illustrated in A, an autoradiogram was produced on LKB Ultrofilm-. H (this IS the same autoradiogram as illustrated in Fig. 5). The parallel white lines indicate the area measured in A. Calibration bar = 1 mm. (For further details, see Needler et al., 1984.)

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fashioned from a photosensitive diode, mounted m a small handheld manipulator that IS attached through a variable resistor and linear amplifier circuit to a digital multimeter The output of the densitometer is proportional to the amount of light falling onto the surface of the diode. To use this apparatus, the autoradiogram is first positioned m a photographic enlarger m a darkroom and treated as though it were a photographic negative The image is prolected onto an easel, and the photodiode is positioned m various spots on the easel so that it samples various regions of the image A diagram illustratmg the apparatus m use IS given m Fig 9.

In any densitometric assessment of autoradiograms, the nuclear emulsion can be calibrated with known amounts of radiolabel so that reasonably accurate determmations of bmdmg can be arrived at and expressed m standard units of femtomoles of hgand per milhgram of tissue protein This IS done by using tissue standards of homogenized brain tissue mixed with known amounts of radiolabeled ormthme or formaldehyde (see Unnerstall et al , 1982). The standards are frozen and sectioned m a cryostat m the normal manner and mounted on glass slides The slides are then apposed to film at the same time as the experimental tissue sections The standards, which can be reused many times, provide a range of optical density values when the resultant autoradiograms of them are measured by densitometry Since the amount of label m adjacent sections of the standards can be determined by “wlpmg” and counting m a scmtillation counter, a calibration curve can be generated that plots optical density against counts per minute (cpm) As long as subsequent measurements of optical density m experimental autoradiograms fall within the linear portion of the calibration curve, any optical density value can be converted to equivalent cpm values Assummg homogeneity, if the area and thickness of the experimental section are known, the amount of protein per unit volume of tissue m a section can be determined From the equivalent cpm and protem values, it is possible to compute bmdmg m an autoradiograph of an experimental section m the conventional units of fmol/mg protein. Of course, the determmation of protein values of a whole section is not always necessary. For example, if protein content per unit area of tissue section remains constant, then comparisons of different areas m terms of optical density values alone may be sufficient. However, if protein concentration vanes for different areas of tissue, or during ontogenetic development, and different

Localization ofAmine Receptor Sites in Neural Tissue

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a 0 wb +===+c

A

Fig. 9. A simple method of performing densitometric measurements on an autoradiogram prepared from LKB film is illustrated in A. A photographic enlarger (a) fitted with an appropriate condenser lens (b) is used to project an image of the autoradiogram (c) onto an easel (e). A simple PIN silicone photodiode (d), mounted in a Plexiglas manipulator, is moved about the easel so that the projected image impinges on it. Apertures of various sizes can be fitted over the photodiode so that a known area of the autoradiogram is sampled at any one time (taking the magnification of the enlarger projection into account). The small output of the light-sensitive photodiode is fed through a simple linear amplifier or attenuator (f) into a digital multimeter (g) in which a voltage readout is obtained that is proportional to the amount of light striking the photodiode. This can be calibrated by means of autoradiographic standards prepared from brain homogenates mixed with known amounts of radioactivity (see text). B is a photograph of the photodiode manipulator; an aperture is not fitted to the device so that the diode (arrow) is clearly visible.

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stages of development are being exammed autoradlographlcally, it becomes important to determine the amount of protein m each condition or area to adlust optical density values appropriately (see Shaw et al , 1984b). One must keep m mind, however, that such protein adjustments are for total protein, and not only for proteins associated with synaptic membranes. The reader who wishes to obtam further details about any of the procedures outlined above 1s referred to excellent review articles by Kuhar (1983), Murrm (1981), and Wamsley and Palacios (1982, 1983).

4. Conclusions The ever-increasing popularity of m vitro autoradlographlc techniques m studies on the brain distribution of amme receptors can be attributed to several factors. The techniques are very sensitive and provide powerful ways of determining where and m what concentrations receptors for various amines occur. Relatively specific hgands, labeled with tritium or other radlolabels suitable for autoradiographic use are now readily available commercially, and the new in vitro methods are cost effective since they do not require vast quantities of these expensive compounds The techniques are relatively simple to perform, and do not require much m the way of expensive laboratory equipment As methods continue to advance, the resolution obtainable from the techniques will continue to improve, and, hopefully, soon it will be feasible to undertake such studies at the electron microscope level so that locallzatlon of receptors m individual regions of single cell membranes may be achieved In the meantime, many types of investigations are now making use of the techniques described above to study normal physiology and pharmacology of receptors, to examme pathologlcal tissues for receptor anomalies, and to test the effects of drugs or other expenmental manipulations on receptor populations

Acknowledgments R.A.L., H.A.R., and K.M.M. thank the Medical Research Council of Canada and Supply and Services Canada for financial support. C.S. was supported by grants from the National Institute of Health (IFYE405393-01) and the Klllam Foundation. We thank Dr.

Localrzatlon ofAmIne Receptor Sites m Neural Tissue M. Wllkmson for valuable comments use of previously unpublished data

and M.C.

Needler

403 for the

Note Added in Proof Recent evidence indicates that quenching of trltium beta particle emlsslons by white matter of the central nervous system can be slgmflcantly different than that of grey matter The result of this may be an erroneous comparison of actual bmdmg of trltlated llgands in different bran-t regions. The enhanced quenching by white matter over grey matter may be ameliorated by tissue defattmg, but unfortunately this also causes loss of label to various extents, depending upon the hgand used. Since the label loss caused by defattmg 1s predictable, however, adjustments can be made to compensate for it (for a dlscusslon of the method, see Herkenham and Sokoloff, 1984, BUU~ Res. 321, 363368.) An alternative method of dealing with this phenomenon m cases where interpretation of results would otherwise be a problem would be to use a llgand labeled with a source of radioactlvlty that 1s not quenched to such a sigmflcant extent by white matter, such as 1251

References Burt D R (1978) Criteria for receptor ldentlflcatlon, m Neurotransmltter Receptor B~~dzrzg (H I Yamamura, S.J Enna and M J Kuhar, Eds ), pp 41-55 Raven Press, New York Chan-Palay V (1978) Autoradlographlc locallzatlon of gammaammobutyrlc acid receptors m the rat central nervous system by using (3H)musclmol Proc Nat Acad. Scr (USA) 75, 1024-1028 Greenamyre J. T , Young A B., and Penney J B (1983) Quantitative autoradiography of H-L-glutamate bmdmg to rat brain Neurosci Lett 37, 155-160 Herkenham M. and Pert C B. (1982) Light mlcroscoplc localization of brain opiate receptors a general autoradlographlc method which preserves tissue quality. 1 Neuroscl. 2, 1129-1149 Hollt V. and Schubert I’ (1978) Demonstration of neuroleptlc sites m mouse brain by autoradlography Bram Res 151, 149-153 Hunt S. I’ and Schmidt J. (1978) The electron-mlcroscoplc autoradlographlc locallzatlon of alpha-bungarotoxm binding sites within the central nervous system of the rat Bram Res 142,152-159. Kuhar M J (1983) Autoradlographic localization of drug and

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neurotransmitter receptors, in Handbook of Chemrc0l Neuroanatoq (A Blorklund and T Hokfelt, Eds ), pp 398415 Elsevier, Amsterdam Kuhar M. J and Yamamura H 1. (1975) Light autoradiographic localization of cholmergic muscarmic receptors m rat brain by specific bmdmg of a potent agonist. Nature (Lond.) 253, 560-561 Kuhar M. J., Murrm L C , Malouf A. T , and Klemm N (1978) Dopamme receptor bmdmg m viva the feasibility of autoradiographic studies. Life Scl 22, 203-210. Meibach R C , Maayam S , and Green J P (1980) Characterization and radioautography of 3H-LSD bmdmg by rat brain slices m vitro the effect of 5-hydroxytryptamme Eur 1 Pkarmacol 67, 371-382 Mendelsohn F. A 0 , Qumon R , Saavedra J M., Aguilera G , and Catt K J, (1984) Autoradiographic localization of angiotensm II receptors m rat brain Pm Nut Amd Scl (USA) 81, 1575-1579 Murrm L C. (1981) Neurotransmitter receptors. neuroanatomical localization through autoradiography Int Rez~ Neurubrol 22, 111-171 Needler M. C , Shaw C , and Cynader M. (1984) Characteristics and distribution of muscimol bmdmg sites m cat visual cortex Bruzn Res 308, 347-353 Palacios J. M , Young W S., and Kuhar M J, (1979) Autoradiographic localization of H,-histamine receptors in brain 3H-mepyramme prelimmary studies Eur. 1 Pkarmacol 58, 295?kg Palacios J. M and Kuhar M J (1980) Beta-adrenergic receptor localization by light-microscopic autoradiography Science 208, 13781380 Palacios J M., Niehoff D L , and Kuhar M. J (1981a) Receptor autoradiography with tritium-sensitive film potential for computerized densitometry Neuruscz Left 24, 111-116 Palacios, J M., Niehoff D L., and Kuhar M J (1981b) 3H-spiperone bmdmg sites m brain. autoradiographic localization of multiple receptors Bratn Res 213, 277-289 Penney J. B , Pan H. S , Young A. B , Frey K A , and Dauth G W (1981) Quantitative autoradiography of muscimol receptors. Science 214, 10361038 Pert C B , Kuhar M J , and Snyder S H (1975) Autoradiographic localization of the opiate receptor m rat brain Life Scl 16, 1849-1854 Polz-Telera G , Schmidt J , and Karten H J (1975) Autoradiographic localization of alpha-bungarotoxm bmdmg sites m the central nervous system Nature (Lond ) 258, 349-351 Rambow T C , Bleisch W V , Biegon A , and McEwen B S (1982) Quantitative densitometry of neurotransmitter receptors, ]

Neuroscl Methods 5, 127-138 Robertson H A , Leslie R. A , and Murphy K.M (1983) Autoradiographic localization of monoamme receptor sites m the dorsal vagal complex of the dog Sue Neuruscz Absfr 9, 112 Rogers A W (1973) Techmques of Auturadzugraphy Elsevier, Amsterdam Roth L J , Drab I M , Watanabe M , and Dmerstem R J (1974) A correl-

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atlve radioautographic, fluorescent, and histochemical technique for cytopharmacology Mol Pharmacol 10, 986-998 Rotter A , Birdsall N J M , Burgen A S V., Field I’ M , Hulme E C., and Ralsman G (1979) Muscarmic receptors m the central nervous system of the rat 1 Technique for autoradiographic localization of the bmdmg of ‘H-propylbenzilylcholme mustard and its dtstributlon m the forebrain Blazll Res Rev 1, 141-165 Salpeter M M , and Salpeter E E (19711 Resolution m electron microscope radroautography II. Carbon’ . 1 Cell Bzol 50, 324-332. Shaw C., Needler M C , and Cynader M. (1984a) Ontogenesis of muscarmic acetylcholme bmdmg sites m cat visual cortex reversal of specific lammar distribution during the critical period DL’z~ Brnru Res 14, 295-299 Shaw C , Needler M C , and Cynader M (1984b) Ontogenesis of muscimol bmdmg sites m cat visual cortex Brazn Res BuIl 13, 331-334 Silver J and Billiar R B. (1976) An autoradiographic analysis of (7H)-alpha-bungarotoxm distribution m the rat brain after mtraventricular mlection ] Cell Blol 71, 95&963 Skinner J E. (1971) Neuroscle?zce. A Laboratory Manunl W.B Saunders, Philadelphia Stumpf W E and Roth L. G (1966) High resolution autoradiography with dry-mounted freeze-dried frozen sections. Comparative study of SIX methods usmg two diffusable compounds, (3H)-estradlol and (3H)-mesobilirubmogen I Hzstochem Cyfochem. 14, 274-286 Unnerstall J R , Niehoff D L , Kuhar M J , and Palacios J M (1982) Quantitative receptor autoradiography usmg (7H)Ultrofilm apphcation to multiple benzodiazepme receptors I NellroscJ Methods 6, 59-73 Unnerstall J. R , Kopaltlc T A., and Kuhar M. J (1984) Dlstrlbutlon of alpha-2 agonist bmdmg sites m the rat and human central nervous system analysis of some functional, anatomic correlates of the pharmacologic effects of clomdme and related adrenergic agents. Brnm Res Reu 7, 69-101 Wamsley J K , Zarbm M A., Nigel J. M. Birdsall N J. M , and Kuhar M J (1980) Muscaruuc cholmergic receptors autoradiographic locahzation of high and low affu-uty agonist binding sites. Brnm Res. 200, 1-12 Wamsley J K , Palacios J M., Young W S., and Kuhar M. J (1981) Autoradiographic determmation of neurotransmitter receptor distributions m the cerebral and cerebellar cortices ] Hlstochem Cytochem 29, 125135 Wamsley J K and Palacios J M. (1982) Receptor mapping by histochemistry, in Handbook of Neurochemsfry 2 Experimental Neurochemistry (Laltha A., Ed ) pp. 27-51 Plenum, New York. Wamsley J K and Palacios J M (1983) Apposition techniques of autoradiography for microscopic receptor localization, m Current

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Methods In Cellular NeurobzoloRy Vol 1 Anatomical Techniques. (Barker J L and McKelvy J F , Eds.) pp 241-268 John Wiley and Sons, New York Waser I’ G and Luthl U (1962) Uber die Flxlermg von “C-curarm m der Endplatte Helv Physlol Acta 20, 237-251 Yamamura H I., Enna S J , and Kuhar M J (1978) Neurotransmztter Receptor Bzndmg. Raven Press, New York Young W S and Kuhar M J (1979a) A new method for receptor autoradlography (3H)-oplold receptors m rat brain Bram Res. 179, 255-270 Young W. S and Kuhar M J (1979b) Noradrenerglc alpha-l and alpha-2 receptors* autoradlographlc vlsuallzatlon. EUY j Pharmacol 59, 317-319. Young W. S. and Kuhar M. J. (1980a) Noradrenerglc alpha-l and alpha-2 receptors. light mlcroscoplc autoradlographlc locallzatlon Proc Nat, Acad. Scz (USA) 77, 1696-1700 Young W S and Kuhar M J (1980b) Serotonm receptor locallzatlon m rat bram by light mlcroscoplc autoradlography EUY ] Pharmacol 62, 237-239 Zarbm M A , Wamsley J K , and Kuhar M J. (1981) Glycme receptor. light mlcroscoplc autoradlographlc locallzatlon with H-strychnine ] Neurosct 1, 532-547 Zlvm J A and Waud D R (1982) How to analyze binding, enzyme and uptake data the simplest case, a smgle phase Life Scl 30, 1407-1422.

Chapter 10

Turnover Rate Assessments of Cerebral Neutrotransmitter Amines and Acetylcholine J. KORF 1. Introduction The aim of the present review is to summarize the methods used to estimate the turnover of various neurotransmitters of low molecular weight m the intact animal brain First, some mtroductory remarks and defmltions are given. The followmg description of the terms used is derived mainly from Atkins (1969), Robertson (1957) and some references therein. The chemical compound whose quantitative behavior will be studied is called a substance and the behavior of the substance is studied m a system Thus, the turnover of the substance acetylcholme (ACh) may be studied in a system, such as the rat strratum or the whole mouse brain In vivo studies of the central nervous system are usually considered to be an open system, meanmg that a precursor enters the system and metabohtes leave the system. The behavior of a substance is not necessarily uniform m the system, and, therefore, a system may contam several pools or compartments of that substance. A compartment is defined as a quantity of a substance that has distinguishable and uniform kmetics of transformation The boundaries of a compartment may, but do not necessarily, conform to anatomical boundaries, and conversely, anatomical boundaries are not necessarily divisions of the compartments. Thus, for example, one may distmguish two compartments of dopamine (DA) m the mgrostriatal system that consist of two types of nerve cells, or alternatively each nerve cell contams two compartments. In an 407

anatomical sense, the two compartments m the striatum consist of hundreds of thousands of nerve terminals. In addition, m each compartment translocation of a given substance (e.g , due to release or uptake) may occur that does not necessarily have implications for the (apparent) compartmental analysis. The size of a compartment is usually given m mass units Often it is assumed that a system or its compartments are m a steady state This term mdicates that the rate of removal of a substance is equal to its rate of entry mto the compartment or system. In the present review, a substance enters the compartments by biochemical conversion (synthesis) or by translocation (transport). For instance, m the case of ACh, the substance is enzymatically formed from cholme (Ch), but the substance Ch can enter the compartment either by translocation or transport from the blood or by the hydrolysis of phosphohpids or ACh. The rate of removal from the compartment is determined either by an enzymatic process (degradation of the substance) or by translocation (such as active transport or diffusion) Under steady-state conditions, the rate of entry or the rate of removal from a compartment equals the turnover rate All these terms have the unit mass/time. Turnover rate will be used to denote the quantitative aspects, whereas the term “turnover” indicates m a qualitative sense the process of transference or transformation of a given substance between compartments Turnover time is the (theoretical) time interval required for the renewal of all molecules of the substance present m a compartment. Turnover time is similar to the average life of a molecule of a substance wrthm a compartment Rate constant (k) is used to express the velocity of the entry or exit of substances m the compartment relative to the size of the compartment (thus the turnover rate) A rate constant is then the ratio of the turnover rate m a certain compartment and the amount of the substance m that compartment during steady-state conditions It follows that the turnover time (t) is the reciprocal of the rate constant Another measure for turnover time is the term “half life” (f,,2), which indicates the time mterval required to renew half of a compartment Assummg firstorder kinetics f//2 = In M = 0 693/k Under steady-state conditions, compartments can be shown by usmg tracer substances, which are labeled forms of the substance having either a radioactive or nonradioactive specific isotope The ideal tracer is not distinguished by the biological system under study and rt can be used m very small amounts, so the steady-

Amlne

and Acetylcholrne

Turnover

409

state condition is not disturbed. With neurotransmitters, usually the radioactive isotopes 3H or 14C or the stable radionuclides ‘H, r3C, 180, or 15N have been used. Recently, positron-emitting radionuclldes have been explored to detect cerebral metabolic processes of neurotransmitters in vivo The specific activity is defined as the ratio of the number of labeled to unlabeled molecules of a substance present m a system. Usually the labeled molecules are indicated by units mdicative for their presence, e.g., CI or mass. In turnover studies of cerebral neurotransmitters, the sequence of the conversions is often considered linear. This means that there is a simple sequence of precursor * substance -+ metabolites. The precursor is often assumed to be derived from blood or from an mtracellular compartment close to the site of synthesis, and the metabolites are formed from the substance and eventually leave the central nervous system This scheme is apphcable to biogemc ammes, such as noradrenalme (NA), DA, adrenalme (A), and 5-hydroxytryptamme (5HT), but not to other neurotransmitters, such as the ammo acids and ACh With ammo acids, recurrent labeling processes may occur. So y-ammobutyric acid is converted to metabolites that may be reused for the synthesis of the substance With ACh, the metabolite Ch may be reused as a precursor Both with tracer and nonsteady-state approaches, recurrent or cyclmg processes complicate the mterpretation of the observed alterations m substance content or specific activity m terms of turnover rates. For the determination of the turnover rate with tracer techniques, the compartments of the precursor, substance, and products should be identified (m kinetic terms) In particular, the behavior of the tracer m the compartment(s) of the immediate precursor and products and of the substance itself should be known. As will be shown in later sections, this mformation is usually not available. In addition to tracer studies under steady-state conditions, several mvestigations are based on nonsteady-state approaches. In such cases, drugs are used to block one of the conversions or translocations of the substance, its precursor or its products The ideal drug used should produce a specific blockade of one of the processes involved and should not interfere with any other conversion of the substance, its precursor, or its metabolites, or with other regulatory processes mvolved m the metabolic fate of the substance. The latter Includes not only regulatory processes directly involved m the syntheses or metabolism of the substance, such as receptor-mediated processes, availability of precursor,

410

Korf

and physlologlcal actlvlty of the neurons that use the given substance as a transmitter, but also processes interacting mdlrectly with the metabohc fate of the substance that may include other innervating neuronal systems and physiological parameters, such as body temperature, oxygen supply, or cerebral blood flow and behavioral responses, such as mob&y

2. Scope of the Review The purpose of the present review 1sto introduce students, either inexperienced or more advanced, to the various approaches available for turnover rate measurements m VIVO. In particular, an attempt will be made to describe the underlying and often not explicitly explained assumptions that have been made to extrapolate experimental results in terms of turnover rates The review 1s limited m that it describes only methods used for the estimation of turnover rates m VIVO. There 1s msufflclent space here to permit a detailed review of the effects of pharmacological and physlologlcal mterventlons on the turnover rates of the various substances mentioned For the same reason, neither the criteria that must be fulfilled m order for a substance to be considered as a neurotransmitter or a neuromodulator nor the chemical methods for these estlmatlons will be given here The substances treated include only low molecular weight neurotransmitters or neuromodulators, thus excluding neuropeptides. The review may be helpful in choosing the best available method for turnover rate measurements of a particular substance and it may serve as a reference source for orlgmal papers on the sublect

3. Turnover Methods for 5-Hydroxytryptamine 3.1. Some Biochemical Features The sequence of the formatlon and metabolism of 5-hydroxytryptamme (serotonm, 5-HT) IS usually considered as* Tryptophan (TP) + 5-Hydroxytryptophan (5HTP) + 5-Hydroxytryptamme (5HT) + 5-Hydroxymdoleacetic acid (5HIAA) The precursor TP enters the site of synthesis, the serotonerglc nerve ending or cell body, from the surrounding glla cells, which

Amlne

andAcety/chobne

Turnover

411

derive TP from the circulation and possibly from protein digests. Thus, Tl? in the blood is probably not directly transferred mto the serotonergic neural element. Accordmgly, the specific activity of TP in the circulation differs from that in precursor pool for 5HT synthesis, as will be described further m the section on steadystate methods The enzymatic conversion of TP to 5HTI’ by the enzyme tryptophan-5-hydroxylase,which is not saturated, and the levels of both 5HT and 5HIAA are increased by TP loading of the animal or by several drugs enhancing TP accumulation m the brain (Wurtman et al., 1981; Tagliamonte et al , 1971a, 1971b, 1973; Van Wilk et al., 1979; Korf et al, 1972). It has been suggested, that the nonprotein-bound TP m the circulation is particularly important for maintammg brain levels (Gessa and Tagliamonte, 1974; Taghamonte et al , 1973, Van Wilk et al., 1979). It should, however, be realized that total plasma TP (both free and protembound) is also of significance, since at the site of ammo acid uptake m the brain, extraction of free TP will rapidly induce a new equilibrium of bound and free TP m the capillaries (Partridge, 1979).The TP levels m the blood are also mfluenced by the diet and the liver TP pyrollase (Madras et al , 1973; Hutson et al , 1976, Badawy, 1982), an enzyme whose activity undergoes circadian variation Availability of TP not only influences the metabolism of 5HT, but also the release of the amme m viva. The release of 5HT from the brain of rats stressed by immobilization is enhanced, this enhanced release is further elevated followmg TP loading, but attenuated by concomitant admmlstration of valme, an ammo acid competing with the uptake of TP m the brain (Joseph and Kennett, 1983) The rate of synthem of 5HTP is dependent on the availability of 02, during hypocapma the synthesis is reduced although the TP levels are not affected (Carlsson et al , 1977, Davis et al., 1973a, b,c) The turnover rate of cerebral 5HT has been estimated by both steady-state and nonsteady-state methods. Both approaches will be discussed

3.2. Nons teady-Sta te Methods Drugs are known to block the synthesis, degradation processes of the precursors, the substance, and the Blockade of the formation of 5HTP produced by pacetamide or p-chlorophenylalanme results m a

or transport metabohtes a-propyldolowering of

412

Korf

5HT-levels because degradation of 5-HT is still proceeding (Corrodi and Fuxe, 1968, Koe and Weissman, 1966) The rate constants obtained with this approach are low relative to other methods (0.211 hP’, Corrode and Fuxe, 1968) In 1972, Carlsson and coworkers introduced cerebrally actmg inhibitors of the aromatic ammo acid decarboxylase (5HTP5HTdecarboxylase) (Carlsson et al , 1972) as tools to estimate turnover rate It IS assumed that the rate of 5HTP accumulation reflects the rate of hydroxylation of TP under nondisturbed conditions. Two inhibitors were used, namely NSD-1015 (3-hydroxybenzylhydrazme HCl) and Ro4-4602 [N’(dl-seryl)-N2-(2,3,4-trihydroxybenzyl)hydradro HCl] The rates of synthesis thus obtained were relatively low. Possible sources of errors with this method are mcomplete mhibition of the enzyme (which is not very likely because of the linear accumulation of 5HTP), competition of the drugs with the uptake of TP m the brain or m nerve terminals, of peripherally formed 5HTP m selective accumulation serotonergic nerve endings (Korf and Venema, 1975) and feedback inhibition of 5HT synthesis by hydroxymdoles (see n/so Curzon, 1981). Despite these uncertamties, the method has been widely used for the investigation of drug and other effects on cerebral 5HT synthesis This method is also useful for small brain areas (Tappaz and Pu~ol, 1980), as summarized m Table 1. Monoamine oxidase inhibitors (pargylme, phemprazme) produce a linear increase of 5HT and an exponential decrease of 5HIAA over time These alterations may reflect turnover rate. The validity of this technique relies on the assumptions that apart from 5HIAA no other metabolites are formed, that the rate of synthesis of the substance is not slowed down by the accumulation of 5HT, that the efflux of 5HIAA is not altered by the drug, that all accumulated 5HT is synthesized m the brain, that the declme of 5HIAA levels is monoexponential and that the efflux of 5HIAA IS far from saturated (Costa and Neff, 1970) In early studies, turnover rates rather similar to those determined with decarboxylase inhibitors were calculated from the results with the monoamme oxidase mhibitors, suggesting that the above mentioned conditions are met. More recent data, however, suggest that the accumulation of 5HT and the decline of 5HIAA are biphasic (MorotGaudry et al , 1974) Accordingly, turnover rates of four times or more those of Neff and Tozer (1968) in rat bram were estimated by Morot-Gaudry et al (1974) m mouse brain. Also, high mltial rates of accumulation or decrease were found by Schutte (1976) m the rat brain Van Wilk and Korf (1981) showed that 5HT may be

Amme

andAcety/chohne

Turnover

413

sublect to post mortem metabolism to 5HIAA, and that this IS prevented by the monoamme oxidase mhibitor pargylme So the imtial rise of 5HT levels or the decline of 5HIAA followmg pargylme admmistration can be attributed to artificially low control levels. With the advent of highly sensitive methods for the determmabon of 5HT, the accumulation of the amme followmg MAOmhibition was measured m several brain areas (Neckers and Meek, 1976) 5HIAA is transported out of the brain and cerebrospmal fluid by a probenecid [p-(di-propylsulfamoyl-benzoic acid)]-sensitive mechanism Probenecid given at high doses produces a linear accumulation of 5HIAA m rodents and other species, the turnover rate thus calculated IS higher than that obtained with the decarboxylase mhibitors and similar to that with the monoamme oxidase mhibitors (according to Neff and Costa, 1970, but not according to Morot-Gaudry et al , 1974). The probenecid method assumes that 5HIAA is the exclusive metabohte of 5HT, that the drug does not interfere with the synthesis of 5HT, and that all transport of 5HIAA is indeed blocked Although some 5-hydroxytryptophol has been detected m the brain, its levels are low (approximately 0.060 nmol g-l, Cheifetz and Warsh, 1980), so 5HIAA can be considered as by far the most significant metabolite of 5HT It is, however, likely that probenecid stimulates the synthesis of 5HT, because of the increased availability of TP m the brain (Perez-Cruet et al., 1971; Tagliamonte et al., 1971a,b, Van Wilk et al , 1979, Schubert, 1974) That probenecid mhibits vu-tually all transport of 5HIAA out of the brain can be concluded from the finding that pargyline did not only prevent further accumulation of 5HIAA, but also prevented a decrease of 5HIAA levels (Neff and Tozer, 1966) A summary of the various turnover rates IS shown m Table 1 Taken together, the results indicate that the various nonsteady-state methods produce rather similar turnover rate values, provided that no postmortem artifacts occur. Probably the method based on the exponential decline of 5HIAA following monoamme oxidase mhibition requires the fewest assumptions and may, therefore, be the method of choice.

3.3. Steady-State

Methods

Labeling of cerebral 5HT has been achieved with radioactive 5HT (mtracerebrally administered), TP or 5HTP. Labeling of 5HT by the radioactive amme is problematic because the tracer does not

Monoamme mhlbltors,

Blockade

oxldase accumulation

of 5HT

of 5HTP decarboxylase

Method

Reported

Rat/whole brain Rat/whole bram Rat/whole brain Rat/n raphe dorsahs N raphe centralis N mterpendunculans N tegmentls dorsalls AI group N locus ceruleus A2 group N raphe magnus Hypothalamus Striatum Hippocampus Cortex Cerebellum Mouse/whole brain Rat/whole brain Rat/whole brain Rat/whole bram Rat/dorsal raphe Median raphe Caudate nucleus Hippocampus

10 16 14 34 3 19 0 5 43 4 20 3 47 3 01 2 92 2 83 3 31 1 77 1 43 1 00 0 21 2 44 25 4 6, 1st phase 1 4, 2nd phase 28 1 16 9 1 75 1 69

Reference

Neckers

and Meek,

1976

Morot-Gaudry et al , 1974 Neff and Tozer, 1968 Schutte, 1976

Carlsson et al , 1972 Carlsson et al , 1977 Van Walk et al , 1979 Tappaz and Pu~ol, 1980

5-Hydroxytryptamme

Turnover rate, nmol g-‘h-’

TABLE 1 Rates of Cerebral

Species/ brain area

Turnover

3H-TP 3H-TP + computer

Isotopic labelmg 14C-5HTP 14C-TI’ mfusion/pulse 3H-TP

srmulatron

label

accumulation

Probenead,

5HIAA

mhrbitors,

Monoamme oxldase declme of 5HIAA

Rat/whole brain Whole brain Tele-drencephalon Bramstem Telencephalon Mouse/whole bram

Mouse/whole brain Mouse/whole brain Mouse/whole bram Rat/whole bram Whole brain Rat/dorsal raphe Median raphe Caudate nucleus Hippocampus Mouse/whole brain Whole brain Whole brain Whole brain Whole brain Whole brain Dorsal raphe Median raphe Caudate nucleus Hippocampus Mouse/whole brain 1.75 1519 12 22 1 85 54

7 09 12 48 23 2 35 19 6 11 5 1 32 1 61 8 1.7 40 2.1 1.4 0 46 92 11 5 18 19 16 and Meek,

1976

et al , 1974

Lane et al , 1977 Tracqul et al , 1983b

1975 Baumann, , 1969, Lm et al Costa and Neff, , Neff et al 1971

Morot-Gaudry

Morot-Gaudry et al , 1974 Van Walk and Korf, 1981 Tracqui et al , 1983b Neff and Tozer, 1966 Lm et al , 1969 Schubert, 1974 Neckers and Meek, 1976

Neckers

Morot-Gaudry et al , 1974 Van Wilk and Korf, 1981 Tracqui et al., 1983-1983b Neff and Tozer, 1968 Schutte, 1976

1970

P t;

Korf

416

mix well with all the endogenous 5HT pools and also non-5HTcontammg structures may accumulate the label (Aghalaman and Bloom, 1967, Richards, 1977) The half-life of mtracerebrally mlected radioactive 5HT m the brain 1s about 4-5 h (Aghalaman et al., 1966, Costa and Neff, 1970). Baumann (1975) studied the decline of radioactive 5HTP, 5HT and 5HIAA m the brain followmg mtraclsternal qectron of 14C-5HTP. The half-life of ‘“C-5HTP was 3.5 h, and a turnover rate for 5HT of 1.75 nmol g -’ h-’ was calculated. This method assumes that all labeled 5HTP accumulates m serotonerglc nerve endings and that at the longer times (l-7 h after mtracrsternal inlectlons) the declme of 5HTP-radloactrvrty IS caused only by 5HT synthesis The two assumptrons are rather unlikely as specrflc uptake has not been demonstrated and the turnover time of the 5HTP pool m the brain must be rather high (small size). Warsh and Stancer (1976) pretreated rats with a peripherally acting decarboxylase inhibitor and studied the central and perrphera1 labelm pattern of 5-hydroxymdoles followmg intravenous mJectlon of 5 *C-5HTP This approach has to be further elaborated before defmrtrve conclusrons on its validity can be drawn Several studies have been devoted to measuring 5HTturnover using labeled TP, either Infused IV or given as pulse label Constant rate mfusron for 20-60 mm of 14C-TP reveals relatively sample kinetics when rt 1s assumed that the substance (5HT) IS stored m a single open compartment (Lm et al , 1969; Costa and Neff, 1970) During the mfuslon the speclfrc actrvrty of 5HT(SA,,r) can be described by: , 1 K -~.Illl - k+/“) 1+ S&FIT = (1) k SHT - k,,(k> ‘I’ kTI’ I where

ksHT = k7 I’ = K =

fractional rate constant of the 5HT compartment; fractronal rate constant of the TP compartment; the apparent rate of entry of radroactrve TP mto the plasma compartment and t = time of mfuslon.

These experiments revealed turnover rates m the whole rat brain of about 1.5-l 9 nmol g-‘h-‘. The same assumptions were applied m experiments of Costa and coworkers using pulse qectrons of “H-TP. This method was preferred because rt avoided possible stress effects (Neff et al., 1971) The fractional rate constant (ksHT) was calculated by the “fmrte difference method.” This method 1s as follows (Neff et al., 1971), “To estimate the synthesis rate of the monoammes, we

Amine and Acetylchobne Turnover

417

measured the specific activity of the ammo acid (TP) and the monoamme (5HT) m tissues at various times during four hours after mlectmg TP (or TYR m the case of catecholamme turnover studies) iv. Then the average specific activities of the ammo acid and the monoamme values were plotted on semilogarithmic graph paper and the best-fit lme connecting the points was drawn by eye.“ The graph was divided mto consecutive 20 mm intervals and ksHT (or kNA, k,,,J calculated for each interval with: S&HT(~

k5HT = (SAY,r

-

S&HT(I,)

t, - t, -

S&HI),,

+

(SAP

-

S&tr),,

(2)

2 This equation was derived from the simple equation

dS&tu

=

&S&r

-

%HT)

dt by equating dt = t, - f2. This assumptron 1s permissrble at the declining part of the time course of the specific activities of TP and 5HT. The fractional rate constants of 5HT m the tele-diencephalon and bramstem were 0 61 and 0.75, respectively, equivalent to turnover rates of 1.2 and 2 2 nmol g -‘h-i Rather similar specific activity time courses of changes m TP and 5HT after mtravenous tracer inJections of TP were found by Schubert (rat, 1974); Lane and Aprison (rat, 1978), Schutte (rat, 1976) and Tracqui et al (mouse, 1983a,b). It IS therefore tempting to conclude that cerebral 5HT is, indeed, confined to a single open compartment For further evaluation of this concept, the labeling of the metabohte should also be considered, and some discrepant results have appeared Schubert (1974) observed that the maximal specific activity of 5HIAA coincides with the maximal specific activities of 5HT (at t = 110 mm), while Lane and Aprison (1978) found that the specific activity of 5HIAA was always higher than that of 5HT for the whole observation period of 3 h Schutte (1976) did not develop the curve for a sufficiently long time to permit conclusions on this issue Tracqui et al (1983a,b) showed curves m the mouse brain, with lower specific activity of 5HIAA as compared with 5HT during the time of sampling for 90 mm The question of which peripheral or central pool(s) of TP serves as the precursor pool for 5HT synthesis is, from the kmetic point of view, not answered. Lane and Aprison (1978) deter-

418

Korf

mined the specrfrc activrty of 5HTP over time and observed that in most rat brain areas (cerebral cortex, striatum, hrppocampus, diencephalon and bramstem, but not cerebellum) the maximum specific activity of 5HTP comcides with that of the curve for TP in that area (at about 20 min), thus mdrcatmg a simple precursor-product relationshrp. The levels of endogenous 5HTP, however are probably too high, and if so, then the maximum SAsHTP exceeds the TP curve severalfold. Tracqm et al (1983a,b) designed a kmetlc model of 5HT metabolism m the whole mouse brain This model consists of three TP compartments, one of which serves either as the precursor for 5HT or for the central precursor pool of TP mvolved m the synthesis of 5HT. The substance, 5HT, IS confined to two compartments, correspondmg to 25% and 75% of total 5HT. In the smaller compartment, 5HT is synthesized and 1s the precursor pool of 5HIAA. The larger compartment represents a storage pool with exchange of 5HT to the other compartment In the rat brain, evidence for compartmentation is also described by followmg the loss of radroactrvrty of 5HT (previously labeled with TI’ during electrical simulation of serotonergrc fibers (Shields and Eccleston, 1972). In this study, the specific activity of 5HT was lower m strmulated rats, as compared with nonstimulated animals. In the model of Tracqul et al. (1983a,b) 5HIAA 1s also confined to two compartments In the smaller compartment, 5HIAA is synthesized and may be transported out of the brain from that compartment The authors tried several alternative models, but none was adequate Despite the elegance of the approach,the model should be further developed, as the turnover of 5HT m this model is srmllar to that of earlier studies m which postmortem artifacts have not been ruled out (see sectron 32)

3.4. Conclusions Several methods are presently available for the estimation of the turnover of 5HT The nonsteady-state methods reveal turnover rate values m the whole brain of 2-3 nmol g-‘h-’ or of 6-9 nmol The latter value may be affected, at least m part, by postg-‘h-’ mortem artifacts The isotopic methods result in turnover rates below 3 nmol g- ‘h-’ except m one study in which higher rates were given In that’ study, computer-simulated models were shown for the mouse brain, suggestmg rt 1s unlikely that 5HT metabolism can be explained m terms of single open compartments.

Amlne

and Acetylchohe

419

TUFf?OVeF

4. Turnover Methods For Dopamine 4.1. Some Biochemical

Features

Dopammerglc neurons innervate widely dispersed brain regions, but the emphasis of this section 1s on DA turnover m the rat striatum. The sequence of DA formatron and metabolrsm IS generally assumed to be as follows: Tyrosine

(TYR) + H

-

3,4-Drhydroxyphenylalanme

-+

Conlugates

DA -+ 3,4-Dlhydrclxyyhenyl~c~tlc

ad /

+ 3-Methoxvtvranme , -

(DOPA)

(DOPAC)

(HVA)

(3MTA)

The precursor TYR orrgmates m the crrculatron and from cerebral protein digest, and rt enters the dopaminergic nerve ending probably after diffusion from other cells (such as glra cells) and extracellular space (as u-r the case of TP). Two possible routes of DA metabolrsm have been described. After the actions of monoamme oxrdase and aldehyde dehydrogenase, DOPAC 1s formed, which is further methylated to HVA or conlugated to sulfonyloxy-DOPAC HVA IS also formed from 3MTA by oxidatrve deammatron and may m some species be further conjugated (at least u-r part) before transport out of the brain According to Wurtman and coworkers, the synthesis of DA IS dependent on the avarlabrllty of TYR m the cuculatron under physrologrcal condmons (Wurtman et al , 1981, and references therem) Thus increased metabohsm of DA was found followmg TYR loadmg of rats Others, however, were unable to fmd mcreased levels of the metabolrtes of DA in the rat strlatum even when the dopammergrc neuJons were activated by drugs or electrical stimulatron (Westermk and Wrrix, 1982, Korf et al , 1976) Under particular condmons, when the aromatrc ammo acid decarboxylase actrvrty IS blocked (for measurmg DOPA synthesis, see below), TYR loading may increase TYR hydroxylatron (e g , Wurtman et al., 1974; Carlsson and Lmdqvrst, 1978). However, under this experimental condmon, the rate of DOPA accumulation 1s substantially decreased The actrvmes of tyrosme-3-hydroxylase and of monoamme oxldase are Influenced by CO2 and O2 tension (Davis and Carlsson, 1973a,b,c; Carlsson et al., 1977) Thus, hypercapma and

Korf

420

hypoxia may cause an increase in the DOPA no changes in DA levels were found.

syntheses;

however,

4.2. Nonsteady-State Methods Followmg mhrbltlon of tyrosme hydroxylase by a-methyl-ptyrosme (or its methyl ester) a more or less exponential decline of the levels of strratal DA were found (Costa and Neff, 1970, and references therein). Assummg an exponential decline, turnover values of about 20 nmol g’h-’ (e.g , Doteuchr et al , 1974, calculated from the second part of the disappearance curve of Javoy and Glowinskr, 1971, Costa and Neff, 1970, and references therein) were found These estimatrons are based on data obtamed followmg mhibitron of tyrosme hydroxylase for 2 h or longer At early time intervals, some lrregularltles m the decline of DA were mrtrally observed by Javoy and Glowmskr (1971) and later confn-med by Doteuchr et al (1974) and Paden (1979) Accordmg to the first two authors, the mrtral rapid decrease following a-methyl-p-tyrosme admmlstratron had to be ascribed to the existence of a small pool wrth a high turnover rate (70 nmol g-‘hP1), whrle Doteuchr et al. (1974) suggested that metabohtes of a-methyl-p-tyrosme (p-hydroxyamphetamme and p-hydroxynorephedrine) caused a partial depletion of strlatal DA. The essential prerequisite of the mterpretatron of Glowinskr and coworkers, that a-methyl-p-tyrosme inhrbrts the synthesis of DA within a few minutes after admmrstratron, could not be substantiated using 3H-TYR to measure the formation of the amme (Paden, 1979). The formatron of DOPA, which reflects DA synthesis under steady-state conditrons, was studied by measuring the rate of the accumulation of DOPA following admmrstratron of decarboxylase mhlbltors (see also section 3.2) The rate of DOPA accumulatron m the rat strratum 1s approximately 10 nmol gP’h-’ (Carlsson et al., 1972, Walters and Roth, 1974, Westermk and Spaan, 1982). This value IS less than those obtamed with other turnover methods (see below, Korf, 1981; Sharman, 1981) This discrepancy may be caused by low TYR uptake or by DA receptor-mediated feedback mhrbrtron of DA synthesis as a consequence of DA accumulatron caused by the decarboxylase mhlbltors, which also inhibit monoamme oxrdase (Carlsson et al , 1976). The levels of DOPA (m the brain of rats not treated with decarboxylase inhibitors) are altered by various drugs Generally, drugs that increase the metabohsm of DA also increase DOPA levels, whereas drugs with the opposite effects on DA metabolism reduce the levels of DOPA (Westermk et al., 1982)

Amlne

and Acetylchobne

Turnover

421

Inhlbltlon of monoamme oxldase results m increases of DA and 3MTA and decreases of DOPAC and HVA. Turnover rates of DA have been estimated from the increases of the amines or decreases of the acidic metabolites. According to Javoy et al. (1973), the increase m DA 1s blphasic (rapid initial increase, suggesting a high rate of synthesis), but others have not confirmed this fmdmg (Kehr, 1976, Westermk and Spaan, 1982) As with the 5HT mcreases after admmlstratlon of monoamme oxldase mhlbltors (sectlon 3.2), postmortem degradation may result m abnormally low DA levels m control animals (Le Roy Blank et al., 1979). From the rate of 3MTA accumulation followmg mhlbltlon of monoamme oxldase, DlGuillo et aL(1978) estimated 3MTA turnover Under control condltlons virtually all DA 1s metabolllzed to DOPAC, so 3MTA accumulation after monoamme ox’dase mhlbltlon may better reflect DA turnover A rate of 18 nmol g -‘h-l was calculated from the m’tlal rapid appearance of 3MTA Using a rapidly acting mhlbltor of catechol-O-methyltranferase (tropolone), Westermk and Spaan (1982a) estimated the turnover rate of 3-MTA to be about 6 nmol gg’h-‘. Others have, however, doubted whether 3MTA 1s a slgmflcant metabohte of DA, since extremely low levels were found m the rat strlatum under well controlled condltlons (Westenberg et al , 1983) The decline of strlatal DOPAC levels followmg mhlb’tlon of monoamme oxldase IS monoexponentlal, and, assuming that further metabolism m the rat by 0-methylatlon and coqugatlon occurs (Elchlsack et al , 1977, Dedek et al , 1979) and that ellmmatlon from the brain IS not affected, the rate of formation of this metabollte can be calculated. Under these condltlons, the decline of HVA 1s not exponential, but it becomes so after mhlbltlon of catechol-0-methyltransferase (Westermk and Korf, 1976, Dedek et al , 1979) These experiments provide a model which shows that all HVA IS derived from DOPAC, and that about two-thirds of DOPAC 1s converted to HVA, but that one-third of DOPAC leaves the brain conlugated (Dedek et al., 1979). The contribution of 3MTA according to this model 1s modest, which fits with more recent turnover studies of 3MTA (Westermk and Spaan, 1982) Rather slmllar turnover rates of DOPAC and HVA were found with probenecld, provided that not only the accumulation of the free metabolltes but also of the conjugates of DOPAC and HVA was included (Dedek et al , 1979) Turnover rates of rat strlatal DOPAC are about 20-30 nmol g P’llm ‘, of HVA 16 nmol gP’hP ‘, and of 3MTA 2 nmol g ‘h-’ (Dedek et al , 1979, Westermk and Spaan, 1982a,b, Westermk et al , 1982) The con~u-

422

Korf

gates of DOPAC and HVA are formed at a rate of 5 nmol g’h-’ (Dedek et al., 1979). The latter results were obtained by computer fitting by assuming the sequence of DA metabolrsm as descrrbed in section 4.1.

4.3. Steady-State Methods When labeled TYR mfusrons or bolus mlectrons are used, DA becomes rapidly labeled. As described m section 3 3, turnover may be calculated assuming simple open compartment kmetrcs. It has been reported that the specrfrc activity of striatal DOPA follows that of TYR (Doteuchr et al , 1974), thus suggestmg that SATYR or SADoF* are equivalent for further computatron However, u-r this report, the levels of endogenous DOPA are probably at least two times too high (compare levels with e g., Westermk et al , 1982), and, rf so, the specific activity of DOPA 1s at least twice as high as that of strlatal TYR The concept of a single open compartment of DA was not substantiated when the specrfrc actrvrtres of the DA metabolrtes were measured followmg intravenous or mtracisternal mlection of radioactive TYR. Gropettr et al , (1977) observed higher specific activmes of DOPAC and 3MTA than of DA itself after labeling with mtracerebrally apphed TYR. Van der Krogt et al , (1981) showed that the specific actrvmes of DOPAC and HVA were already substantrally higher a few minutes following mtravenous inJectron of 3H-TYR. Thus DA-containing neurons have a rather small pool u-r which DA 1s preferentially synthetrzed (Papeschr, 1977) and degraded and from which DA may be released upon electrical strmulatron of the nigrostrratal pathway (Korf et al., 1976; Murrm et al., 1976) or by other stimuli (Nreouillon et al , 1977)

4.4. Conclusions Several nonsteady-state methods reveal rather similar turnover rates for DA. Accordingly, the turnover rate of DOPAC reflects DA turnover (at least in the rat stnatum), although the contribution of 3MTA is questionable The steady-state methods are stall under development, as compartmentation IS not yet fully understood and no models to describe the labeling patterns of the precursor/product relatronshop have been developed. A summary of reported turnover rates for DA IS shown m Table 2.

Amine

and Acetylchollne

Turnover

423

5. Turnover Methods for Noradrenaline 5.1. Some Biochemical Features In the central nervous system, DA is the immediate precursor of NA, while the major metabolites are normetanephrme (NMN), 3,4-dlhydroxyphenylethylene glycol (DOPEG; DHPG), 3-methoxy-4-hydroxyphenylethylene glycol (MOPEG; MHPG) and the sulfonyloxy conlugates of these alcoholic metabolites. At present, there 1s no model available to describe the precise sequence of the conversions, but it may occur as shown below.

The formation of DA from TYR 1s generally believed to proceed as m dopaminerglc neurons (section 4.1). In the urine vamllylmandelic acid (VMA) is a major metabollte of NA, formed directly from the amme or from MOPEG (Blombery et al , 1980) In the rat and human brain, however, VMA 1s a minor metabollte of NA, possibly not orlgmatmg m noradrenerglc neurons (Karoum et al., 1976; Langer, 1974,

Inhlbltlon of DA)

of synthesis

Method

(decline

Turnover Species/ brain area

Dopamme

1 79 1 83 2 42 1 29 21 7 25 6 96 3 1 70 26 0 26 32 11 29 06 55 19 5 02 05 48 35 30 20 07

Turnover rate, nmol g-‘h-’

TABLE 2 Rate Values of Cerebral

Rat/whole brain Whole bram Different rat species Whole brain Striatum Striatum Striatum 1st compartment 2nd compartment Raticaudate nucleus Dorsal septal nucleus Lateral septal nucleus Penventricular nucleus Paraventricular nucleus Nucleus hypothalamus anterior Arcuate nucleus Median eminence CA2 Dorsal raphe nucleus A9 region A10 region A6 region A2 region Nucleus commlssuralls

Reported

Versteeg

et al , 1978

Wlderlov and Lewander, 1978 Costa and Neff, 1970 Papeschl, 1977 Doteuchl et al , 1974 Paden, 1979 Javoy and Glowmskl, 1971

Reference

oxldase

of dopamme

of DOPAC)

(declme

Blockade of egress metabolltes “H-TYR qectlon ‘H-TYR ml ection

of DOPAC)

of 3MTA)

(decline

(accumulation

Inhlbltlon of monoamme (accumulation of DA)

Inhlbltlon of decarboxylase (accumulation of DOPA)

Ratistnatum Rat/whole brain

Ratistrlatum Nucleus accumbens Tuberculum olfactorlum Frontal cortex Striatum Ratistrlatum Tuberculum olfactorlum Frontal cortex Ratistrlatum Striatum Strlatum Tuberculum olfactorlum Ratistnatum Llmblc areas Striatum Tuberculum olfactorlum Frontal cortex Rat/nucleus accumbens Hypothalamus Hlppocampus Occipital cortex Brain stem Cerebellum Striatum Ratistnatum 10 2 21 2 21 9 14 15 6 (15 2) 20 33 0 65 170 18 75 45 23 17 25 5 (18 4) 38 (32 9) 0 76 30 0 0 77 0 12 0 16 0 28 0 11 20 21 21 21 7 3 33 and Spaan,

and Korf,

and Spaan,

1976

1982

1982

et al , 1979 et al , 1979 Doteuchl et al , 1974 Lane et al , 1977

Dedek Dedek

Westermk and Spaan, 1982, Westermk et al , 1984 Westermk et al , 1984

Westermk

Westermk

Javoy et al , 1973 DlGlullo et al , 1978

Westermk

Walters and Roth, 1974 Westermk and Spaan, 1982, Westermk et al , 1984

2 3 2 2

g 2

3

? 2

rb 2

426

Korf

Nrelsen, 1976; AdPr et al , 1978). The contributron of NMN to the turnover of NA remains to be established (Kehr, 1981). Evidence has appeared that the synthesis rate of NA or its metabolites IS independent of clrculatmg TYR, either under normal conditrons or m reserpmlzed rats (Orshl and Wurtman, 1982). However, the levels of endogenous DOPA are increased by TYR (Westermk et al., 1982). There are two rate-lrmltmg enzymes mvolved in the synthesis of NA, namely tyrosme hydroxylase and dopamme+hydroxylase. The content of the Immediate precursors of NA (DA and DOPA) in noradrenergrc neurons are less than 5% of the NA content (Brschoff et al , 1978, Westermk et al , 1982). Therefore, these are ideal precursors with which to study the labeling patterns with radioactrve TYR The synthesis of NA IS dependent on 02/C02 pressure (see also section 4.1 for further details).

5.2. Nonsteady-State

Methods

Followmg mhrbrtron of NA synthesis erther at the level of tyrosme hydroxylatron or at the level of dopamme B-hydroxylatron, the content of NA declines exponentrally. The mhlbrtors used are a-methyl-p-tyrosme (Costa and Neff, 1970, Brodre et al , 1966, Papeschr, 1977), drethyldrthrocarbamlde (Goldstem, 1966) or FLA 63 (brs[4-methyl-1-homoprperazmylthrocarbonyl] drsulfrde, Svensson and Waldeck, 1969) Turnover rates u-r the rat brain of approximately 650 pmol gP1hP’ have been found. The rate of hydroxylation of TYR can be assessed wrth DOPA decarboxylase mhrbrtors, provided that there IS no coexrstence of dopammergrc mnervatron (drugs, see sectron 3.2) The rate of hydroxylatron has been determined to be about 480 pmol g- rh- ’ m the hemispheres (Carlsson et al., 1976). Such a figure may not mdrcate the rate of hydroxylation m noradrenergrc neurons /?er se, because the contrrbutron of dopammergrc neurons 1s not known It IS, however, surprrsmg that this turnover rate IS rather low as compared wrth other methods. Attempts have been made to quantify the rates of formatron of the varrous metabolrtes of NA. The turnovers of unconlugated MOPEG and of MOPEG-sulfate were estimated either from pargylme-induced (exponential) decline or from probenecrdproduced increase (Meek and Neff, 1973, Karoum et al , 1976; Nielsen and Braestrup, 1976, Ader and Korf, 1979, Lr et al , 1981, 1983, Kohno et al , 1981) The turnover rates of MOPEG and Its conjugate are about half that of the turnover of NA determined by synthesis mhrbrtlon. Therefore m addrtron to the 0-methylated

Amrne

and Acefylchol~ne

Turnover

427

products, DOPEG and its sulfate conlugate also contribute at least half the fraction of NA metabolites (Scatton, 1982; Warsh et al., 1981; Li et al., 1981; Jackman et al., 1982). Li et al. (1981, 1983) showed that u-r the hypothalamus, the midbrain, the brainstem, and the cerebral cortex more conlugated DOPEG than MOPEG accumulated following probenecid. Therefore, both conlugates are the final NA metabolites that leave the brain by active transport Free MOPEG IS probably the only precursor of MOPEG sulfate, as the rates of formation determined for the two metabolites are the same usmg the pargylme method or probenecid accumulation (Ad& and Korf, 1979; Meek and Neff (1973). Free MOPEG may be derived from NMN or DOPEG Labelled DOPEG or NMN are both converted to MOPEG (Eccleston and Ritchie, 1973, Gale and Maas, 1977, Schanberg et al., 1968) The formation of MOPEG from radioactive NA is enhanced by conditions favoring extraneuronal metabolism [after destruction of NA-containing neurons or after treatment with desmethylimipramme (Glowinski and Baldessarmi, 1966; Braestrup and Nielsen, 1975, Nielsen and Braestrup, 1977; De Met and Halaris, 1979)]. Whether, however, MOPEG is formed exclusively from extraneuronal NA is doubtful, as after electrical stimulation of cerebral noradrenergrc neurons or after various drug treatments which enhance firing activity of these neurons, the levels of DOPEG are Increased (Nielsen, 1976; Scatton, 1982; Warsh et al., 1981, Jackman et al , 1982). Despite the uncertamties mentioned, a tentative scheme for NA metabolism m the rat brain has been given above. Unlike the case of DA, the scheme has not been rigidly tested.

5.3. Steady-State Methods The approaches used for turnover rate measurements of NA under steady-state conditions are based on labeling of NA with mtracerebrally applied radioactive NA or with systemically applied radioactive precursors. The first method, used by Iversen and Glowinski (1966), requires that the label mixes completely with endogenous NA so that metabolism of the labeled and endogenous NA is similar (Costa and Neff, 1970). The decline of labeled NA is monoexponential and the turnover rate calculated from the apparent fractional rate constant and endogenous levels of NA is about 210 pmol gg’h-’ in the rat cerebral cortex, substantially lower than that obtained with various other methods The second method, developed by, e g , Sedvall et al (1968),

428

Korf

Neff et al. (1971), and Nielsen (1976), required knowledge of the kinetic behavior of the immediate precursor of NA (DA m NAcontammg neurons) for turnover rate calculations. This IS not as yet fully possible. Some approxlmatlons were therefore made (as m the case of DA or 5HT turnover methods with labeled precursors) As an index for turnover rate, the conversion index was used. This is the ratio of the specific actlvlties of the substance (NA) and TYR m brain tissue, obtained after constant infusion of the tracer (Sedvall et al , 1968, Costa and Neff, 1970). As the size of the precursor pool IS unknown, the absolute turnover rate cannot be calculated but, provided that this pool does not change after various drug treatments, alterations in turnover rates may be noticed. This assumption has recently been challenged, as drug treatment may change cerebral levels of TYR and DOPA (Westermk et al., 1982, Westerink and Wu-lx, 1982). Moreover, single compartment kmetlcs have to be assumed Similar assumptions were applied m an attempt to calculate absolute turnover rates of NA (Costa and Neff, 1970). During constant infusion of TYR (K), the specific activity of the ammo acid increases according to* SA TYR where

=

LL(l

kTYR = the rate constant

The specific

activity

-

e-hq

of plasma

of cerebral

(4) TYR

NA (SAN,)

can be described

by* SA NA where

&A

K =

-

k TYR

1+

1 k NA

IS the fractional

-

(kTyRe-k”Af - kNAe-hf) kTYR

rate constant

of cerebral

NA.

1 (5)

The experimental procedure requu-es immoblllzatlon of the amma1 during the infusion (for 20-60 mm), so stress effects are likely to occur Such an artifact may be overcome by constant infusion through the jugular vein by an implanted cannula, as used for DA by Van Valkenburg et al (1983) and Lane et al. (1977). The advantage of the above procedure 1s that there IS only one time interval to be measured. An alternative approach described by Costa and coworkers 1s to follow specific actlvitles of the precursor and amines after a bolus mlection of radloactlve TYR (as outlined m section 3 3 for 5HT) Again the mathematical equations were slmpllfled to the

Amine and Acetylchobne Turnover

429

fume difference method (formula 2, section 3.3). By taking mtervals of 20 mm (t2 - ti) Neff et al. (1971) found kNA values of 0.17 0 35 h-’ for the tele-diencephalon and brain stem The calculated turnover rates for these brain areas were 0.59 and 0.83 nmol g-‘h-l, respectively. The radioactivity curves of MOPEG (free t conlugated) and DOPEG sulfate were determined followmg mtracerebral mlection of radioactrve TYR (Nielsen, 1976). The rate constants for drsappearance of the two metabohtes were rather similar, mdicatmg the rates of formatlon are equally fast From these k values and the levels of the conlugates found by Li et al. (1983), the rates of formation of the NA metabohtes were calculated by this reviewer, and they appeared to be close to those calculated from the probenecid method (section 5.2). In the experiments of Nielsen (1976), the specrfrc activities of NA and of the two metabolrtes (MOPEG and DOPEG sulfate) are almost the same l-2 h after admmrstration of the 3H-TYR mtraventrrcularly, suggesting that Indeed the system can be described as containing single open compartments. Almost equal rates of formatron of the two sulfonyloxy conlugates were also found by Eccleston and Ritchie (1973) and Sugden and Eccleston (1971) after mtracisternal mlectron of 14C-NA or 3sS042P for labeling.

5.4. Conclusions The highest turnover rates of NA are found by the a-methyl-ptyrosme method, by the labellmg method with mIection or infusion of 3H-TYR or by measuring the formation of the two conlugates. In all these cases turnover rates of about 600-800 nmol were determined for whole rat brain o-’ As adrenaline is probably metabolrzed to the same conlugates, these values may be somewhat too high On the other hand the contribution of this amme to the metabolite formation m, e.g., the telencephalon is probably negligible (see section 6). A malor advantage of the a-methyl-p-tyrosme method is that using the current highly sensitive methods for NA detailed regional turnover measurements can be performed (Versteeg et al , 1975; 1978). A summary of turnover rates is shown m Table 3

6. Turnover Methods For Adrenaline Cerebral adrenaline sized from NA

(A) is stored m separate neurons A 1s syntheby the enzyme phenylethanolamine-N-

Synthesis

mhlbltlon (decline of NA)

Method

Reported Turnover rate, pm01 gP ‘h-’

Species/ brain area

Rat/median eminence Arcuate nucleus area Rat/dorsal septal nucleus Lateral septal nucleus Nucleus mterstltlahs strlae terminalis Supraoptlc nucleus Periventrlcular nucleus Medial forebrain bundle Paraventricular nucleus Nucleus hypothalamus anterior Arcuate nucleus Median eminence Parafascicular nucleus Ventral thalamlc nucleus Lateral posterior thalamlc nucleus

Rat/whole brain

4500 9900 5400 4350 2800 4500 1700 2650 1600 550

213 420 562 497 480 1095 3248 610 3110 2600

Noradrenalme

TABLE 3 Rates of Rat Cerebral

Various rat species/ whole brain

Turnover

Versteeg et al , 1978

Papeschl, 1977 Wlderlov and Lewander, Versteeg et al , 1975

Costa and Neff, 1970

Reference

1978

MOPEG or MOPEG sulfate (decrease followmg monoamme oxrdase mhrbrtron) MOPEG, MOPEG sulfate, DOPEG sulfate (disappearance followmg MAO mhibmon) Pulse qectron of 3H-TYR

DOPEG sulfate (accumulatron followmg probenecrd)

Ratltele-drencephalon

Posterror thalamrc nucleus Subrculum Gyrus dentatus CA2 DorsaI raphe nucleus A8 regron A9 region A10 region A6 region Al region Nucleus tractus solltaru A2 region Nucleus commrssuralrs Rat/hypothalamus Midbrain Brain stem Cerebral cortex Hrppocampus Strratum Cerebellum Rat/whole bran-r Rat/whole brain Rat/whole brain Rat/whole brain

590

1700 2100 2400 4800 2600 1540 2080 1640 10870 2000 3500 7980 1270 2800 734 650 372 325 284 104 202 193 240 502

Neff et al , 1971

Karoum et al , 1976 AdPr and Korf, 1979 Nielsen and Braestrup, Nielsen and Braestrup,

Lie et al , 1981, 1983

1976 1976

MOPEG sulfate (accumulation followmg probenecld)

Constant Infusion of 3H-TYR Pulse qectlon of 3H-TYR Synthesis mhlbltlon (accumulation of DOPA) MOPEG sulfate (accumulation followmg probenecld)

Method

3 (colztrnued)

200 320 1030 600 530 580 550 310 240 220 50 951 297 393 281 222 212 53

Rat/whole brain Rat/whole brain Hypothalamus Thalamus Pons + medulla Midbrain Amygdala Hlppocampus Basal ganglia Cerebral cortex Cerebellum Rat/hypothalamus Midbrain Brain stem Cerebral cortex HIppocampus Striatum Cerebellum oblongata

830 710 350 487

Turnover rate, pm01 g-‘/z-’

Brain stem Rat/whole brain Rat/whole brain Ratlhemlspheres

Species/ brain area

TABLE

et al , 1981

LI et al , 1981, 1983

Kohno

Costa and Neff, 1970 Lane et al , 1977 Carlsson et al , 1976

Reference

6 %

AmIne

and Acetylchobne

Turnover

433

methyltransferase, and the metabolism of A leads to the same metabolltes as NA (probably MOPEG and DOPEG and their conjugates, (e.g , Fuller, 1981, Ader and Korf, 1979). Nonsteady-state methods for A turnover have been described, based on synthesis inhibition by c-w-methyl-p-tyrosme (Versteeg et al 1978) or by FLA 63, a dopamme-@hydroxylase inhibitor (Scatton et al., 1979) or on specific inhibition of phenylethanolamme-N-methyltransferase (e.g., Fuller et al 1981) The turnover rate of A in two hypothalamic nuclei (penventricular nucleus and paraventncular nucleus) and m the A2 region of the brain stem have been estimated to be approxrmately 0.3, 4 6, and 0 15 nmol g-‘h-‘, respectively (Versteeg et al., 1978, a-methyl-p-tyrosme method). Similar fractional constants were found by Scatton et al. (1979) with FLA 63 (0 2 h-‘), leading to calculation of turnover rates for the hypothalamus of 25 pmol gg’h-‘, for the Al region of 55 pmol g-‘h-l and for the A2 region of 82 pmol g-‘h -I. Fuller et al. (1981) found a rate constant in the hypothalamus of 0 18 h-’ using 2,3-dichloro-cu-methylbenzylamme, an mhrbitor of methylatlon. Taking these data together, all the mentioned methods give rather similar fractional rate constants. The resultant turnover values of A vary, which may be due, lntev alla, to differences in dissection of the rather small brain areas to which A is confined

7. Turnover Rate Methods for Acetylcholine 7.1. Some Biochemical Features Cholme (Ch) is both a precursor and degradation product of acetylcholme (ACh), so no simple linear metabolic relationship as is the case for the mdoleammes or catecholammes, seems applicable. This more complicated sequence of reactions lrmrts the possrbilitles for both steady-state as well as nonsteady-state methods for turnover rate measurements. In addition, the methods for the determmatron of Ch and ACh are in general rather complicated as compared with those of the ammes ACh 1s synthesized from Ch by the enzyme cholme acetyltransferase, which couples Ch and acetyl-Co enzyme A. The ongm of Ch, serving as a precursor for ACh synthesis, is not well established, at least four sources have been proposed, namely phospholrpids m the brain, phosphatidylcholme or lysophosphatidylcholme m the circulatron, ACh followmg hydrolysis by acetylcholme esterase, and blood Ch (Ansell, 1981, Freeman and Jenden, 1976, Tucek, 1983). The acetyl moiety of ACh 1s derived

434

Korf

from glucose and pyruvate, the importance of citrate as a source has been questioned (detailed discussion by Tucek, 1983, 1985, Lefresne et al., 1973, Rospars et al , 1977, Sims et al., 1982; Hrdina, 1974). The synthesis and content of ACh m the synaptosomal fraction has been proposed to depend on cholmergic activity (Jenden et al , 1976, Murrin and Kuhar, 1976; Kuhar and Murrm, 1978). In particular, the sodium-dependent high affinity uptake system for Ch is enhanced or depressed at higher or lower activeties (either induced pharmacologically or electrically) of cholmergic neurons, respectively (seeKuhar and Murrm, 1978). A low affinity uptake of Ch into ACh-containing nerve endings is, m addition to high affinity uptake, also important for the synthesis of ACh (reviews Haubrich and Chippendale, 1977, Kuhar and Murrin, 1978). In vitro, ACh is compartmentalized, as only a fraction of ACh m nerve-endings is released followmg depolarization and this fraction is derived from precursors added to the mcubatron medium (Collier, 1969, Suzkiw and O’Leary, 1982, Richter and Marchbanks, 1971, Molenaar et al , 1973). The relative sizes of the vesicular and cytoplasmlc pools of ACh in synaptosomes have been estimated to be 4 6 (Suzkiw and O’Leary, 1982) By allowmg post-mortem degradation of ACh, two pools also become apparent The ACh that is degraded withm the first 10 seconds comprises 3040% of all the ACh The remaining ACh is more stable postmortem (Norberg, 1977, Cedar and Schuberth, 1977). As the enzyme cholme acetyltransferase is not saturated m vivo and because the acetylation reaction is, at least m prmcrple, reversible, the question arises as to whether systemic admmistration of Ch increases cerebral ACh levels and/or synthesis. Increased ACh levels were reported by some workers after Ch chloride admmlstration or by mcreasmg dietary content of Ch (Cohen and Wurtman, 1975,1976; Haubrich et al , 1975, Haubrlch and Chippendale, 1977) but were not found m more recent reports (Flentge and Van den Berg, 1979, Consolo et al , 1979, Brunello et al., 1982, Trommer et al., 1982, Wecker and Schmidt, 1979). It is, as yet, uncertain whether precursor loading mcreases the ACh levels under experimental conditions with an enhanced utilrzation of the transmitter. Jope (1982), Wecker et al. (1978), Schmidt and Wecker (1981) and Trommer et al (1982) found that administration of phosphorylcholme and choline prevented depletion of rat striatal ACh by atropme, pentylenetetrazole or

AmIne and Acetylchobne Turnover

435

fluphenazme These observations have been challenged recently (F Flentge, personal communication). Of paramount importance for turnover measurements are postmortem artifacts. Without adequate measures to prevent postmortem changes, the levels of ACh can decrease to less than one-half normal values and the Ch levels can rise severalfold. Thus turnover rate measurements are possible only by using mlcrowave irradiation or rapid freezing techniques to prevent these alterations.

7.2. Nonsteady-State

Methods

Two approaches have been applied: the inhibition of synthesis or blockade of degradation. Followmg blockade of ACh synthesis with hemlcholmium-3 (by local appllcatlon), decreased ACh levels were found (Dommo and Wilson, 1972, Schmidt and Buxbaum, 1978) that were nearly exponential By recalculatmg the data of Schmidt and Buxbaum (1978), and assummg first order kmetlcs and a single compartment of ACh, approximate turnover rates (m nmol g-‘h-l) were obtained for the following brain regions: striatum (140), midbrain (37.6), hypothalamus (20), and hippocampus (60). The speclflclty of this drug m mhlbitmg ACh synthesis has been questioned as it blocks predommantly neuronal uptake of Ch (Rommelspacher and Kuhar, 1974, Ansell, 1981). If so, then these experiments show the dependency of ACh synthesis on the recapture of extracellular Ch Inhibition of ACh esterase by paraoxon or dlchlorvos produces an accumulation of ACh, which is, in most brain areas, maximal wlthm 5-15 min (Stavinoha et al , 1976; Wecker and Dettbarn, 1979). The experiments with dichlorvos show maximal apparent turnover rates for several rat brain areas (in approximate nmol g-‘h-’ values, recalculated from the data of Stavmoha et al., 1976)-striatum, 350, hippocampus, 190, cerebral cortex, 130, cerebellum, 22; medulla-pons, 100; midbrain, 54; and thalamus, 50 With paraoxon, the followmg turnover rates (nmol g-‘h-l) were calculated* stnatum, 144; hippocampus, 22; and cerebral cortex, 30 These values were higher when the rats were pretreated with Ch The originators of the latter method conclude that the Ch pool for ACh synthesis 1sdepleted as the result of the esterase inhibition, and that under such circumstances elevation of circulatmg Ch stimulates ACh formation. On the other hand, the data with dlchlorvos indicates that synthesis of ACh can proceed at a much higher rate, without signs of precursor pool depletions.

436

Korf

7.3. Steady-State Methods 7.3.1. Pulse Labeling Attempts to assess the turnover rate of cerebral ACh have been made following mtravenous admmistratron or mfusion of labeled Ch or phosphorylcholme or after mtracerebral mlectron of Ch. Intravenous admmistration of 3H-Ch produced specific labeling patterns for Ch and ACh m several brain areas, consistent wrth smgle compartment kmetics (Nordberg, 1977, Nordberg and Sundvall, 1976). These data were analyzed with various procedures [graphical method of Zilversmit, 1960 ; the fmrte difference method of Neff et al , 1971, from the slope of the lme obtained by plotting SAch - SAACh against d(labeled ACh)ldt, according to Jenden et al., 1974, from the ratio of radioactivities of ACh and et al., 1969, and from the method of Saelens et S&h, Schuberth al., 1974, which is aimed to correct for the size of the precursor pool] Apparent mean turnover rates (nmol gg ‘h-l) were* striaturn, 2500, hippocampus, 1200; and cortex, 1600. The highest and most consistent values were found when early time pomts were used, indicating that during the first 2 mm the assumptions may be correct. Vocci et al. (1979) determined the time course of the specific activities of ACh and Ch followmg a tracer mjection 3H-Ch of big h s p ecific activity. They calculated the turnover rate of ACh with the fume difference method (Neff et al., 1971) and for the ratio of the activities of Ch and ACh multipled by the tissue Ch levels (Schubert et al., 1969). Two turnover rates were thus obtained in whole mouse brain* m the first phase 20 nmol g-‘h-’ (whole mouse brain) and m a later phase 6.5 nmol gg’h-‘. These authors suggest two compartments of ACh m whole mouse brain, which IS in lme with a study of Jenden et al. (1974) A serious problem in the labeling methods is the identification of the precursor pool of Ch Schubert et al (1969), Saelens et al (1974) and Atweh and Kuhar (1976) observed that during the first minute the ratio of radioactive ACh/Ch was linear after a bolus mlection of 3H-Ch Assuming that all free Ch m the brain IS mvolved m the synthesis of ACh, the rate of ACh formation can be calculated by multiplymg this ratio and tissue Ch. Saelens et al. (1974) calculated the size of the precursor pool by assummg that at maximal SAAC,, the Ch m this pool had the same SA. Surprrsmgly, the size of the Ch pool thus calculated comprised virtually all tissue Ch The turnover rates calculated by Nordberg (1977) with this method are rather low The size of the tracer dose affects the shape of the specific activity curves of the precursor and the substance m the mouse brain, as was shown

Amine and Acetylcholrne Turnover

437

with dh-Ch by Karlen et al (1982) This observation indicates that the tracer does not mix umformly with Ch m the brain and that ACh is derived from a specific Ch pool Turnover rates were calculated from the initial mcorporation of dh-Ch m ACh, which was linear in the first mmute. Maximal rate constants of about 45 h-i (striatum) and 80 h-’ (whole mouse brain) were calculated, corresponding to turnover rates of 3825 and 2040 nmol g -‘h-l, respectively, (assummg a single ACh compartment). These values are rather similar to those reported by Nordberg ei al. (1977) A posslble reason for the dose dependency of ACh labeling is, according to Karlen et al (1982), that at the higher dosages, the high affmlty uptake of Ch mto cholmergic neurons is no longer active If so, then the extracellular precursor pool of Ch for ACh synthesis is very small and can be directly influenced by Ch m the circulation Intraventricularly applied tracer doses of Ch labeled Ch and ACh maximally within 5 mm (Buccafusco, 1982). Tracer Ch declined exponentially m the cerebral cortex. From the mitral nearly logarithmic increase of specific activity of ACh and the exponential decrease of the specific activrties of Ch, presumably also m the precursor pool, the turnover rate of ACh was calculated The followmg rates (nmol gP’h-l) of ACh in various brain regions were thus found: cerebral cortex, 720, midbrain pons, 912, medulla, 636; striatum, 2052, and hippocampus, 774

7.3.2. lnfwon of radioactive precursors, By mfusion such as Ch or phosporylcholme, at a constant rate, the labelmg pattern of cerebral Ch and ACh is expected to be relatively simple Phosphorylcholme was chosen by Racagm et al. (1975a) because of the assumption that this compound enters the brain before rt IS hydrolyzed, and thus serves as a precursor pool of ACh which is not mixed with that produced after hydrolysis of ACh It is, however, unlikely that a malor part of phosphorylcholme enters the brain unhydrolyzed (see Ansell, 1981) Racagni et al (1975a) assumed that the labeled precursor accumulates linearly m the blood and m the precursor pool relevant for ACh synthesis, and the followmg Eq (6) was derived.

In this equation, ti is time elapsed after the onset of the infuand SAo,r, the specific activities of ACh and Ch m sion, S&CM tissue, respectively, and kA and ks the fractional rate constants of Ch (in the Ch pool relevant for ACh synthesis) and ACh, respec-

of synthesis

Pulse labeling calculated) Pulse labeling

14C-ACh

3H-Ch

(two

phases

Pulse labeling 3H-Ch (several calculation procedures)

(Paraoxon)

degradation

(hemlcholmum-3)

Blockade of acetylcholme (Dlchlorvos)

Blockade

Method

Reported Species/ brain area

TABLE 4 Rate Values

Mouse/whole

brain

Turnover rate, nmol g-‘h-’ 140 60 38 20 320 180 200 150 100 70 60 20 144 22 30 2220-3540 30&3600 36&6240 3300 1260 1620 1200-1500 300-600 390

of Acetylcholme

Rat/stnatum hlppocampus mldbram hypothalamus Rat/Stnatum Hlppocampus Cerebral cortex Midbrain Medulla-pons Thalamus Hypothalamus Cerebellum Rat/stnatum Hlppocampus Cerebral cortex Mouseistrlatum (range) Hlppocampus (range) Cerebral cortex (range) Mouselstrlatum hlppocampus cerebral cortex Mouse/whole brain

Turnover

and

and Sundvall

Nordberg

Sealens et al (1974)

Vocci et al (1979)

(1977)

from Wecker (1979)

(1977)

and

from Stravmoha

Nordberg

Calculated Dettbarn

Recalculated al (1975)

Calculated from Schmidt Buxbaum (1978)

Reference

et

of phosphoryl

of 3H-Ch

of deuturated

of deuterated

of phosphoryl

Infusion

Infusion

Infusion

Infusion

3H-Ch)

Ch)

(CH,-‘4C)-cholme

choline

phosphorylcholme

(Me-‘4C)cholme

(mtraventncular

Pulse labelmg

Infusion

(d4-substituted

Pulse labelmg

Cortex Midbrain Brain stem Cerebellum Mouse/whole brain striatum Ratistnatum Hlppocampus Cerebral cortex Mldbram-pons Medulla Rat/stnatum Hlppocampus Occipital cortex Llmblc cortex Brain stem Ratistrlatum Cerebral cortex Rat/nucleus accumbens Hlppocampus Cerebral cortex Ratistrlatum Hlppocampus Frontal cortex Parietal cortex Hypothalamus Ratistrlatum Hlppocampus Cortex Brain stem 400 375 340 80 3825 2040 2052 774 756 912 636 1300 520 200 200 92 1428 216 820 460 150 860 180 120 100 70 760 530 190 250 Zsllla

et al (1977)

et al (1976)

(1982)

et al (1976)

et al (1981)

et al (1977)

Brunello

Zsilla

Eckernas

Racagm

Buccafusco

Karl&n et al (1982)

440

Korf

trvely The fractional rate constant of ACh (kR) can thus be determined at a single time interval, when the fractional rate constant of Ch(kJ IS known By applying smgle open compartment analysis [assuming that SA Cl, 1s simrlar inside and outside cholinergrc neurons and correctmg for the nonlinear part of the radroactrvrty curves early during phosphoryl-(Me-14C)-cholme mfusron] turnover rates m various brain areas were calculated These values were rather similar to those obtained with the finite-drfference method of Neff et al. (1971) Apparent turnover rates (nmol gP’h-l) were: strratum, 1300, brain stem, 86, occrprtal cortex, 150, and limbrc cortex, 200 This method has been applied to several pharmacologrcal and physrological studies (Murray et al , 1982, Brunello et al., 1982, Zsilla et al , 1976, Moron1 et al., 1978) The exchange of Ch of the cu-culatron and the brain has been studied in some detail with intravenous mfusrons of Ch m rats (Freeman et al , 1975, Char et al , 1975, Racagm et al , 1975a). Lower levels of Ch were found m the blood of the carotid artery than in the posterror facial veins, suggesting the productron of cerebral Ch (Freeman et al , 1975, Char et al., 1975) and confirming proposals by others (Ansell, 1981) The maximum amount of Ch that could be infused without disturbing the steady-state concentration of Ch was 1 pmol/kg-’ mu--’ (Racagm et al., 197513). Eckernass et al. (1977) installed steady-state levels of Ch by comfusron of labelled Ch and cold Ch They calculated turnover rates of 1428 and 216 nmol g-‘h-’ m the strratum and cerebral cortex, respectrvely. Very similar rates were found wrthout the cold Ch, thus showing that plasma levels of Ch are rrrelevant to the ACh synthesis rate Srmrlar conclusrons were reached more recently by Brunello et al. (1982)

7.4. Conclusions The nonsteady-state methods result m relatively low turnover rates of ACh, which may be caused by exhaustion of the precursor pool, by feedback regulatory mechanisms, and/or by mcomplete mhrbrtron of the esterase. The rsotoprc methods result m turnover rates at least 2 or even 5 times higher A serious problem of the latter methods IS the lack of knowledge about the kinetic behavior of the precursor pool and possrble compartmentatron of ACh in the brain or bram regrons A summary of reported turnover rates is shown m Table 4 Thus sectron shows clearly that. “many experimental designs and methods of data analysis have been proposed for the assessment of ACh turnover, all of which have severe limrtatrons and

Amlne

andAcely/choOne

rest on unproven 1977).

441

Turnover

and sometimes

incorrect

assumptions”

(Jenden,

8. Turnover Methods for Other Amines 8.1. Tryptamine The occurrence of tryptamme (T) m the brain of humans and several other animal species has been established with a variety of techniques including radioenzymatic, spectrofluorimetric, dansylation, and mass spectrometric assays (Saavedra and Axelrod, 1972, Philips et al., 1974, 1978; Sloan et al., 1975, Snodgrass and Horn, 1973; Warsh et al , 1979). The endogenous levels of this amine, as reported by the various mvestigators, differ by several orders of magnitude. Snodgrass and Horn (1973) and Saavedra and Axelrod (1972) found levels of approximately 0.5 and 0.1 nmol g-’ wet weight of rat brain, respectively, whereas Sloan et al., (1975) reported brain levels of the rat, cat, dog, and the guinea pig of about 0.1-O 3 nmol g-i The lowest, and because of that probably most reliable, levels were provided by Philips et al (1974, 1978), namely l-3 pmol g-’ wet wt of rat brain tissue The brain levels of the amme increase postmortem (Philips et al. 1978, Snodgrass and Horn, 1973) High T levels were found m the caudate nucleus, the putamen and the thalamus m man (Philips et al , 1978) In the rat brain, the highest levels were observed m the striatum, hypothalamus and the spinal cord (Snodgrass and Horn, 1973), but Sloan et al (1975) reported low levels m the caudate and hippocampus of the cat and dog bram The content of brain T depends on the availability of TP (Warsh et al., 1979, Saavedra and Axelrod, 1973). Followmg mhibition of monoamme oxidase with pargyline, T accumulates lmearly for 90 mm at rates of 1 nmol gP h-’ (Warsh et al., 1979) or 0.160 m rat bram and 0.040 nmol g-‘h-’ m rat spinal cord (Durden and Philips, 1980). The maIor metabolite of T is mdoleacetic acid (IAA) (Wu and Boulton, 1973). The levels of plasma IAA are (almost seven times those m the brain (Young et al , 1980), which may affect the results of drug studies But assuming that after probenecid all accumulated IAA is derived from cerebral T, an apparent turnover rate for T of 17 pmol g-‘h-’ can be calculated (Young et al., 1980). In contrast to this low value, Warsh et al. (1977) found with probenecid a synthesis rate of 160 pmol g-‘h-i, which is close to

442

Korf h

that found 5).

using pargylme

to mhibit

T metabolism

(see also Table

8.2. Histamine Histamme (HA) IS localized m both neurons and mast cells. In the rodent brain, approximately 50% is confined to either cell type (Maeyama et al., 1983; Dismukes and Snyder, 1974; Taylor and Snyder, 1971; Martres et al., 1975). The synthesizing enzyme, histidme decarboxylase, is exclusively localized in neurons, so the increased levels of the amine followmg histidme loading are therefore probably confined to these cells (Verdi&e et al , 1977). The turnover rate of mast cell HA is an order of magnitude lower than that of neuronal HA (see below). The malor enzyme mvolved m degradation is histamine-N-methyl-transferase, which is predommantly or exclusively confined to nonhistammergic neurones or glia cells (Bischoff and Korf, 1978) The levels of HA are particularly high m the hypothalamus and three-five times lower m various other brain regions A postmortem decrease has been noticed (Taylor and Snyder, 1971). Both steady-state and nonsteady-state methods for turnover rate measurements have been explored The nonsteady-state methods are all based on the use of inhibitors of the synthesizmg enzyme, such as a-hydrazme histidine, NSD-1055 (4-bromo-3hydroxybenzyloxyamme, broscresme) and cu-fluoromethylMaximal

TABLE 5 Accumulation of Various Trace Ammes Following Pargylme Administration Accumulation, Rat strlatum

Rat whole brain

436"

1520"

Phenylethylamme p-Tyramme nl-Tyramme Tryptamme “Boulton and Juorlo “Durden and PhIlIps ‘Warsh et al (1979) “Young et al (1980), probenecld ‘Warsh et al (1977), probenecld

pmol g-‘h

’

199” 21" 60

160" 1000' 17' 160

(1982) (1980) Indoleacetlc

acid accumulation

followmg

Indoleacetlc

acid accumulation

followmg

Amme

andAcety/chohne

Turnover

443

histidme (Martres et al., 1975; Taylor and Snyder, 1971, Maeyama et al , 1983). In all cases, biphasic decline curves were seen, of which the latter phase presumably reflects the low turnover rate of HA m mast cells, to be disregarded m the turnover rates to be mentioned Turnover rates of about 0 20 nmol g-‘h-’ for the whole mouse brain were estimated (a-fluoromethylhistidme, Maeyama et al , 1983). In the hypothalamus, widely varying values have been reported, ranging from 1500 (Dismukes and Sn der, 1974), to 10 (Taylor and Snyder, 1971), and to 0 20 nmol g-‘h-’ (“midbrain ” Maeyama et al 1983) Attempts to estimate the turnover rate of HA at steady-state conditions have been based on systemic or mtraventricular inlections of labeled histidme. Followmg intraventricular qection m rats, a rapid increase m the labeling of HA and N-methylhistamme was noticed (Taylor and Snyder, 1971, Dismukes and Snyder, 1974). The specific activity of the precursor and HA was similar from 50 min post-mlection onwards. The authors, however, did not correct for mast cell HA Moreover, these authors considered the mmal, rapidly declmmg content of hypothalamic precursor as u-relevant for the turnover calculation. So very high turnover rates were calculated. Recalculation of the data of Dismukes and Snyder (1974) by the present author points to substantially lower turnover rates. Mouse brain HA turnover rate was calculated with mtravenously administered 3H-histidme by Verdi&-e et al. (1977) Maximal specific activities of the precursor and substance in the brain were reached after 5 and 20 mm, respectively. The latter comcides with both plasma and brain histidme specific activity. Correcting for mast cell HA, a half-life was estimated from the conversion index (= ratio of the radioactivity of HA and the specific activity of histidme) correspondmg to about 36 min, and an approximate turnover rate of 180 pmol g-‘h-’ for whole mouse brain was calculated. Such a value is close to those reported by Maeyama et al (1983), but substantially lower than those reported by Snyder and his coworkers.

8.3. Other Amines Several phenylethylammes, such as p- and m-tyramine, octopand phenylethanolamme have been amme, phenylethylamme, detected in the brain of humans and of rodents (Boulton et al., 1973, Philips et al., 1978, Jones et al , 1983, Saavedra and Axelrod, 1973; Harmar and Horn, 1976, Mosnaim et al , 1973) Most of these ammes accumulate following high doses of monoamme oxi-

444

Korf

dase mhlbltors. Assuming that the accumulation IS due to the central production, rates of formatlon of these ammes can be calculated Some of these rates are shown m Table 5 (see Boulton, 1982 for a recent summary)

Acknowledgments Mrs. M Alkema manuscript

and Mrs

W

van der Meer

typed

the

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m rat brain

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and Acetykhollne

Turnover

447

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448

Korf Increase

m rat bram acetylcholme

induced

by cholme or dean01 L$e

SC1 17, 975-980

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Jope R S. (1982) Effects of phosphatldylcholme admmlstratlon to rats on choline m blood and choline and acetylcholme m brain J Pharmacol Exp Ther 220, 322-328 Joseph M.H and Kenneth G A (1983) Stress-induced release of 5-HT m the hlppocampus and its dependence on increased tryptophan avallablhty an ~1 UVIVOelectrochemical study Brnm Res 270, 251-257. Karlen B , Lundgren G., Lundm J., and Holmstedt B (1982) On the turnover of acetylcholme m mouse brain influence of dose size of deuterlum labelled choline given as precursor Blochem Pharmacol 31, 2867-2872

Karoum F , Neff N H , and Wyatt R J (1976) Dlstrlbutlon and turnover rate of vamllylmandellc acid and 3-methoxy-4-hydroxyphenylglycol m rat bram 1 Neurochem 27, 3535

Amrne andAcetylchoOne Turnover

449

Karoum F., Neff N.H , and Wyatt R J (1977) The dynamics of dopamme metabohsm n-t various regions 01 rat brain. Euv 1, Pharmacol 44, 311-318 Kehr W. (1976) 3-Methoxytyramme as an indicator of impulse-induced dopamme release m rat bram m vlvo. Naunyn-SchmledeberX’s Arch, Pharmacol 293 209-215 Kehr W (1981) 3-Methoxytyramme and normetanephrme as indicators of dopamme and noradrenalme release m mouse brain UI UIUO 1 Neural Transm 50, 165-178 Koe B K and Welssman A (1966) p-Chlorophenylalanme a speclhc depletor of brain serotonm ] Pharmac.ol Exp Ther 154, 499-516 Kohno Y , Tanaka M , Nakagawa R , Toshlma N , and Nagasaki N. (1981) Reglonal dlstrlbutlon and production rate of 3-methoxy-4hydroxyphenylethyleneglycol sulphate (MHPG-Sob) m rat brain, 1 Neurodzem 36, 286-289 Korf J (1981) Turnover of neurotransmltters m the brain an mtroductlon, m Central Neurotransmltter Turnover (Pycock C J and Taberner P V , eds ), pp 1-19, Croom Helm, London Korf J , Praag H M van and Sebens J B. (1972) Serum tryptophan decreased, brain tryptophan Increased and bram serotonm synthesis unchanged after probenecld loading Beam Rcs 42, 239-242. Korf J , Grasdljk L , and Westermk B H. C (1976) Effects of electrical stlmulatlon of the mgrostrlatal pathway of the rat on dopamme metabolism 1 Neurochem 26, 579-584 Korf J , Venema K , and Postema F (1974) Decarboxylatlon of exogenous L-5-hydroxytryptophan after destruction of the cerebral raphe system \ Nelrroclzern 23, 249-252 Kuhar M J and Murrm L C. (1978) Sodium-dependent, high affmlty choline uptake (short review) J Neurochem 30, 15-21 Lane J D. and Aprlson M. H (1978) The flux of radioactive label through components of the serotonerglc systern followmg the mlectlon of (7H) tryptophan product-precursor anomalies providing evldence that serotonm exists m multiple ~001s. ] Neurochem 30, 671-678 Lane J D , Co C T and Smith J E. (1977) Determmatlon of slmultaneous turnover of serotonm, dopamme and norepmephrme In the telencephalon of unrestrained, behaving rats Life Scf 21, 1101-1108 Langer S Z (1974) Selective metabolic pathways for noradrenalme m the peripheral and m the central nervous system. Med Bdogy 52, 372-383 Le Roy Blank C , Sasa S , Isernhagen R , Meyerson L R , Wassll D , Wong D , Modak A T , and Stavmoha W B (1979) Levels of norepmephrme and dopamme m mouse brain regions followmg microwave mactlvatlon-rapid post-mortem clegradatlon of strlatal dopamme in decapitated animals 1 Neuroclzrm 33, 21%219 Lefresne P , Guyenet I’ and Glowmskl J (1973) Acetylcholme synthesis

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from (Zr*C)pyruvate m rat striatal slices ] Neurochem 20, 1083-1097. Ll P P , Warsh J J and Godse D D (1981) 3,CDlhydroxyphenylethylene glycol (DHPG) formation the malor route of rat brain Progr Netlro-Psychoplzar?llacoI 5, norepinephrme metabolism 531-535 Li P P , Warsh J J,, and Godse D D (1983) Rat brain norepmephrme metabolism substantial clearance through 3,4-dihydroxyphenylethylene glycol formation \ Netlroche?iz 41, 1065-1071 Lm R C , Costa E , Neff N H , Wang E T , and Ngar S H (1966) In viva measurement of 5-hydroxytryptamme turnover rate m the rat brain from the conversion of C”‘-tryptophan to Cl”-5-hydroxytryptamme 1 Pharnzacol Exp Ther 170, 232-238 Madras B K , Cohen E L , Fernstrom J D., Larm F , Munro H. N., and Wurtman R J (1973) Dietary carbohydrate increases brain tryptophan and decreases serum-free tryptophan Nature (Lond.) 244, 3435 Maeyama K , Watanabe T., Yamatodam A , Taguchi Y , Kambe H., and Wada H (1983) Effect of a-fluoromethyl hrstldme on the hrstamme content of the brain of W/W” mice devoid of mast cells: turnover of brain histamme J Neurochem 41, 128-134 Martres M I’., Baudry M , and Schwartz J C (1975) Histamine synthesis the developing rat brain evidence for a multiple zmpartmentation Bmn Res 83, 261-275 Meek J L and Neff N H (1973) The rate of formation of 3-methoxy-4hydroxyphenylethyleneglycol sulphate m brain as an estimate of the rate of formation of norepmephrme I Pharmacol Exp. Ther 184, 570-575 Molenaar P.C , Nicholson V. J , and Polak R L (1973) Subcellular localization of newly formed (3H)acetylcholme m rat cerebral cortex in ultra 1 Neurochem 21, 667-678 Moron1 F , Malthe-Sorensen D , Cheney D L , and Costa E (1978) Modulation of ACh turnover m the septal-hippocampal pathway by electrical stimulation and lesronmg Brazn Res 150, 333-341 Morot-Gaudry Y , Hamon M , Bourgom S , Ley J. P , and Glowmski J. (1974) Estimation of the rate of 5-HT synthesis m the mouse brain by various methods Naunyn-Schmredeberg’s Arch Pharmacol 282, 223-238 Mosnaim A. D , Inwang E E , Sugerman J H., De Martmi W J and Sabelh H C (1973) Ultraviolet spectrophotometric determmation of 2-phenylethylamme m biological samples and its possible correlation with depression. Blol. Psychdry 6, 235-257 Murray T F., Blaker W D , Cheney D. L., and Costa E (1982) Inhrbmon of acetylcholme turnover rate m rat hippocampus and cortex by mtraventrrcular mlectron of adenosme analogs. 1 Pharmacol EX,V Ther. 222, 550-554. Murrm L E., and Kuhar M.J. (1976) Activation of high-affmity cholme

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uptake UI vitro by depolarrzmg agents Mel Pharmacol 12, 1082-1090 Neckers L M and Meek J L (1976) Measurements of 5HT turnover rate m discrete nuclei of rat brain Life Scl 19, 1579-1584 Neff N H. and Tozer T N. (1968) In uuo measurement of bram serotonm turnover Adv Pkarmacol. 6A, 97-109 Neff N H., Spano I’ F , Groppettl A, Wang C T , and Costa E (1971) A simple procedure for calculatmg the synthesis rate of norepmephrme, dopamme and serotonm m rat bram / Phvwacol Exp Tker 176, 701-710 Nielsen M (1976) Estrmatron of noradrenalme and Its major metabolltes synthesized from (lH) tyrosme m the rat brarn / Nrllrocl~er~ 27, 493-500 Nielsen M. and Braestrup C (1976) A method for the assay of conlugated 3,4-drhydroxyphenylglycol, a major noradrenalme metabollte m the rat brain. ] Ne~l~ocl~em27, 1211-1217 Nielsen M and Braestrup C (1977) Chrome treatment with deslpramme caused a sustamed decrease of 3,4-drhydroxyphenylglycol sulphate and total 3-methoxy-4-hydroxyphenylglycol m the rat brain Nau~zyn-Schmledebel~s’s Arch Pliarmacol 300, 87-92 Nreoullon A , Cheramy A , and Glowmskl J (1977) Releaseof dopamme JJZ ZUI from cat substantra mgra Nnf~vc (Lond ) 266, 375-376 Nordberg A (1977) Apparent regronal turnover of acetylcholme m mouse brain Acta Pkysrol Scmd Suppl 445, 1-51 Nordberg A and Sundvall A (1976) Brosynthesrs of acetylcholme m dlfferent brain regions III UUIUC) followmg alternative methods of sacrifice by mrcrowave rrradratlon Acta PkyswI Scmd 98, 307-317 Orshr T and Wurtman R J (1982) Effect of tyrosme on brain catecholamme turnover m reserpme-treated rats 1 Ncurol Tramm 53, 101-108 Paden C. M. (1979) Disappearance of newly synthesized and total dopamme from the striatum of the rat after mhrbrtron of syntheses evidence for a homogeneous kmetrc compartment. ] Ncurochm 33, 471-479 Papeschr R (1977) The functronal pool of brain catecholammes Its size and turnover rate Psycl~opha~ macolo~y 55, l-7 Pardrrdge W M (1979) Tryptophan transport through the blood-brain barrier JH ZUZXJ measurement of free and albumm-bound ammo acid LJfe SCJ 25, 1519-1528 Perez-Cruet J , Taglramonte A , Taglramonte I’ , and Gessa G L (1972) Changes In brain serotonm metabolism associated wrth fasting and satratron m rats LJfe SCJ 11, 31-39 Philips S A and Boulton A. A. (1979) The effect of monoamme oxldase mhrbrtors on some arylalkylammes in rat strratum ] NcJlrodJerlJ 33, 159-167 Phllrps S R , Rozdrlsky B , and Boulton A A (1978) Evidence for the presence of m-tyramme, p-tyramme, tryptamme and phenyl-

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ethylamme m the rat brain and several areas of the human brain Bml Psychu7fry 13, 51-57 Racagnl G , Trabuccl M , and Cheney D L (1975a) Steady state concentrations of choline and acetylcholme m rat brain parts during a constant rate mfuslon of deuterated choline Natlnyll-SchmlcdeberX’s Arch Pharmacol 290, 99-105 Racagm G., Cheney D L , Trabuccl M , Wang C , and Costa E (1975b) Measurement of acetylcholme turnover rate m discrete areas of rat brain Lzfe Scl 15, 1961-1975 Racagnl G , Cheney D L , Zsllla G , and Costa E (1976) The measurement of acetylcholme turnover rate m brain structures Nruropharmacology 15, 723-736 Richards J G (1977) Autoradlographlc evidence for the selective accumulation of (3H) 5HT by supra-ependymal nerve terminals. Braw Rcs 134, 151-157 Richter J A. and Marchbanks R M (1971) Synthesis of (3H)-acetylcholme pools by subcellular fractions of cerebral cortex slices incubated with (“H)cholme ] Neurochem 18, 705-712 Robertson J S (1957) Theory and use of tracers m determining transfer rates m blologlcal systems Physzol Rev 37, 133-154 J (1977) Effect Rospars J P , Lefresne I’ , Beaulom J C , and Glowmskl of external ACh and of atropme on ‘“C-ACh synthesis and release m rat cortical slices Nulllzyn-SchlnIedeber~‘s Arch PharmacuI 300, 15?+161 Rommelspacher H. and Kuhar M J (1974) Effect of electrical stlmulatlon on acetylcholme levels m the central cholmerglc nerve terminals Brain Res 81, 243-251 Saavedra J M. and Axelrod J (1973) Demonstration and dlstrlbutlon of phenylethylanolamme m brain and other tissues Proc Nafl Acad Scl (USA) 70, 769-772 Saavedra J M , and Axelrod J (1973) Effects of drugs on the tryptamme content of rat tissues J Pharmacol Exp Ther 185, 523-529 Saelens J K , Slmke J P , Schuman J., and Allen M I’ (1974) Studies with agents which influence acetylcholme metabolism m mouse brain Arch Intern Pharmacodyn Ther 209, 250-258 Scatton B (1982) Brain 3,4-dlhydroxyphenylethyleneglycol levels are dependent on central noradrenerglc neuron activity L$e SCI 31, 495-504 Scatton B , Pelayo F , Dubocovlck M L , Langer S Z , and Bartholml G (1979) Effects of clorudme on the cerebral adrenaline turnover and the adrenaline release m nucleus tractus soIltar of the rat, m Presynaptlc Receptors, Adu Blosc~ 28, (Langer S Z , Starke K and Dubocovlck M L , eds) pp 231-236, Pergamon, Oxford Schanberg S M , Schlldkraut J J , Breese G R , and Kopm I J (1968) Metabolism of normetanephrme-H3 m rat bran-ldentlflcatlon of conjugated 3-methoxy-4-hydroxyglycol as the major metabollte Blochem, Pharmacol 17, 247-254

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Schmidt D E and Buxbaum D M (1978) Effect of acute morphme admmrstratlon on regional acetylcholme turnover m the rat Bmrn Res 147, 194-200 Schmidt D E and Wecker L. (1981) CNS effects of choline admmlstratlon evidence for a temporal dependence Neuroplzannncolo‘~y 20, 535-539 Schubert J , Sparf B., and Sundvall A. (1969) A technique for the study of acetylcholme turnover m mouse brain I?I ZX?IU 1 Ncwockern 16, 693-700 Schubert J. (1974) Labelled 5-hydroxytryptamme and 5-hydroxymdoleacetlc acid formed m vlvo from 3H-tryptophan m rat bram effect of probenecld. Actlz Pkyslol Stand 90, 401-408 Schutte H H (1976) Het metabohsme van serotonme m rattehersenen Thesis, Umverslty of Gronmgen. Sedvall G C , Welse V K , and Kopm I. J (1968) The rate of noreplnephrme synthesis measured ~YI UIZXI during short intervals influence of adrenerglc nerve impulse activity j Pknmncol Exp Tker 159, 274282 Sharman D F (1981) The turnover of catecholammes, m Cerltral Neurotmmmltter Tu~~~over (Pycock C J and T#lberner P V , eds ) pp.20-58 Croom Helm, London Shields P J and Eccleston D (1972) Evidence for the synthesis and storage of 5-hydroxytryptamme m two separate pools m the brain 1 Neurockeln 20, 881-888 Sims N. R , Marek K L , Bowen D M , and Davison A. N. (1982) Productlon of (“C)acetylcholme and (‘%Z)carbondloxlde from (U-l’C)glucose m tissue prisms from aging rat brain. 1 Neuuockcm 38, 48%492 Sloan J W , Martin W R , Clements T H , Buchwald W F , and Bridges S R (1975) Factors influencing brain and tissue levels of tryptamme species, drugs and lesions 1, Neurockm 24, 523-532 Snodgrass S R and Horn A S. (1973) An assay procedure for tryptamme m brain and spinal cord using its [?H]-dansyl denvatlve. ] Nrurockem 21, 687-696 Stavmoha W B , Modak A T and Wemtraub S T (1976) Rate of accumulation of acetylcholme m discrete regions of the rat brain after dlchlorvos treatment 1 Ncurockem 27, 13751378 Sugden R F and Eccleston D (1971) Glycol sulphate ester formation from (‘“C) noradrenalme m brain and the influence of a COMT mhlbltor 1 Neurockem 18, 2461-2468 Suzklw J B and O’Leary M E (1982) Differential labeling of depot and active acetylcholme pools m nondepolarized and potasslumdepolarized rat brain synaptosomes 1 Ncurockem 38, 1668-1675 Svensson T H and Waldeck B (1969) On the slgmflcance of central noradrenalme for motor activity experiments with a new dopamme P-hydroxylase mhlbltor Eur ] Pkarnmol 7, 278-282 Tagllamonte A , Tagllamonte I’., Perez-Cruet J , and Cessa G L (1971a)

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Increase of brain tryptophan caused by drugs which stimulate serotonm synthesis Nature New Blol 229, 125-126 Taghamonte A , Taghamonte I’ , Perez-Cruet J , Stern S , and Gessa G. L (1971b) Effect of psychotropic drugs on tryptophan concentration m the rat bram ] Phamacol Exp Ther 177, 475480 Taghamonte A , Tagliamonte I’ , Gessa R , Duce M , Maffei C , and Gessa G L. (1971~) Increase of bram tryptophan by probenecid Rlv Farm. Ther 11 207-213 Taghamonte A , Biggie G., Vargm L , and Gessa G L (1973) Free tryptophan m serum controls brain tryptophan level and serotonm synthesis Life Scl 12, 277-287 Tappaz M L. and Pulol J -F (1980) Estimation of the rate of tryptophan hydroxylation 1?1~1uo a sensitive microassay m discrete rat brain nuclei. J Neurochem 34, 933-940 Taylor K M and Snyder S H (1971) Histamme m rat brain sensitive assay of endogenous levels, formation in uluo and lowermg by mhibitors of histidme decarboxylase. / Phamacol Exp Ther 179, 619-633. Tracqui P , Brezillon I’ , Staub J F , Morot-Gaudry Y , Hamon M , and Perault-Staub A M (1983a) Model of bram serotonm metabolism I Structure determmation-parameter estimation Am 1 Physrol 244, R193-R205 Tracqui I’., Morot-Gaudry-Y , Staub J, F., Brezillon I’ , Perault-Staub A M , Bourgom S , and Hamon M (1983b) Model of bram serotonm metabolism. II Physiological interpretation Anz ] Physfol 244, R206-R215. Trommer B A , Schmidt D E , and Wecker L (1982) Exogenous choline enhances the synthesis of acetylcholme only under condmons of mcreased cholmergic neuronal activity. I Neurochem 39, 1704-1709 Tucek S (1983) The synthesis of acetylcholme, m Handbook of Neurochewstry, 2nd edmon, vol 4 (Laltha, A , ed.), pp 219-249 Plenum Press, New York Tucek S (1985) Regulation of acetylcholme synthesis m the brain / Neurochern 44, 1 l-24 Van der Krogt J A , Van Valkenburg C F M , and Van der Leden A (1981) Simultaneous analysis of dopamme synthesis and breakdown m rat brain by HPLC-ECD after intravenously admmlstered ‘H-tyrosme Abstract 438 8th Meeting Int Sot Neurochem , Nottmgham, U K Van Valkenburg C F M , Van der Krogt J A , and Moleman I’ (1983) Dopamme turnover compartmentation m rat brain methodological aspects 5th Catecholamme Symposium, Goteborg, abstract 501 Van Wilk M , Sebens J B , and Korf J (1979) Probenecid-induced mcrease of 5-hydroxytryptamme synthesis m rat brain, as measured by formation of 5-hydroxytryptophan PsyclzoF7harmacolo;yy 60, 229-235. Van Wilk M and Korf J (1981) Post-mortem changes of 5-hydroxytryptamme and 5-hydroxymdoleacetic acid m mouse brain and therr

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prevention by pargylme and mrcrowave rrradlatron Neurochem Rcs 6, 425430 Verdi&e M., Rose C , and Schwartz J C. (1977) Turnover of cerebral histamme m a stressful situation Brain Res 129, 107-119. Versteeg D H.G , Van der Gugten J , and Van Ree J. M (1975) Reglonal turnover and syntheses of catecholammes m rat hypothalamus Noture (Lond ) 256, 502-503 Versteeg D H G , Tanaka M , and De Kloet E R (1978) Catecholamme concentration and turnover m discrete regions ot the brain of the homozygous Brattleboro rat deflcrent u-r vasopressm Endoc~~zolu~~!/ 103, 16541661 VOCCI F J , Karbowskr M J , and Dewey W L. (1979) Apparent IH ZUZIO acetylcholme turnover rate m whole mouse bram evidence for a two compartment model by two Independent kmetrc analysrs ] Neurochern 32, 1417-1422 Walters J R and Roth R M (1974) Dopammergrc neurons drugmduced antagonism of the increase m tyrosme hydroxylase actrvlty produced by cessation of rmpulse flow 1 Pha~rrracal Exp Ther 191, 82-91. Warsh J J and Stancer H C (1976) Brain and peripheral metabolrsm of 5-hydroxytryptophan-“C followmg peripheral decarboxylase mhrbltron \ Pharmacol Exp Ther 197, 545-555 Warsh J J , Chan P W , Godse D D , Coscma D V , and Stancer H C (1977) Gas chromatography-mass fragmentographlc determmatron of mdole-3-acetic acid m rat brain ] Neurochem 29, 955-958 Warsh J J , Coscma D V , Godse D D , and Chan P W (1979) Dependence of brain tryptamme formation on tryptophan avallablllty ] Nelrrocheln 32, 1191-1196 Warsh J J , Lr P I’, Godse D D , and Chueng S. (1981) Brain noradrenerglc neuronal actrvlty affects 3,4-dlhydroxyphenylethyleneglycol (DHPG) levels Life Scl 29, 1303-1307 Wecker L., Dettbarn W -D , and Schmrdt D E (1978) Cholme admmlstratron modlfrcatlon of the central actions of atropme. Sc~rlce 199, 86-87. Wecker L and Dettbarn W -D (1979) Relatlonshlp between cholme avarlabrlrty and acetylcholme syntheses m discrete regions of rat brain ] Neurochel?l 32, 961-967. Wecker L and Schmidt D E. (1979) Central cholmergrc functron relatronshrp to choline admuustratlon Llfc Scr 25, 375-384. Westenberg H G , Merger L A , Vulto A G , and Versteeg D H G (1983) Srmultaneous determmatron of dopamme, serotonm and their metabolrtes by llqurd chromatography post-mortem changes 5th Catecholamme Symposmm, Goteborg, Abstract 508 Westermk B. H C and Korf J. (1976) Turnover of acid dopamme metabolltes m strratal and mesolrmblc trssue of the rat brain Eur ] Pharmacal 37, 249-255 Westermk B H C and Spaan S. J (1982a) Estrmatron of the turnover of 3-methoxytyramme m the rat strratum by HPLC with electrochem-

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lcal detection. lmpllcatlons for the sequence m the cerebral metabolism of dopamme I. Neurochem 38, 342-347 Westermk B H. C and Spaan S. J. (1982b) Simultaneous determination of the formation rate of dopamme and its metabollte 3,4-dlhydroxyphenylacetlc acid (DOPAC) m various rat brain areas Brazn Res 252, 239-245 Westermk B H C and Wlrlx E (1982) On the slgmflcance of tyrosme for the synthesis and catabolism of dopamme m rat brain evaluatlon by HPLC with electrochemical detectlon j Neclrochem 40, 758-764 Westermk B H C , Van Es T P and Spaan S J (1982) Effects of drugs interfering with dopamme and noradrenalme blosynthesls on the endogenous 3,4-dlhydroxyphenylalanme levels m rat brain 1 Neurochem 39, 44-51 Westermk B H C , Bosker F and Wlrlx E (1984) Formation and metabolism of dopamme m rune areas of the rat brain modlflcatlons by haloperldol ] Neurochem 42, 1321-1327 Wlderlov E and Lewander T. (1978) Inhlbltlon of the in zxuo blosynthesls and changes of catecholamme levels m rat brain after alpha-methyl-p-tyrosme time and dose-response relatlonshlps Natlnyn Schmredeberg’s Arch Pharmacol 304, 111-123 Wu P H , and Boulton A A (1973) Dlstrlbutlon and metabohsm of tryptamme m rat brain Can ] Blochem 51, 1104-1112 Wurtman R. J , Heft1 F , and Melamed E (1981) Precursor control of neurotransmltter synthesis Pharmacol Rezl 32, 315-335. Wurtman R. J , Larm F , Mostafapour S , and Fernstrom J D (1974) Bram catechol synthesis control by brain tyrosme concentration Science 185, 183-184 Young S N , Anderson, G. M , and Purdy W C (1980) Indoleamme metabolism m rat brain studied through measurements of tryptophan, 5-hydroxymdoleacetlc aad, and mdoleacetlc acid m cerebrospinal fluid ] Neurochem 34, 309-315 Zllversmlt D B., Entenman C , and Flshler M. C (1943) On the calculation of “turnover time” and “turnover rate” from experiments mvolvmg the use of labeling agents ] Gcrz Physlol 26, 325-331 Zllversmlt D B (1960) The design and analysis of isotope experiments Am ] Med 29, 832-848 Zsllla G , Cheney D L , and Costa E (1976) Regional changes m the rate of turnover of acetylcholme m rat brain followmg dlazepam or musclmol. Naunyn-Schnuedeberfs Arch Pharmacol 294, 251-255

Chapter 11

Neuronal Transport of Amines In Vitro GLEN

B. BAKER

AND

LILLIAN

Es.DYCK

1. Introduction Investigations m vitro on the transport of biogemc ammes have done much to expand our knowledge of neurotransmission in general, of the role of ammes m nervous function, and of the actions of a wide variety of psychotropic drugs In this review, we will be describing techniques that are employed to study uptake and release and hope to demonstrate how these have been applied to mdividual ammes and utilized to study mteractions among various neurotransmitters m the central nervous system

1.1. Uptake and Release of Neurotransmitters Accordmg to the conventional concepts of neurotransmission, an action potential generated m the cell body of a neuron travels down the axon, reaches the nerve terminal, and causes a transient influx of extracellular Ca” This results in the exocytotic release of neurotransmitter molecules from storage sites into the synaptic cleft After acting on postsynaptic receptors, the action of the neurotransmitter can be terminated by diffusion away from the synaptic cleft, metabolic degradation, or an active reuptake process mto the nerve termmals. In the case of the putative neurotransmitter ammes dopamme (DA), noradrenalme (NA), and 5-hydroxytryptamme (5-HT, serotonm), the latter process appears to be the prmcipal mechanism of synaptic mactivation. In contrast, the prmcipal mechanism of mactivation of acetylcholme (ACh) is not reuptake, but degradation by acetycholme esterase. The cholme produced by the enzymatic hydrolysis of ACh is, however, taken up mto the nerve ending by a high-affinity proc457

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ess Demonstration of the presence of these active reuptake processes is often included as one of the criteria that a substance must fulfill to be considered a neurotransmitter Although neurotransmitters can be taken up and released m a number of different ways from nerve endings, neuronal cell bodies and @al cells, only one uptake process and one release process are considered to be involved m conventional neurotransmission The uptake process by which a set of nerve endings accumulate their neurotransmitter is thought to exhibit these characteristics. (1) the uptake occurs against a concentration gradient, i.e., is a concentrative process requirmg an expenditure of energy, (2) the uptake system exhibits a high affuuty (low K,,,) for only the transmitter employed by its nerve endings, and (3) the uptake appears to be carrier-mediated with Naf being cotransported The release process mitiated by depolarizing of the presynaptic membranes is thought to occur by a secretion of the neurotransmitter from the storage granules, or vesicles, withm the nerve endings. This secretion is thought to depend upon an increase m mtracellular Ca2+ levels caused by the membrane depolarization In addition to these transport processes, transmitters can be taken up and released m other ways. A transmitter may enter nerve endings, cell bodies, or glial cells by a nonspecific, passive diffusion In addition, a transmitter may enter neuronal cell bodies, glial cells, and nerve endings of another transmitter type by an active, lowaffinity uptake system. Transmitters can be released m two other ways distmct from the depolarization-induced mode. First, a transmitter can wash out by diffusion, and second, a more specific release, or efflux, can be mmated by reversal of carnermediated uptake, or influx. This latter type of release, for example, seems to be involved m the way in which &hetamine stimulates catecholamme release.

1.2. Tissue Preparations Used to Study Uptake and Release Processes A perusal of the literature on uptake and release of biogemc ammes (or their precursor amino acids) indicates that a wide variety of tissue preparations have been used m vitro to investigate neuronal transport of these substances. These preparations melude slices, prisms, minces, homogenates (with or without removal of nuclear debris by centrifugation or by sieving), P2 fractions (crude mitochondnal fractions containing myelm fragments, synaptosomes, and mitrochondria), purified synaptosomes (nerve terminals), and purified synaptic vesicles The preparation used may be the result of several considerations*

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Speedand ease of prepauatzon. For example, homogenates, slices, minces, prisms, and P2 fractions can be prepared more quickly than purified synaptosomes or synaptic vesicles Although brain slices are easy to prepare and may more closely resemble the normal physiological environment (because of their greater retention of cellular integrity), they can swell during incubation and have a large extracellular space. These factors may make it difficult to get a good estimate of the kmetic parameters of specific uptake processes, because of the difficulty of correcting adequately for these other factors that might contribute to the measured uptake. On the other hand, synaptosomes, which offer a more concentrated sample of nerve endings, are more fragile and require great care during their washing and collection onto filters (Wheeler, 1978) The mechanlsvzs under study. The researcher may wish to study uptake and/or release of amines m vesicles m the absence of transport across the nerve terminal membrane; m this case the nerve terminal would be osmotically shocked in order to obtain the enclosed vesicles which would then be isolated Conversely, it may be desirable to study transport across the nerve terminal membrane, with interactions from vesicular transport mmimized, m such cases, uptake has been studied in tissue prepared from animals pretreated with reserpme. Cost and avadabrlzty of equipment (e.g., superfuszon chambers, transfer holders, tzssue choppers, ultracentrzfuges). For example, if one chooses to use homogenates, minces, prisms, or subcellular preparations of neural tissue, then release has to be studied using superfusion chambers because the tissue cannot be easily and rapidly separated from the medium by a transfer technique. On the other hand, superfusion or transfer techniques can be used to study release from brain slices.

1.3. Experimental Conditions for Studying Uptake and Release Uptake and release have been studied m brain m VIVO as well as m vitro, but this review will concentrate on m vitro procedures. In general, in vitro techniques have become more popular than m vivo methods because of their greater convenience and ease of mterpretation. Uptake can be studied in vivo by admmistermg either the radiolabeled ammes or their labeled precursor ammo

460

Baker

and Dyck

acids, dissectmg out tissue, and performing autoradrography or isolating the labeled ammes from the brain tissue In viva procedures employed to investigate release have mcluded mtraventricular perfusion and perfusions of specific brain areas usmg a variety of instruments, including combmed push-pull cannulae, two separate inflow and outflow cannulae, and perfusion cups (Barthohm et al., 1976; Chmeh and Moore, 1974; Nieoullon et al., 1977; Soubrie et al , 1983). Most of these experiments have mvolved measurement of radiolabeled amme (and/or its metabolites) m the perfusate after admmlstration of the radiolabeled amme itself or the radiolabeled ammo acid precursor. The development of highly sensitive analytical techniques such as highresolution mass spectrometry, radioenzymatic procedures, and electrochemical detectors now permits analysis of endogenous amines in the perfusates of m viva or m vitro experiments (Kant and Meyerhoff, 1978, Hefti and Melamed, 1981; Philips et al., 1982, Dyck et al., 1982; Hutson and Curzon, 1983, Wightman and Dayton, 1982; Joseph and Kennett, 1983). A malor difficulty with these m viva experiments IS the mterpretation of findings. Smce uptake and release are both gomg on contmuously, a drug that causes mhibition of reuptake will result m increased amme m the synaptic cleft, and this may be interpreted as a releasing effect in vivo. It has been claimed, however, that a number of m vitro techniques can differentiate true release from such an apparent release caused by inhibition of reuptake (Raiteri et al , 1974, 1975, Heikkila et al., 1975, Hunt et al , 1979), however, even in vitro the ability of some of these various techmques to differentiate between mhibitron of uptake and stimulation of release is controversial. In addition to the theoretical considerations mentioned above, there is the problem of deciding upon the experimental conditions to use when studying uptake. Conditions that may vary from one publication to another include composition of mcubation medium, substrate concentrations, time of mcubation, and method of isolation of tissue after incubation with the radiolabeled amine Generally, the incubation media used do not differ greatly, and an exammation will indicate that though most are similar with regard to pH and concentrations of Na ‘, Ca2 ‘, K’ , eg , and Mg*+, they may differ m the method used for buffermg, use of sodium phosphate or Tris buffer. In addition, m many mstances an appropriate inhibitor IS included m the buffer to prevent degradation of the substrate during uptake and/or release studies Whether an inhibitor is used or not, the radioactive mate-

Amrne

Transport

In Ktro

461

nal taken up or released should be analyzed to ensure that it has not been significantly metabolized to another compound. The substrate concentrations selected for the incubations will depend upon the type of experiment. If a kinetic analysis is undertaken, a wide range of substrate concentrations is used If the effects of drugs, etc. on the neurotransmitter-specific uptake system is to be examined, the concentration of the radiolabeled neurotransmitter chosen should be close to the K,,, derived for this uptake system. It should be noted here that a widely employed graphical analysis of determining kinetic constants (in which the Lmeweaver-Burk double-reciprocal plot is resolved into two uptake systems) is algebraically incorrect A mathematically correct method estimates the rate constant for nonsaturable diffusional uptake and then uses this constant to calculate saturable uptake (Stahl and Meltzer, 1978). In general, mcubation times should be kept as short as reasonably possible and m any case limited to a time interval in which uptake of the amme is linear. With regard to isolation of the tissue after mcubation with radiolabeled amme, methods used include rapid coolmg followed by centrifugation, centrifugation at room temperature in a microfuge, and rapid filtration using a vacuum filter system. Although this matter has not been investigated extensively, Raiteri and Levi (1973) did study various conditions of tissue isolation and found that rapid coolmg can lead to significant loss of radiolabeled catecholammes from the tissue In addition, the type of filter used can affect the uptake values (Wheeler, 1978). It should be emphasized that if the tissue sample is solubilized by the addition of alkaline digesting agents (such as Protosol from NEN or NCS from Amersham), the sample must be neutralized before its radioactivity is determined by liquid scintillation counting If it is not neutralized, spurious counts from chemilummescence will occur. Moreover, a toluenebased cocktail rather than a water-miscible cocktail is preferable in this case. In addition, if Milhpore or Gelman filters are used, they are also solubilized by the alkaline digesting agents and produce a brown color that may cause significant quenching. Paper filters, which do not cause this problem, may be preferable When the release of a transmitter is the focus of experimental investigation, the tissue preparation is first preloaded with the labeled transmitter of interest. The experimental conditions chosen here are similar to those above, except that the incubation time is usually much longer so that more label is mcorporated into the tissue In addition, the concentration of transmitter incubated with the tissue may be higher to increase the amount taken up;

Baker

and Dyck

however, the concentration chosen should not be so great that it approaches the rate constant of nonspecific uptake systems, because that would lead to less specific labelmg. The first phase of all release studies involves “washing out” poorly bound labeled transmitter by superfusmg or suspending the tissue m a physiological buffer. After the rate of this basal, or spontaneous, release has reached a plateau, various types of stimuli are then applied to the tissue to see whether they cause an increase m the rate of release. When one is attempting to determine whether a particular compound is a transmitter, then the releasing stimulus is a depolarizmg one (i.e., electrical depolarization, high concentrations of K+, or the addition of the alkaloid veratridme to the buffer). In order to assess whether the resulting release, if any, is Ca2+-dependent, Ca2+ can be omitted from the buffer and EGTA added to chelate any other Ca2’ present Alternatively, Ca2+ influx can be antagonized by supplementing the buffer with extra Mg2+ or adding Ca2+-channel blockers, such as verapamil and D-600 A different approach to assess whether the observed release is of a secretory nature is to add drugs, such as colchicme, that disrupt contractile elements m the nerve ending When one is mvestigatmg the release of a compound that has already been identified as a neurotransmitter, then the stimulus applied after a steady rate of washout is reached may be one that is thought to cause carrier-mediated efflux-such as a structural analog of the transmitter, or the transmitter itself. Usually, fairly high concentrations (micromolar) of such compounds are needed to stimulate release. An Interesting possibility that might occur in brain slice experiments, but which has not been adequately discussed yet m the literature, is that the addition of high concentrations of some transmitters might depolarize mterneurons in the preparation and cause release of endogenous compounds that might affect the release of the labeled material. It has also been reported that accumulated amme can be more easily lost (washed out) from vesicles when the preparation IS centrifuged rather than filtered (Lentzen and Philippu, 1977) In order to assist those readers who would like an overview of some of the problems mentioned above, we have compiled a list of references (seeTable 1) that deal with a number of aspects of m vitro studies on uptake and release, includmg the use of different types of tissue preparations, the use of a variety of apparatus, and methodologies for isolating and perfusing tissue Included are examples of investigations of each of the classes of amines mentioned subsequently m this chapter, 1 e , catecholammes,

Amlne

Transport

Methodological

463

In V&o

TABLE 1 Aspects of Uptake and Release E*lcperiments In Vitro

Reference

Factors mvesttgated

Snyder et al , 1970

Review of uptake and subcellular distribution of catecholamines and 5-HT m brain Ionic and metabolic requirements for release of ‘H-monoammes evoked by field stimulation Review of role of transmitter uptake mechanisms Release of endogenous HA, together with hJA and 5-HT from slices of cerebral hemispheres Isomers of amphetammes, deoxypipradrol, and methylphemdate and mhibmon of uptake of tritxated catecholammes mto cerebral cortex slices, synaptosomal preparations from cerebral cortex, hypothalamus, and striatum, and mto adrenergic nerves of rabbit aorta Synaptosomal uptake of 5-HT m discrete brain regions followmg midbrain raphe lesions Synthesis and release of radioactive ACh m brain slices Effects of cations on uptake of 3H-DA by homog enates of corpus striatum Studies on effects of sudden coolmg on radiolabeled ammes taken up by synaptosomes Studies on the distmction between uptake mhibltion and release of 3H-DA m brain slices Description of a superfusion apparatus that elimmates reuptake effects when studying release m synaptosomes

Farnebo,

Iversen,

1971

1971

Atack and Carlsson,

1972

Ferris et al., 1972

Kuhar et al , 1972

Grewaal and Quastel, Harris and Baldessarml,

Raiterl

Heikkila

Raiteri

and Levi, 1973

et al , 1975

et al , 1974

1973 1973

464

Baker TABLE

Reference Redburn

Baumann

and Maltre,

de Bellroche

Richter,

1976

1976

1976

Shore,

1976

White,

1976a

Carruba

et al , 1977b

Lane and Apnson,

Lentzen

1976

et al , 1976

Levi and Ralten, Paton,

1 (cont~~tted) Factors

et al , 1975

and Phllhpu,

1977

1977

and Dyck

mvestlgated

Studies on stimulus-secretion coupling m vitro using a rapld perfusion apparatus to mamtam efflux of transmitters from tissue samples Studies to determine if mhlbltlon of DA uptake by drugs IS a mlsmterpretation of in vitro experiments Automated separation and analysis of DA, its ammo acid precursors and metabolltes appllcatlon to measurement of specific radloactlvltles of DA m strlatal synaptosomes Review of synaptosomal transport processes A book containing chapters by several authors on mechanisms of neuronal and extraneuronal transport of catecholammes Study of characterlstlcs of release of ACh by superfused bram shces The role of storage granules m the functlonal utilization of newly synthesized DA Synaptic vesicles and rapid bmdmg of newly accumulated NA withm synaptosomes Study of mazmdol and amphetamines as mhlbltors of uptake of, and releasers of, 3H-DA m strlatal slices Ca2+-dependent release of endogenous 5-HT and catecholammes from nerve endings Uptake of tyramme mto synaptic vesicles prepared from caudate nucleus (mttnued)

Amlne

Transport

ln Vitro

465 TABLE

Reference Moisset,

Factors 1977

Ross and Kelder,

et al , 1977

Vargas

et al., 1977

Chow and Abramson,

de Bellroche

Haycock

Horn,

1978

and Bradford,

1978 et al , 1978

1978

Review of compartmentation of synaptosomal DA Glucose and release of ammes from striatal tissues Alkaline earth stimulation of neurotransmrtter release from synaptosomes

1978

Kant and Meyerhoff,

investigated

Factors contributmg to modulation of uptake of NA by cortical synaptosomes Efflux of ‘H-!j-HT from cortical synaptosomes Properties of uptake of ‘H-NA by storage vesicles prepared from whole brain Nature of K’-induced release of 3H-NA from cortical slices prepared from normal and reserpmized brain Effects of pH changes and charge characteristics on synaptosomal uptake of NA

1977

Seidler

Dorris,

1 (corztrntred)

Review of characteristics neuronal DA uptake 1978

Release of ertdogenous DA from brain regions

of NA and

Raiteri et al., 1978a

Review of uptake and release of DA m synaptosomes

Raiteri

et al , 1978b

Investigation autoreceptors

Slotkm

et al., 1978a

Uptake of %I-NA and “H-5-HT into synaptic vesicles prepared from whole brain and brain regions

Slotkm

et al , 1978b

Uptake of 3H-DA by synaptic vesicles prepared from whole brain and brain regions

of presynaptic and DA release

466

Baker TABLE

Reference Barker,

de Langen

and Mulder,

de Langen

et al , 1979

Ferris

and Tang,

1979

1979

Fischer

and Cho,

Heuser

et al., 1979

Hunt

1979

et al , 1979

Manukhm

1 (contznued) Factors

1979

and Volma,

Paton,

1979

Petrali

et al , 1979

Ralterl et al , 1979

1979

and Dyck

investigated

An overview of choline avallablhty and transport and regulation of acetylcholme synthesis Compartmental analysis of the accumulation of 3H-DA m strlatal synaptosomes The nature of the releasable pool of DA m synaptosomes prepared from striatum Effects of isomers of amphetamines, methylphemdate, and deoxyplpradrol on uptake of 3H-NA and 3H-DA by synaptic vesicles prepared from whole brain, stnatum, and hypothalamus Strlatal homogenates and chemical release of DA evidence for an exchange diffusion model Study of synaptic vesicle exocytosls using quick freezmg Use of synaptosomes and fieldstimulated brain slices to dlfferentlate DA uptake and inhibitors and releasing agents Study on reverse transsynaptlc regulation of neuronal NA uptake Book contammg chapters by several authors on release of catecholammes from adrenerglc neurones Study on uptake of WI- and p-TA into slices of caudate nucleus and hypothalamus Use of nomlfensme to differentiate two release mechamsms for DA m synaptosomes

Amine Transport In Vftro

467 TABLE

Factors

Reference Redburn

et al , 1979

Tuomrsto

and Tuomlsto,

Tzeng and Slekevltz,

Annunzrato

Arbllla

et al , 1980

1980

Biggs and Johnson,

1980

Cerrlto

1980

Hardy

1979

1979

and Langer,

Evonruk

1 (cutr t~n~eli)

and Rarterr, and Slotkm,

et al , 1980

Levi et al , 1980

1980

mvestlgated

Reserpme and Ca2+-dependent release of ‘H-NA from synaptosomes depolarized with K’ or veratrldme Comparison of 3H-DA intake into crude partially purified and purified synaptosomal preparations from striatum Black widow spider venom and release of neurotransmrtters, depletion of synaptic vesicles and bmdmg to membrane, studres on a cerebral cortex preparation Kmetrcs of DA uptake m synaptosome:; prepared from medran eminence and other dopammerglcally innervated brain areas Influence of rnhlbltron of MAO on release of ?H-DA evoked by K’ and amphetamine from substantla mgra and corpus strlatum Electrically-Induced release of “H-HA from hypothalamus Newly recaptured DA and regulation of DA btosynthesls pH and uptake of “H-NA into storage vesicles prepared from brain, heart, and adrenal medulla Differential release of transmitters from nerve termmals from basal ganglla and substantra mgra Reevaluation of veratrldme as a tool for investigatmg depolarlzatlon-evoked release of neurotransmrtters from nerve endings

Baker

468 TABLE

and Dyck

1 (continued)

Reference

Factors

Logan,

Extrusion of Na+ from cortical synaptosomes Na’ , K ‘-ATPse and uptake of NA by synaptosomes Revrew of neuronal transport of catecholammes Review of neuronal transport of 5-HT Study on structural requrrements for uptake mto serotonmerglc neurones Revrew of uptake of catecholammes by storage vesrcles Uptake and metabohsm of catecholammes m synaptosomes contrrbutlon of MAO Storage and release of NA m synaptosomes prepared from hypothalamus Release of DA mduced by electrrcal stlmulatron of mrcrodlssected caudate-putamen and substanha ntgra Ontogenesis of uptake and deaminatlon of 5-HT, DA, and PEA m isolated perfused lung and lung homogenates Selected neurotoxrc chemicals and synaptosomal monoamme uptake and K’-dependent phosphatase Ca2+-sensrtive accumulation of NA m cerebral cortex Release of DA from dendrites m substantra mgra Covalent mteractlon of 3H-DA with bran-r proteins m VIVO and with the DA reuptake site 1x-rvitro

1980

Logan and O’Donovan, Paton,

1980

Ross,

1980

Ross and Ask,

Slotkm

1980

and Barels,

Urwyler

West

1980

and von Wartburg,

and Flllenz,

Aceves

1980

and Cuello,

Benhararl

et al , 1981

Burrows

et al., 1981

Cheramy

1981

and Youdlm,

Bracken

Davies

1980

et al., 3981 and Abood,

1981

1981

1980

mvestrgated

(conffnued)

Amine

Transport

469

in titro TABLE

1 (cunt~zued)

Reference

Factors investigated

Enrus and Cox, 1981

GABA enhancement of 3H-DA release from strlatal slices dependence on slice size Physlcochemlcal propertles of phenylethylammes and their uptake mto synaptic vesicles prepared from caudate nucleus Na+, K+-ATPase and voltage mdependent release of cytoplasmlc neurotransmitters Dependence on extracellular Ca2+ of electrically mduced release of “H-NA from cortex slices

Lentzen

and Phllllpu,

O’Fallon

et al , 1981

1981

Orrego and Sanchez-Armass,

Rowe11 and Duncan,

Taklmoto

1981

1981

Subsynaptosomal dlstrlbutlon and release of 3H-ACh synthesized by cerebral cortical synaptosomes Study on effects of sympathomlmetlc ammes on release of ‘H-NA from different intraneuronal storage compartments

et al , 1981

Thompson

et al , 1981

Accumulation and release of 3H-DA m stnatal slices from young, mature, and senescent rats

Tsudzukl.

1981

Study showing that newly synthesized ‘“C-DA failed to accumulate immediately m synaptic vesicles m synaptosomal cytoplasm

Cavmess and Wghtman,

Chesselet

et al , 1982

1982

A rapid superfusion technque to differentiate release of DA from strlatal tissue evoked by sympathomlmetlc ammes from release evoked by K’ In vlvo and m vitro studies on presynaptic regulation of strlatal DA release

470

Baker and Dyck TABLE

1 (co~tr~~~cd)

Reference

Factors

Cuello,

1982

Fischer

et al., 1982

Llang

and Rutledge,

Mulder,

Pollard

1982 and Nlckolson,

Schoepp

and Azzaro,

Gershten

et al., 1983

Llang and Rutledge,

Pastuszko

1982

et al , 1982

Schoemaker

Mmchm

Review of storage and release of ammes, ammo acids, and peptldes m dendrites Naf, K’-ATPase and DA release from striated slices Carrier-mediated efflux of DA from corpus striatum Review article on subcellular locallzatlon, release, and termmatlon of actlon of amine, ammo acid, and peptlde neurotransmltters m the CNS Transmitter release and parallel changes m ultrastructure and NA content of nerve terminal5 in rat vas deferens

1982

Sanders-Bush,

1982

1983

et al , 1983

et al , 1983

investigated

1982

Review article on regulation of storage and release of 5-HT Interaction between influx and efflux of DA m strlatal synaptosomes Types A and B MAO and metabolism of “H-DA released from striatal slices Accumulation of 3H-DA by synaptic vesicles m membrane lmpermeable medium Ca2’-independent release of 3H-DA by veratrldme m corpus strlatum from rats treated with pargyline or reserpine Electroconvulslve shock and uptake and release of NA and 5-HT m brain slices DA uptake m strlatal synaptosomes exposed to peroxldatlon

Amlne

Transport

In Wro

471

Factors

Reference Saankoskl,

Seyfned,

1983

1983

Shalaby

et al , 1983

Taklmoto

et al , 1983

Thureson-Klein,

Wood

and Wylhe,

Wustmann

Dyck,

1983

et al , 1983

1984a

Cooper

and Meyer,

Cunnane,

Klein

1983

1984

1984

and Thureson-Klem,

Whlttaker,

1984a,b

1984

mvestqqated

Functional development of adrenerglc uptake mechanisms m human fetal heart Study on DA uptake-mhlbltory vs DA-releasing properties of fencamfamine Release of DA from coaggregate cultures of mesencephallc tegmenturn and corpus strlatum Effects of reserpme, KCl, and d-amphetamme on depletion of ‘H-DA from osmotically defined storage sites A commentary that discusses exocytosls from large and small dense coned vesicles m noradrenerglc nerve terminals Critical assessment of uptake of NA m synaptosomal preparations Hypoxia and DA release from strlatal slices of rats at different ages Review article on neuronal transport of trace ammes Review on mechanisms that may be involved In release and modulation of release of neuroactive agents Review article on mechamsms of neurotransmitter release from sympathetic nerves Orgamzatlon and function of noradrenerglc vesicles Review articles on the preparation and use of synaptosomes and synaptic vesicles

472

Baker

and Dyck

5-HT, ACh, choline, trace amines, histamine, and polyammes References m the table are listed chronologically, with authors listed m alphabetical order within a given year. The table references can also be found m full, alphabetically, m the References section

2. Uptake and Release of Specific Amines 2.1. Catecholamines Of all the biogemc ammes, the catecholammes noradrenalme (NA) and dopamme (DA) have probably been studied the most. The neuronal and nonneuronal transport of NA was studied extensively m the 1960s. There is now ample evidence demonstrating that NA is accumulated by noradrenergic neurones by a two-stage process The first stage involves passage of NA across the neuronal plasma membrane, and the second stage mvolves uptake of NA Into, and the subsequent bmdmg of NA m, mtraneuronal vesicles. Although the term uptake IS often used to include both processes, in strict terms the first process is uptake and the second is storage. It has been demonstrated that highaffuuty uptake of NA m both the periphery and the CNS is a saturable process that obeys Michaelis-Menten kmetics (Paton, 1976); the apparent K,,, m this process for (-)NA is of the order of 0 l-l PM in a variety of tissues The neuronal uptake of NA has been demonstrated to be temperature-sensitive and Na ‘-dependent (Paton, 1976). The uptake process for NA has the characteristics of a carrier-mediated process (White, 197610) It appears that the carrier can mediate both the uptake and release of the amme (White, 197613; Raiteri et al., 197713) It has been proposed that extracellular Na+ could facilitate the bmdmg of the amine to the carrier, increase the rate of translocation of the amme-carrier complex, or increase the total number of carrier sites available for uptake (Paton, 1980) The nature of efflux of NA (and DA) has been the subject of a large number of studies m recent years. It has been proposed that depolarizatron of the membrane of the nerve terminal by electrical stimulation, by a high-K+ medium, or by the addition of veratridme results m exocytotic release directly mto the synapse, whereas release caused by sympathomimetic ammes results m release from the vesicular storage site mto the axoplasm of the neuron followed by carrrer-mediated efflux (release) mto the synapse (Raiten et al , 1977b; 1979), the matter contmues to be a controversy (Trendelenburg, 1979) As with NA,

Amrne

Transport

473

In vitro

DA IS accumulated by Its neurons m the CNS by a process that 1s saturable and obeys Mlchaelis-Menten kinetics; the high affuuty transport (K,,, of 0.1-l FM) 1s temperature sensmve, energydependent, and Na+-dependent (Horn, 1976; Paton, 1980). Experiments by Rarteri et al (1979) suggest that with DA, as with NA, strmulatron produced electrically or by a high-K+ medium leads to exocytotrc release into the synapse, whereas sympathomimetrc ammes cause release into the axoplasm that IS followed by carrier-mediated release of DA into the synapse. A large number of psychotropic drugs are known to have rather marked effects on neuronal transport of catecholammes. These include trrcyclrc antidepressants, cocaine, amphetammes, methylphenldate, and some “second-generation” antrdepressants (e.g., nomlfensine). Whereas many of these drugs appear to inhibit catecholamme reuptake only, some (e.g., amphetamme, other sympathomrmetrc ammes) have a true releasing effect. Some of the catecholamine uptake inhibitors are more selective for NA (e.g , desmethylrmipramme) than for DA, and vice versa (e.g., benztropme) Selected examples of studres on the effects of psychotroprc drugs and sympathomrmetic ammes on the transport of a variety of brogemc amines are comprled m Table 2 later in this chapter

2.2. 5Hydroxytryptamine

(5HT, Serotoninr)

A high-affuuty, saturable, Na’-dependent, and temperaturesensrtrve neuronal uptake of 5-HT has been demonstrated (Ross, 1980). As with the catecholammes, this accumulation appears to be a two-stage process, involving transport through the neuron membrane and binding to mtraneuronal storage granules (Baumgarten et al., 1978, Ross and Renyi, 1967). Carrier-mediated uptake and efflux have both been demonstrated for 5-HT (Ross, 1980; Ross and Kelder, 1977). Research thus far indicates that binding and storage of 5-HT in vesicles are similar to that for NA and DA, 1.e., u tillzing a mechanism dependent on ATP-Mg2+. A number of drugs and sympathomlmetic ammes with CNS activity are known to mfluence neuronal transport of 5-HT. These drugs include tryptamme derivatives, amphetamine and some of its analogs, trrcyclrc antrdepressants, and some “second-generation” antidepressants (e g., trazadone, zimelidme). Table 2 mcludes a hst of references describing the effects of such substances on neuronal transport of 5-HT. As noted prevrously with the catecholamines, those drugs that stimulate a carrier-mediated ex-

change of 5-HT cause a true release of 5-HT. In addition, several

h

so-called specrfic serotonm-uptake mhibltors have been discovered (e.g., zimehdme, citalopram, chlorimipramme). A relatively large body of evidence now suggests that there are at least two pools of DA, NA, and 5-HT m nerve termmals“storage” and “functional” pools. Much remains to be learned about the precise nature and physiological implications of these proposed pools (Mulder, 1982), but some detailed discussions are available to the interested reader (e.g., Glowmski, 1975, Patrick and Barchas, 1976; Bustos et al., 1978; Salzman and Roth, 1980; Cooper et al., 1982, Delanoy et al., 1982; Mulder, 1982; Cunnane, 1984; Whittaker, 1984b). 2.3. Choline and Acetylcholine Unlike the catecholammes and 5-HT, the postsynaptic action of acetylcholine (ACh) is not terminated by an active reuptake of Ach but by its enzymatic degradation. The cholme (Ch) produced by degradation of ACh by acetylcholinesterase is taken up into the nerve ending by a specific neuronal transport system and serves as a precursor for ACh synthesis (Murrm, 1980). It has now been demonstrated (Carroll and Buterbaugh, 1975; Dowdall and Simon, 1973; Yamamura and Snyder, 1972; Haga and Noda, 1973, Kuhar et al., 1975) that two kinetically distinct transport systems for Ch exist. The high-affmlty system (IX,,, of 0 5-10 pM)ls Na’dependent, has a regional distributron similar to other cholinergic markers, and is associated with an efficient conversion of Ch to ACh. The low-affinity system (K,,, of 50-200 PM) is very weakly Na+-dependent and does not appear to be related to the regional distribution of cholmergic neurons or to efficient synthesis of ACh Experiments have shown that the high-affinity Ch transport system is dependent on Na ‘, K ‘, and Cl- (Diamond and Kennedy, 1969; Kuhar et al., 1975; Yamamura and Snyder, 1973, Carroll and Goldberg, 1975; Murrin and Kuhar, 1976; Simon and Kuhar, 1976; Kuhar and Zarbin, 1978). Other than Naf and Kt, no other cations, mcludmg Ca2’, appear to be required for highaffmity Ch uptake (Murrm, 1980), however, depolarizationinduced increase m high-affinity Ch uptake has been shown to be Ca2+-dependent (Collier and Ilson, 1977, Murrm and Kuhar, 1976, Murrin et al., 1977, Roskoski, 1978). It has been proposed that the rate of activity of high-affinity Ch transport is controlled by the cytoplasmic concentration of ACh m chohnergic nerve endings (Jenden et al , 1976; Weiler et al., 1978), however, this concept is still controversial (Klemm and

Amlne Transport In V&o

475

Kuhar, 1979, Murrm, 1980). There has been considerable research to determine the nature of possible couplmg of high-affinity Ch transport to cholme acetyltransferase (ChAT). Some workers have suggested a direct physical couplmg (Barker and Mrttag, 1975; Collier et al , 1977), though others have proposed an indirect krnetrc coupling m which the Ch uptake is controlled by ACh concentration m the nerve terminal and the conversron of Ch to ACh IS governed by levels of ACh (Barker and Mittag, 1975, Suszkiw and Polar, 1976). The nature of this couplmg and the possibility of tissue difference m the mode of couplmg contmue to be active areas of research (Murrm, 1980). Another factor that deserves consideration 1s the proposal that high-affmrty Ch transport may be a mechamsm for removing a cholmergic agonist, Ch, from the synaptic cleft Though Ch 1s a weak cholinergic agonist, its synaptic levels may be concentrated enough to activate these receptors; if so, it would be important to remove Ch from the synapse (Palacros and Kuhar, 1979) Although ACh 1s not taken up from the synaptic cleft, there is a large body of m vitro evidence mdicatmg that the ACh synthesized in the neuron can be released by electrical strmulation, depolarizmg agents, and a variety of psychotroprc drugs and putative neurotransmrtters or neuromodulators (Grewaal and Quastel, 1973; Richter, 1976; Rowe11 and Duncan, 1981, Corner1 et al., 1981, Cantrill et al , 1983; Cubeddu and Hoffmann, 1983; Leventer and Johnson, 1983). The evidence in favor of quanta1 release of ACh at the neuromuscular lunctlon (Katz, 1971) and the subsequent discovery of isolation techniques for synaptosomes and synaptic vesicles (Gray and Whrttaker, 1960, 1962; De Robertis et al , 1962, 1963) led to a great deal of research on the characteristics of ACh release from nerve terminals m the CNS (Murrin et al., 1977, Mulder, 1982, Cunnane, 1984; Whittaker, 1984a,b). In addition, ACh release has been studied extensively using brain slices (Molenaar et al., 1973, Richardson and Szerb, 1974; Mulder et al , 1974; Richter, 1976). As with the catecholamines and 5-HT, there 1sgood evidence for the existence of more than one pool of ACh (Molenaar et al., 1973; Richter, 1976; Marchbanks, 1975; Cooper et al , 1982, Whittaker, 1984a,b) Despite the large volume of literature on this subject, there is still considerable disagreement on the relative importance of cytosollc and vesicular ACh u-rrelease of ACh caused by nerve stimulation. There are a number of excellent commentaries on this sublect, and the reader is referred to these (Israel and Dunant, 1979, Marchbanks and Wonnacott, 1979; Suszkrw and Whittaker, 1979;

476

Baker

and Dyck

von Schwarzenfeld et al., 1979; Zimmerman, 1979; Collier, 1984; Cooper and Meyer, 1984; Marchbanks, 1984; Vizi, 1984; Whittaker, 1984a,b,c; Zimmerman, 1984). Thesleff and Molgo (1983) have recently described a new type of ACh release at the neuromuscular Junction. This release is unaffected by nerve terminal depolarization and transmembrane Ca2+ fluxes, and these workers have proposed that it is partly responsible for the spontaneous mmrature endplate potentials observed m murme skeletal muscle m conditions such as botulmum poisonmg, nerve terminal degeneration, or treatment with 4-ammoqumolme.

2.4. Histamine and Polyamines Although the dramine histamine (HA) fulfills several of the criteria for a substance to be considered a neurotransmrtter (Green et al., 1978; Schwartz et al., 1980), a high-affinity, active uptake system has not been demonstrated for this amme. However, it has now been demonstrated that radiolabeled histidme, the precursor of HA, can be taken up by nerve terminals, and that the radiolabeled HA formed subsequently can be released from the tissue by depolarizmg agents and by neurotransmitters or neuromodulators (Verdiere et al., 1975; Biggs and Johnson, 1980; Fewtrell et al., 1982; Foreman and Jordan, 1983) Less work has been done with the polyamines (e g., spermine, spermidine, putrescme, cadaverme) than with other biogenic amines. It has been known for some time that the polyamines are involved mtimately with growth and replication of dividing cells (for review, see Seller, 1981, 1982) In recent years, there has been active interest in the possible role of polyamines as neuromodulators in the peripheral and central nervous systems. There have been a variety of reports of uptake and/or release of radiolabeled polyammes m neuronal tissue (Pateman and Shaw, 1975, Salzman and Stepita-Klauco, 1981, Harman and Shaw, 1981, Smith and Wyatt, 1981; Smith et al , 1982) Seiler and Deckardt (1978) found active uptake of ornithine, a precursor of polyamines, into synaptosomes and suggested that the previously reported active uptake of putrescme by bram slices (Laltha and Sershen, 1974) probably represented uptake into cell compartments other than nerve endings. Seller and Deckardt (1978) favored the hkehhood of local putrescine synthesis m nerve endmgs. Law et al. (1984) studred the effects of polyammes on uptake of some neurotransmitters by rat forebrain synaptosomes. They found inhibition of uptake of Ch, DA, GABA, and glycine by some polyamines, but the I& values determined were very high (all greater than 200 PM)

Amine Transport In V&o

477

2.5. Trace Amines The trace amines nz-tyramine (m-TA), p-tyramme (y-TA), B-phenylethylamme (PE), phenylethanolamine (PEOH), p-octopamme (OA), and tryptamme (T) are present m nerve tissues from a number of different animal species (Boulton, 1979; Boulton and Juorio, 1982, Philips, 1984; Williams et al , 1984) Their presence m such tissues and their structural similarities to catcholand mdoleamme transmitters have led to the suggestion that they also function as neurotransmitters In Invertebrates some of the trace ammes are present in kg/g amounts and seem to function as transmitters, however, m this chapter, we shall discuss only those studies that have used neural tissue from vertebrates. Previous studies of the transport of T, PE, and PEOH have shown they are taken up poorly by a variety of tissue preparations (Stacey, 1961, Ross et al., 1968; Ross and Renyi, 1971; Baldessarmi and Vogt, 1971; Born et al., 1972; Wu and Boulton, 1973, Pletscher, 1976, Costa et al., 1977, Greenberg and Whalley, 1978, Schroder et al., 1979, Osborne, 1980, Ross and Ask, 1980, Dyck, 1984b). Their uptake, m general, seems to be mediated mainly by diffusion rather than an active process. Similarly, the release of these compounds appears to occur mainly by a rapid spontaneous washout and depolarizmg stimuli do not increase their rates of release (Snodgrass and Iversen, 1974; Saldate and Orrego, 1977, Hery et al , 1979, 1983, Dyck, 198413). Such transport properties are consistent with the hpophillc properties of these trace ammes and seem to rule out at least a traditional type of transmitter function, because there do not appear to be speaalized systems to regulate their uptake and release and thereby regulate a neurotransmitter function. In contrast to the above lipophillc trace amines, the more polar trace amines (m-TA, p-TA, OA) generally have been found to be actively accumulated by various nerve tissue preparations (Commarato et al., 1969, Pletscher, 1976, Costa et al., 1977, Hicks, 1977; Lentzen and Phillipu, 1977, 1981, Dyck, 1978, Petrall et al., 1979; Wu et al , 1980; Blanch1 et al , 1981; Johnson et al., 1982, Dyck, 1984a) It has been suggested that m-TA, p-TA, and OA might act as alternative or cotransmitters since most studies have shown that both their uptake and release characteristics (Kopm et al., 1965, Baldessarmi and Vogt, 1972; Stoof et al., 1976, Hicks, 1977; Saldate and Orrego, 1977; Dyck, 1978; Dyck and Boulton, 1980) are compatible with conventional concepts of neurotransmission. Whereas some evidence seemed to indicate that specific m- or p-tyrammergic neurons exist (Nilsson and Holm-

478

Baker and Dyck

gren, 1976; Petrah, 1977, 1980; Juono and Jones, 1982), more recent data indicate that both radiolabeled and endogenous m- and p-TA are stored in rugrostriatal dopammergic nerve termmals (Dyck et al , 1982; Dyck, 1984a). Additional evidence of their coexistence m catecholammergic neurons has emerged from studies on the effects of inhibitors or activators of tyrosme hydroxylase on the endogenous levels of m- and p-TA (Juono, 1977,1979, Duffield et al, 1981) and on the synthesis of these ammes from radiolabeled precursors (Dyck et al , 1983) These studies have shown that the levels of m- and p-TA are controlled by the activity of tyrosme hydroxylase-an enzyme that is a marker for catecholammergic neurons. Even though these ammes may coexist with catecholamines, two different classes of drugs, the methylphemdate-like stimulants and the dipropyl substituted ami notetralms, stimulate release of m- and p-TA but not DA (Dyck, 198413). The mechanism of this preferential release is unknown, but probably mdicates a different intracellular storage of the tyrammes compared to DA The effects of Ca2+ removal on veratridme-induced release of p-TA and DA support this suggestion (Dyck, 198413).

3. Extraneuronal

Transport of Biogenic Amines

In this review, we have concentrated on the neuronal transport of biogemc ammes, however, it is also known that extraneuronal uptake (uptake-2) of some of these compounds also occurs In 1965, Iversen described a novel, low-affinity uptake process m heart; this process was subsequently characterized as extraneuronal uptake. Extraneuronal uptake of NA and adrenalme (A) was thought to explain a number of observations that had been made previously m heart and salivary gland (Raab and Gigee, 1955; Stromblad, 1959, Herttmg et al , 1962) Burgen and Iversen (1965) characterized this uptake process, determining uptake kmetics and affinity for a variety of ammes Extraneuronal uptake occurs m a variety of organs and species (see Hendley, 1976; Gillis, 1976, Gillespie, 1976, Almgren and Jonason, 1976; Trendelenberg, 1971, 1980) This extraneuronal uptake of catecholammes is a saturable transport process that obeys Michaelis-Menten kinetics. The transport system has a low affinity (high K,,,), but a high capacity (V,,,,,) for catecholammes (Bomsch, 1980) It is also characterized by low tissue/medium values, rapid efflux mto amine-free perfusion fluids, high affinity for isoprenaline, and an almost complete resistance to depletion

Amlne Transport In V&o

479

TABLE 2 In Vitro Studies on Effects of Some Sympathomlmetlc Ammes and Psychotropic Drugs on Neuronal Transport of Blogemc Ammes Reference Farnebo

and Hamberger,

Clofalo

and Lucero,

Hendley

Horn

and Snyder,

1972

1972

1973

and Baldessarml,

Hltzemann Ciofalo, Howes

Komlskey Banerlee

Pollard

1971

et al , 1972

Balfour,

Harris

Factors

1973

and Loh, 1973 1974

and Osgood,

1974

and Buckner, et al , 1975

et al , 1975

1974

studled

Field-stimulated brain slices and drug-induced changes m release of ‘H-monoammes Narcotics and narcotic antagomsts and synaptosomal 3H-NA uptake Ephedrine, methylphemdate, and phenyl-2-plpendyl carbmol and catecholamme uptake by synaptosomes RIgId analogs of amphetammes and catecholamme uptake by synaptosomes Nicotine and uptake and retention of 14C-NA and 14C-5-HT by brain homogenates Amphetamine analogues and uptake of 3H-catecholammes by homogenates of corpus strlatum and cerebral cortex Morphme and transport of DA mto bram slices Methadone and “H-5-HT uptake by synaptosomes A”-Tetrahydrocannabmol and uptake and release of 14C-DA m crude strlatal synaptosomal preparations Lithium and adrenerglc amme uptake m synaptosomes Cannabmolds and neurotransmltter uptake m synaptosomes Chlorpromazme and uptake of NA and 5-HT m synaptosomes from squid brain

Baker and Dyck

480 TABLE

2 (continued)

Reference

Factors

Ralterr

d-Amphetamine and release and mhrbrtron of reuptake of blogemc amines in synaptosomes Trrcyclrc antidepressants and mhrbrtron of uptake of 3H-NA and 14C-5-HT m slices and crude synaptsosome preparations of mrdbram-hypothalamus Oral admmlstratron of trlcyclrc antidepressants and uptake of 3H-NA and 14C-5-HT m slices of midbrain-hypothalamus Fenfluramme and accumulation of 5-HT and other neurotransmrtters into synaptosomes Neuroleptrcs and combmatrons of d-amphetamme and neuroleptlcs on 3H-DA uptake by strratal homogenates Amphetamine and p-hydroxyamphetamme and DA efflux from striatal tissue d-and I-amphetamine and uptake, release and catabolrsm of NA, DA, and 5-HT m several brain regrons Fenfluramme and blockade of synaptosomal 5-HT uptake and tryptophan hydroxylase actrvrty Effects of a large variety of drugs and sympathomlemetrc amines on uptake of catecholammes and 5-HT m synaptosomal preparations Amphetamine and release of DA from substantra mgra Effects of a variety of drugs on transport of ‘H-NA m whole brain homogenates

et al , 1975

Ross and Renyr,

1975a

Ross and Renyr,

1975b

Belm et al , 1976

Del RIO and Madro-nal,

1976

Fischer

and Cho, 1976

Holmes

and Rutledge,

Knapp

and Mandell,

1976

1976

Koe, 1976

Paden et al , 1976 Pylatuk

and McNerll,

1976

studied

(contwmf)

Amine

Transport

481

In V&-o

TABLE

2 (conttnued)

Reference

Factors studied

Raiteri

Mianserm and imipramme and uptake and release of neurotransmitters in synaptosomes p-Tyramme: release and receptorstimulatmg properties m brain Effects of mdolealkylammes on uptake and release of 3H-5-HT m striatal prisms Amphetamine, methylphemdate, and nomrfensme and transport of biogemc ammes Structure-activity relationships, p-carbolmes and mhibitlon of monoamme uptake mto a synaptosomal preparation Mazmdol, fenfluramme, and chlorimipramme and 5-HT uptake and storage Mazmdol and amphetamine and mhibltion of uptake and release of 3H-DA by stnatal synaptosomes Antidepressants and DA uptake m brain regions Mianserin and m vitro and m vlvo uptake of monoammes Nicotme and release of ammes from hypothalamus in vitro Isomers of cocame and tropacocame and 3H-catecholamme uptake by synaptosomes

et al , 1976

Stoff et al , 1976 Baker et al., 1977

Braestrup,

1977

Buckholtz

and Boggan,

Carruba et al., 1977a

Carruba et al , 1977b

Friedman

et al., 1977

Goodlet

et al , 1977

Gulati

and Shah, 1977

Komiskey

Moore,

et al , 1977

1977

Raiteri et al., 1977a

1977

Amphetamine catecholammes

and its effects on

Amphetamine, p-hydroxyamphetamme and a variety of phenylethylammes and

482

Baker TABLE

Rarteri et al , 1977b

Randrup

and Braestrup,

Ross,

1977

Smith

et al., 1977

Thomas

1977

Bosse and Kuschmsky,

Kruk

1977

and Jones, 1977

Yu and Smith,

Hyttel,

2 (contznued) Factors

Reference

1978

1978

and Zarrmdast,

Marquardt

et al , 1978

1978

and Dyck

studred

transport of “H-blogemc ammes m synaptosomal preparations Effect of desmethylrmlpramme on release of NA from hypothalamic synaptosomes by phenylethylamme derrvatrves New antidepressant drugs and mhrbltron of uptake of blogenlc ammes Reserpme and mhlbrtron of accumulatron of ‘H-DA m strlatum by amphetamine Phencyclldme and uptake of 3H-catecholammes and 3H-5-HT m synaptosomal preparations Clomlpramme and desmethylclomlpramme and uptake of radlolabeled 5-HT and NA m cortical slices Cocame and desmethylrmrpramme on uptake, retention, and metabolism of 3H-5-HT m bram shces Morphine and K +-induced release of 14C-DA from strlatal synaptosomes Psychotropic drugs and mhrbltron of 3H-DA accumulatron m strlatal synaptosomes Morphme and uptake and release of DA m mouse and rat strratal synaptosomes Stereoisomers of methylenedroxyamphetamme and synaptosomal uptake and release of 3H-NA

Amine Transport In V&o

403 TABLE

2 (contra&)

Reference

Factors

Martin

Viloxazme and transport of 3H-labeled DA, NA, 5-HT, and GABA m prisms prepared from striatum Review on tricylics and MAO mhibitors, mcludmg sections on then effects on uptake and release of catecholammes and 5-HT B-Carbolmes and high-affinity uptake of 5-HT, NA, DA, GABA, and cholme mto synaptosome-rich fractions prepared from various brain regions

et al , 1978

Maxwell

and White,

Rommelspacher

Ross and Renyi,

Sherman

1978

et al , 1978

1978

et al , 1978

studied

(+)-Amphetamine and retention of ‘H-catecholammes m slices of normal and reserpn-nzed brain and heart Neuroleptics m striatum

and choline

uptake

Slotkm

et al , 1978b

Study of effects of methadone on uptake of NA and 5-HT m synaptosomes and synaptic vesicles in vitro

Taylor

and Ho, 1978

Inhrbrtion of monoamme uptake by cocame, methylphemdate, and amphetamine

Tseng,

1978

5-HT uptake mhibitors and blockade of [I-methoxyamphetamme mduced 5-HT release

Ferris

and Tang,

1979

Isomers of amphetamine, methylpherudate, and deoxyprpradrol on the uptake of ‘H-NA and ‘H-DA m synaptic vesicles from whole brain, striatum, and hypothalamus

484

Baker and Dyck TABLE

Factors

Reference Kouyoumdllan

Logan,

Miller

et al , 1979

1979

and Friedhoff,

Rommelspacher Subramaman, Slotkm

et al , 1979

1979

et al., 1980

Cameron

and Smith,

de Langen

Gross

1979

and 1979

Trendelenburg,

Baker

2 (co~Wued)

1980

and Mulder,

and Schumann,

1980

1980

studied

Fenfluramme admmlstratlon and synaptosomal uptake of some neurotransmitters Inhlbltlon of catecholamme uptake by pemolme, amphetamine, and methylphemdate Haloperldol and apomorphme and K+ depolarized overflow of “H-DA from strlatal slices Tetrahydronorharmane and depolanzatlon-induced efflux of 5-HT and DA from bram shces P-Carbolme, mdolealkylamme, phenylethylamme, and n-alkylamme derivatives and mhlbltlon of ‘H-NA uptake mto synaptic vesicles Phenylethylammes and release of catecholammes from adrenerglc neurons MAO inhibitors and release of 3H-DA and 3H-5-HT from strlatal prisms Acute and chronic lithium treatment and “H-NA upJake by brain slices Psychotropic drugs and dlstnbutlon of “H-DA mto compartments of strlatal synaptosomes Neuroleptlcs and enhancement of NA release from cerebral cortex

Lai et al , 1980

Clorgylme and I-deprenyl and uptake of DA, NA, and 5-HT by synaptosomal preparation

Maltre

Criteria of selectivity uptake mhlbltors

et al , 1980

for amine

Amlne

Transport

In Wro

485 TABLE

Reference

Factors

McKlllop

and Bradford,

Steranka

and Sanders-Bush,

Arbllla

1980

1980

et al., 1981

Baker and Yasensky,

Brandao

et al., 1981

Demblec

and Cohen,

1981

1981

Freedman

et al , 1981

Glennon

and Rosecrans,

Holtman

and Richter,

Homan

2 (contznued)

and Zlance,

Jones,

1981

Kamal

et al , 1981

1987

1981 1981

studied

Benztropme and nomlfensme and DA uptake and release from striatal synaptosomes Brain DA concentration and synaptosomal uptake long term effects of contmuous exposure to amphetamme Amphetamine and electrical stlmulatlon-induced release of ‘H-DA from caudate nucleus Effects of phenylethylamme, I?-tyramme, and tryptamme on release of ‘H-DA from strlatal prisms Release of NA by TA a kmetlc study Effects of preexposure to uptake mhlbltors on K+-induced release of ‘H-catecholammes from brain Conformatlonally restrained analogues of 5-HT and uptake and bmdmg of 5-HT m rat brain Studies on mechamsms of action of hallucmogemc mdolealkylammes Barbiturates and K+-stimulated release of 3H-ACh &Amphetamine and potassium and serotonm release and metabolism m cerebral cortex tissue Antldepressant and antlcholmerglc drugs and uptake of choline m cerebral cortex slices Electrical stlmulatlon, amphetamine, and p-TA and presynaptlc modulation of release of DA from caudate nucleus

486

Baker and Dyck TABLE

Factors

Reference Kamlya

et al , 1981

Kelly,

1981

Mmchm

and Pearson,

Rapoport

et al., 1981

1981

Rauca et al , 1981

Smart,

1981

Taklmoto

Trelser

et al , 1981

et al , 1981

Ahluwalla

and Smghal,

Ary and Komlskey,

Azzaro

Chou

2 (contrnued)

and Demarest,

et al , 1982

de Boer et al , 1982

1982

1982

1982

studled

Imlpramme and K ‘-evoked ‘H-DA release m rat strlatum Effects of apomorphme and haloperldol on release of ‘H-DA and “H-NA from brain slices Catechol and neurotransmltter uptake and release of brain slices Compartment analysis of TAinduced depletion of NA Choline, hemxholmum-3, and naphthylvmylpyrldme and uptake and acetylatlon of ‘H-cholme m hippocampus sixes Competltlve mhlbltlon of cholme uptake by harmala alkaloids Sympathomlmetlc ammes and release of ‘H-NA from different intraneuronal storage compartments Ll ’ and 5-HT release m hlppocamPus Lithium treatment and withdrawal and uptake of DA mto synaptosomes Phencyclldme and release of ‘H-DA from chopped strlatal tlssue Type A and B MAO inhibitors and synaptosomal ‘H-DA accumulation Caffeine and amphetamine and DA uptake and release m corpus striatum Convulsant and antlconvulsant drugs and release of radlolabeled GABA, glutamate, NA, 5-HT, and ACh from cortical slices (torrfrmred)

487

Amine Transport In V&o TABLE

2 (cor-rtrrl~d)

Reference

Factors

de Jong et al , 1982

Effects of cY-alkyl substitution on mhibition of NA uptake m synaptosomes by phenylethylammes A structure-activity mvestigation of transport sites for hypothalamic and striatal uptake systems Revrew of effects of zimelidme on various neurotransmitter systems m brain Opiates and NA release Release of “H-DA from corpus strtatum effects of amphetamine, fenfluramme and unlabeled DA

Eckhardt

et al , 1982

Hall et al , 1982

Illes, 1982 Liang and Rutledge,

Makriyanms

Miller

Raiteri

1982

et al., 1982

et al , 1982a

Sparatore

Trulson

et al , 1982

and Shore,

Qumaux

1982

Ettects ot a-alkyl side chain and methoxyl ring substitutions on ml-ubition of synaptosomal ‘H-NA uptake by phenylethylammes Amphetamine and amtonelic acid and the disposition of striatal newly synthesized DA Five antidepressants and mhibibon of m vitro and ex VIVO uptake of NA and 5-HT: correlation with reduction of spontaneous fn-mg rate of central monoaminergic neurons Some atypical antidepressants and catecholammes synthesis and release Rigid analogies of imipramme and amitriptylme on uptake of NA, 5-HT, and cholme m synaptosomes

et al , 1982

and Trulson,

studied

1982

Chronic methamphetamme mmistration and 3H-5-HT synaptosomal uptake

ad-

488

Baker

and Dyck

TABLE 2 (contrnuec() Reference Vlckroy

Factors studied and Johnson, 1982

Waldmerer, 1982 Borrom et al , 1983

Cantrlll et al., 1983

Dubocovlch and Weiner, 1983 Goosey and Doggett, 1983 Hadfreld and Nugent, 1983 Leventer and Johnson, 1983

Marlen et al , 1983 Myers and Tessel, 1983 Dyck, 1984a

Dyck, 1984b Robinson and Marsden, 1984

Phenycyclldme and noramphetamme stimulants and release of DA from strlatal slices Antrdepressant drugs and DA uptake and metabolrsm d-Fenfluramme and d-norfenfluramme and presynaptlc 5-HT mechanisms d-Amphetamine and electrrcallyevoked release of “H-ACh from striatal slices Enkephalms and ‘H-DA release from retina Neuroleptlcs and strlatal 3H-DA release Effects of cocame on DA uptake m extrapyramldal and Ilmbrc systems Phencychdme and release of radloactlvlty from stnatal slices labeled with ‘H-cholme Oprords and regional ‘H-DA release Deslpramme and efflux of endogenous DA from hypothalamus Review of neuronal transport of trace ammes, with emphases on tyramine Effects of drugs on transport of ‘“C-tryptamine in slrces Effects of tryptamme on release of 5-HT from brain slices

for lsoprenalme, and an almost complete resistance to depletion by TA admmlstratlon. The DA metabohte 3-methoxytyramme IS accumulated mto strlatal slices by this extraneuronal process 1977) It has been shown that (Gordon and Shellenberger, catecholammes are metabolized prmclpally by catechol-o-

Amlne Transport In V&o

489

methyltransferase (COMT) after their transport into extraneuronal cells Mulder (1982) has proposed that at relatively low amme concentrations specific high-affinity uptake probably prevails in termmation of action of released neurotransmitter, whereas under conditions of high neuronal activity that result m high extracellular amme concentrations, low-affinity uptake mto a variety of cells may contribute substantially to the removal of the ammes

4. Presynaptic Receptors and Interactions Putative Neurotransmitters

Among

The concept of presynaptic receptors IS now widely accepted, and release experiments have given important support to such hypotheses. These receptors may be acted upon by neurotransmitters or neuromodulators other than the one employed by the nerve ending, or they may be “autoreceptors,” bemg acted upon by the neurotransmitter itself Such receptors for biogemc ammes have been studied conveniently by mvestigatmg the release (basal and/or stimulated) of the ammes m the presence or absence of various receptor antagonists or agonists The discovery of presynaptic receptors has given further impetus to an already active area of research, namely the mvestigation of mteractions among various neurotransmitters m the nervous system. An mterestmg approach to identlfymg the selectivity of dopammergic antagonists and agonists for preand postsynaptic receptors has been developed that assesses the abllity of the drug to modulate DA release (presynaptic effect) and its ability to stimulate ACh release from striatal mterneurons (postsynaptic effect) (Stoof et al., 1980). It is now evident that our origmal ideas about neurotransmitters were rather simplistic and that some very complex mteractions among various putative neurotransmitters and neuromodulators exist. Uptake and release experiments have been on the forefront m mcreasmg our knowledge of such mteractions. The experiments reported m this area are numerous, and in Table 3 we have given representative samples of studies m which m vitro transport experiments were employed to investigate interactions among neurotransmitters and to yield mformation about presynaptic receptors. Although the modulation of NA release by autoreceptors and other presynaptic receptors seems to be well established, many contradictory reports have appeared regarding the modulation of

490

Baker

and Dyck

striatal DA release. If DA release is sublect to such modulation, then DA antagonists would be expected to increase the amount of DA released by depolarization. This prediction was fulfilled m the mmal study by Farnebo and Hamberger (1971); however, several authors have not been able to repeat this finding (Seeman and Lee, 1975; Dismukes and Mulder, 1977; Arbilla et al., 1978, Raiteri et al., 197813, de Belleroche and Bradford, 1981), whereas many reports confirming it have also appeared (Westfall et al , 1976; Starke et al , 1978, Reimann et al , 1979, Hope et al., 1979) A number of factors may contribute to this difficulty. First of all, DA antagonists are highly reactive and can bmd not only to receptors in brain tissue but also to glassware and tubing used m the experiments, and can thereby contaminate the equipment (Starke et al , 1978) In addition, there appear to be species differences-the rabbit showmg an increase m DA released m response to an antagonist, but the rat showmg either a decrease or no change m DA release after admmistration of antagonists. Moreover, the type of response depends on the concentration of antagonist used (Miller and Friedhoff, 1979) Low doses increase and high doses reduce DA release. It appears therefore that although a response to DA antagonists occurs, the type of response IS variable. Recently, Lehman et al (1981) noted that the use of an MAO mhibitor during the preloadmg of rat striatal slices with 3H-DA abolished the subsequent presynaptic mhibition of its release b a DA agonist. Similarly, Stoff et al. (1982) observed a decreased YH-DA release m the presence of some DA agonists. However, though the failure of Arbilla et al (1978) to observe presynaptrc modulation of DA release by an antagonist was attributed to the use of an MAO mhibitor during the preloadmg, Brase (1980) observed that a DA agonist mhibited 3H-DA release from striatal slices obtained from an MAO mhibitor-treated rat It is perhaps important to note that when an agonist was used to study feedback regulation of DA release, the expected decrease m DA release was observed, however, when a DA antagonist was used, a variety of responses were observed. Interestmgly, there is little controversy regarding the autoreceptor-mediated mhibition of 5-HT release (Farnebo and Hamberger, 1974, Hamon et al , 1974; Bourgom et al., 1977, Cerrito and Raiteri, 1979, Gothert and Wemheimer, 1979, Baumann and Waldmeier, 1981, Martin and Sanders-Bush, 1982, Mounsey et al , 1982, Gothert and Schlicker, 1983; Suter and Collard, 1983). In most cases, this was tested by studying the effects of a serotonm agonist rather than an antagomst. When an antagonist was examined, it was usually added to antagonize the

Amlne Transport In WFO

491

agonist-induced mhrbition of 5-HT release. In many of these studies, it is assumed that the use of a superfusion technique prevents reuptake; hence, the problem of dlstmgurshmg between a true release and an apparent release (caused by inhibition of uptake) 1s overcome If this assumption is true, then the released transmitter should not be able to feedback onto the presynaptrc sites. In such a superfusion system, then, an antagonist should have no effect, and only an agonist added to the superfusion fluid should feedback inhibit DA release. Other types of receptors may also exist on the dopammergic terminals to regulate release The ability of ACh to regulate NA release in the periphery is well known, for example. ACh, by actmg on mcotmlc and muscarmic receptors, has been found to strmulate the basal release of striatal DA m the CNS as well (Westfall, 1974a, Glorguieff et al., 1976, de Belleroche and Bradford, 1980) In addition ACh can modulate depolarization-induced striatal DA release by acting on a muscarimc receptor, however, whether muscarimc stimulation mcreases or decreases evoked DA release IS unclear (Westfall, 197413, de Belleroche and Bradford, 1980, Raiteri et al , 198213) From a neuroanatomical perspective, rt seems possible that y-ammobutyric acid (GABA), glutamic acid, 5-HT, opiates, and substance I’ could also interact with receptors located on dopammergic terminals m the striatum. The effect of some of these potential modulators has been investigated with respect to altering either basal or K+-stimulated release m striatal tissue preparations 5-Hydroxytryptamme can increase the basal release of striatal DA, since high concentrations are required, this 1s probably the result of a carrier-mediated exchange rather than a presynaptic modulation (Andrews et al., 1978). Lower concentrations of 5-HT inhibit synthesis of DA m a manner compatible with receptor activation (de Belleroche and Bradford, 1980), but it is not clear how larger concentrations of 5-HT release DA (de Belleroche and Bradford, 1980) Recently, rt has been demonstrated that 5-HT (m PM amounts) inhibits K+-evoked striatal DA release (Enms et al,, 1981, Westfall and Titternary, 1982; Westfall, 1982). This effect was attributed to a presynaptic action of 5-HT. Glutamrc acid has been reported to stimulate strratal DA release either by itself or by potentiating K’-stimulated release (Giorgureff et al., 1977; Roberts and Sharif, 1978; Roberts and Anderson, 1979; Rudolph et al , 1983). De Belleroche and Bradford (1980) could not confirm this effect of glutamate on basal DA release Although some evidence suggests that glutamic acid

492

Baker and Dyck

fmdmgs inconsistent with this acts on presynaptic receptors, were seen m animals with kamic acid-induced lesions of the striaturn (Roberts and Anderson, 1979) GABA also affects DA release Some studies have shown that GABA potentiates Kt-stimulated DA release (Starr, 1978, 1979, Kerwin and Pycock, 1979, Stoff et al , 1979); however, Martm and Mitchell (1980) could not replicate this. The thickness of the slice and the location (rostral-caudal dimension) from which it is taken from the striatum can alter the effect that GABA exerts (Starr, 1979; Stoff et al., 1979). This may contribute to the lack of reproducibihty found m studies on the effects of GABA on basal DA release. It has been found to have no effect, to increase or to decrease basal DA release (Giorguieff et al , 1978, Cheramy et al , 1978, Kerwm and Pycock, 1979, Starr, 1978, 1979, Reimann et al , 1982). According to Reimann et al. (1982), GABA mhibits DA release (basal or stimulated) by actmg on a presynaptic receptor, but if GABA is taken up mto the nerve terminals, then it decreases release. Glycme has also been reported to increase the basal and K+-stimulated release of trmated DA (Giorguieff-Chesselet et al , 1979, Kerwm and Pycock, 1979, Martin and Mitchell, 1980) Martm and Mitchell (1980), however, did not observe any effect of glycme on basal release. In summary, although a number of mconsistent reports have appeared regarding the abilities of transmitters (DA itself, and others) to modulate DA release, presumably via presynaptic receptors, these inconsistencies seem to be largely related to experimental techniques In general, ACh, glutamic acid and GABA have been found to enhance the stimulated release of DA 5-Hydroxytryptamme also appears to interact with presynaptic receptors to mhibit DA release For a recent update on the status of presynaptic regulation of neurotransmitter release, the reader IS referred to a review by Chesselet (1984) that was published during the preparation of this chapter.

5. Binding of Antidepressants Amines

and Uptake of Biogenic

This chapter has dealt primarily with transport m neurons, but it should be noted that transport of ammes m platelets has also been studied extensively because of structural similarities between platelets and nerve terminals and because of the availability of

Amine

Transport

In Wtro

493

TABLE 3 Use of m Vitro Transport Experiments to Study Interactlons Among Neurotransmitters and to Investigate Presynaptlc Receptors Factors investigated

Reference Westfall,

1974

Westfall

et al , 1976

Reubl et al., 1977 Starke et al , 1977 Subramaman

Anderson Arbllla Cheramy

and Mulder,

and Roberts,

and Langer,

1977

1978

1978

et al., 1978

Harms

et al , 1978

Kerwm

and Pycock, 1978

Pelayo et al , 1978

Ralterl et al , 1978b Reubl et al , 1978

Starke et al., 1978

Muscarmlc agonists and release of radlolabeled catecholammes by K ’ and electrical stlmulatlon from brain slices Presynaptlc receptors and release and synthesis of 3H-DA by strlatal slices DA and release of 3H-GABA from substantla mgra Presynaptlc receptors and catecholammes Histamine and efflux of radlolabeled catecholammes from brain slices Ammo acids and “H-DA release from striatum GABA and the K+-evoked release of 3H-NA from occlpltal cortex GABA and DA release from nlgrostrlatal dopammerglc neurons Adenosme and depolanzatloninduced release of 3H-NA from slices of neocortex Glycme and ‘H-DA release from dendrites of substantla mgra Cyclic nucleotldes and regulation of NA release from rat pmeal through presynaptlc receptors Dopamme autoreceptors GABA, DA, and substance P and release of newly synthesized ‘H-5-HT from substantla mgra DA receptor agonists and antagorusts on DA release m caudate nucleus (contmud)

494

Baker and Dyck TABLE

3 (contznued)

Reference

Factors

Starr,

GABA and Kf-stimulated 3H-DA release from slices of substantla mgra and corpus strratum Regulation of DA release by presynaptic nicotmic receptors m strratal slices GABA-mediated potentratron of amine release from nigrostriatal DA neurons Presynaptrc DA receptors and electrrcal strmulatron and amphetamine-evoked release of 3H-DA from caudate nucleus GABA and drazepam and ‘H-serotmm release from hlppocampal slrces Presynaptlc and postsynaptrc strratal DA receptors and drfferentral sensmvlty to apomorphme mhlbttron of 3H-DA and 14C-GABA release Presynaptrc control of synthesis and release of DA from stnatal synaptsosomes effects of 5-HT, ACh, and glutamate NA and depolanzatron mduced 3H-5-HT release from slices of hlppocampus

1978

Giorgmeff-Chesselet

Starr,

Arbrlla

Balfour,

et al , 1979

1979

et al , 1980

1980

Brase, 1980

de Belleroche

and Bradford,

Frankhuyzen

and Mulder,

Gothert

and Huth,

1980

1980

1980

mvestrgated

cr-Adrenoreceptor-mediated modulation of 5-HT release from cortex slices

Herttmg

et al , 1980

DA and ACh release m caudate nucleus

Jackrsch

et al , 1980

Presynaptrc dopammergrc of DA release m caudate

Langer,

1980

Presynaptic regulation catecholamme release

control nucleus

of

(cwztmxd)

Amlne

Transport

In Vitro

495 TABLE

Factors

Reference Martin

and Mitchell,

Starr, Vizl,

1980

1980 1980

Wemstock

Aurelha

et al , 1980

et al , 1981

Borawska Corrieri

and Wismewski,

1981

et al , 1981

Enms et al , 1981 Erlavec et al , 1981 Kamal et al., 1981

Blanch1 et al 1982 Fewtrell

et al , 1982

Frankhuyzen

and Mulder,

Illes, 1982 Kalsner, 1982 Maura

3 (cantznued)

et al , 1982

1982

mvestlgated

Ammo actds and K+-Induced release of ‘H-DA from striatum GABA and DA release m substantia nigra Modulation of cortxal release of ACh by NA NA and modulation of the mcrease m striatal DA metabohsm induced by muscarmlc receptor stimulation DA autoreceptors and ‘H-DA release m caudate nucleus Bradykmm and uptake and release of DA by strlatal synaptosomes Inhlbitlon by adenosme denvatives of cholme uptake and ACh release Inhibitory 5-HT receptors that modulate DA release m striatum Substance I’ and hlstamme release Presynaptlc modulation of DA release from caudate nucleus, electrical stimulation, amphetamine, and tyramme GABA and ACh release from brain slices Substance I’ and hlstamme and 5-HT release Charactenzatlon of presynaptic receptors modulatmg ‘H-NA release from bram slices Opiates and NA release Discussron of presynaptx receptors NA inhibits 5-HT release through cxuz-adrenoreceptors on serotonergic nerve terminals

496

Baker and Dyck TABLE

3 (co~~fm~cd)

Reference

Factors investigated

Ralterl et al., 1982b

Presynaptic muscarinic receptors and strlatal DA release GABA and DA release m the caudate nucleus Presynaptlc a-adrenerglc modulatlon of 3H-NA and 3H-5-HT release m brain slices 5-HT and the electrically induced release of ‘H-DA m strlatal slices DA and carrier-medlated release of GABA from retinal horizontal cells Automhlbltlon of brain HA release DA receptors and electrically evoked release of “H-ACh from striatal slices DA receptors and release of ACh and DA from strlatum Enkephalms and “H-DA release from retma a-Adrenoreceptors and release of 5-HT and NA m cortex Histamine release induced by neuropepbdes Transmitter uptake mhlbltlon and effects of cu-adrenoreceptor agonists on 5-HT and NA release m cortex Regional release of “H-DA oplolds and release induced by K’ , mcotine, and L-glutamlc acid DA autoreceptors after chronic haloperldol treatment a-Adrenoreceptor antagonists and release of 5-HT and NA from cortex slices influence of NA uptake

Relmann et al , 1982 Schoffelmeer and Mulder,

Westfall and Tlttermary,

1982

1982

Yazulla and Klemschmldt,

1982

Arrang et al , 1983 Cantrlll et al , 1983

Cubeddu and Hoffmann,

1983

Dubocovlch and Wemer, 1983 Enms, 1983 Foreman and Jordan, 1983 Gothert et al , 1983

Marlen et al., 1983

Nowak et al , 1983 Schhcker et al , 1983

Amine Transport In Vitro

497 TABLE 3 (confznued)

Reference

Factors mvestlgated

Ueda et al., 1983

Westfall Chesselet,

et al , 1983 1984

mhlbltlon and determmatlon of pA2 values Release of endogenous NA and DA from slices of rat hypothalamus presynaptlc medlatlon by alpha2-, beta,-, and beta*-adrenoreceptors Nlcotinlc receptors and release of DA and 5-HT from strlatal slices Overview of presynaptic regulatlon of neurotransmitter release In the brain

platelets for clmlcal studies (Blanch1 et al., 1981; Costa et al , 1977, Lmglaerde, 1981, Paasonen, 1973, I’letscher and Laubscher, 1980, Stahl and Meltzer, 1978; Tuomisto, 1981). Work in this area has increased following the report of a high-affimty binding site of 3H-lmipramme m human platelets (Briley et al., 1979) and m brain tissue (Bnley et al , 1980, 1981; Brunello et al , 1982; DumbrilleRoss et al , 1981; Gross et al., 1981, Hrdina et al., 1982; Kinmer et al., 1981; Langer et al., 1981, 1982; Palkovits et al., 1981). It has been proposed that this binding site 1s closely related, but not Identical, to the uptake site for 5-HT (Barbacaa et al., 1983; Langer et al., 1980; Mocchetti et al , 1982; Paul et al., 1981; Rehavl et al , 1981, Talvenhelmo et al., 1983). Subsequent studies have demonstrated a high-affmlty binding site for 3H-desmethylimlpramine that 1s thought to be associated intimately with the uptake site for NA (Hrdina, 1981; Lee et al., 1982; Ralsman et al., 1982; Rehavi et al , 1982) In addition, 3H-cocaine and 3H-mazindol have been proposed as ligands for the DA transport site (Javltch et al., 1983, Kennedy and Hanbauer, 1983; Reith et al., 1983, Pimoule et al., 1983) These findings will be Important m furthering our understanding of transport mechanisms for ammes, but may have even more far-reachmg clmlcal impllcatlons because it has been suggested that bmdmg of 3H-antldepressants may be a useful pharmacologlcal tool in the diagnosis of affective and neurological dlsorders (Langer and Ralsman, 1983).

498

Baker

and Dyck

6. Typical Protocols Employed in Neuronal Transport Studies In Vitro 6.1. Effects of Drugs on the Uptake of Radiolabeled DA, M, or 5-HT Into Prisms Prepared From Rat Brain Areas This protocol is based on the procedure of Martin et al (1978) Rats are killed by cervical fracture, the brains removed onto an ice-cooled plate, and the appropriate area dissected out The tissue is chopped on a McIlwam trssue chopper to give prisms 0.1 x 0.1 x approximately 2 mm, and dispersed m cold mcubatlon medium contammg 123 mA4 NaCl, 5 mM KCl, 2 7 mM CaC12, 1 2 m&I MgS04, 20 mM Tris-HCl buffer, pH 7.4, and 10 mM glucose, 50 pM pargyline (or 12.5 FM malamide), and 1 mM ascorbic acid. The trssue suspension at a concentratron of 1 mg/mL IS then equrlibrated at 37°C m a shaking water bath for 15 min “H-labeled NA, DA, or 5-HT (NEN) is then added simultaneously with varrous concentrations of drug and the mcubatlon continued for a further 5-10 mm. The tissue IS subsequently separated from the incubation medium either by high-speed centrlfugatlon (1 mm m a Beckman microfuge B) or by rapid filtration using Mrlllpore filters and is washed twice with warm (37°C) mcubation medium The tissue pellet or filter contammg the tissue is dissolved m ethoxyethanol or a srmilar tissue solubillzer, and a lrqurd scmtillation cocktail is added to the counting vial. All samples, mcluding controls (no drugs added) and blanks (incubated at O’C) are run in duplicate or triplicate 6.2. Superfusion Apparatus to Study Effects of a Drug on the Release of Radiolabeled DA, NA, or 5HT in Prisms Prepared from Rat Brain Areas The procedure described here is taken from Baker et al (1980) and is a modrfication of the procedure of Raiteri et al. (1974, 1975) that was used to study release from synaptosomes or P2 fractions The mitral part of the experiment IS carried out as described in the uptake experiments above, except that the drug was omitted during the incubation perrod The tissue 1s subsequently separated from the mcubatron medium by rapid filtratron through a Mlllipore filter contained m a superfusion chamber (Ralteri et al., 1974) thermostatically maintained at 37°C. The tissue is washed by connecting the stem of the chamber to a vacuum line and pouring 2 x 5 mL of mcubatron medmm at 37°C over the tissue. Subsequently, more mcubatron medium 1s added to the

Amine

Transport

In Vitro

499

chamber containing the filter and tissue. The outflow from the chamber IS then attached to a peristaltrc pump and the mcubatron medium IS drawn over the tissue at a rate of 0.5 mL/mm, l-mm fractrons are collected and at fractrons 46 the mcubatron medrum in the chamber IS replaced by medium containing the drug of mterest, and the superfusion IS contained for a further 10 min. The tissue and filter are then removed from the chamber, drssolved m ethoxyethanol, and the radroactrvrty present m each of the fractions and that remaining m the tissue IS determmed by liquid scintrllatron countmg The radioactrvrty present m each of the fractions is then expressed as a percentage of that recovered m all the fractions plus the tissue The results are calculated as the change m release caused by the drug compared to the release obtamed in controls without the drug addmon. The constructron of a superfusron chamber used m such studies 1sdescribed by Rarterl et al. (1974) A typical release curve IS shown m Fig 1.

6.3. Transfer Procedure to Investigate the Release of Radiolabeled p-TA and DA From Rat Striatal Slices Thrs protocol is based on procedures described by Dyck et al. (1980, 1984b). 6.3.1. Preparatron

of Media

The cornpositron of the standard Krebs-Henselert medium 1s as follows. 120 mM NaCl, 4.75 mM KCl, 1 77 mM CaC12, 1 18 mM MgS04, 26 mM NaHC03, 1 2 mM KH2P04, 5 5 & glucose, 58.5 mM sucrose, 1.1 mM ascorbic acid, and 12 5 FM nialamrde or 10 FM pargyline The following modrfred media with these alterations can also be employed (1) low-sodmm medium NaCl replaced by 240 mM sucrose (2) high-potassmm medium: 50 mM KCl, 75 mM NaCl (3) calcmm-free medium. CaC& replaced by 2 mM EDTA or EGTA (disodmm) (4) high-potassmm calcmm-free medrum, as m (2) and (3) above (5) cocame medium 5 PM cocame hydrochlorrde added. 6.3.2. Preparation

of Sbces

Rats are stunned and sacrrfrced by cervical dislocatron. The brain IS removed rapidly, rmsed m the chilled standard medium, and placed on an ice-chilled Petrr dish. The anterior portion of each

Baker

500

and Dyck

9.0 > c, a-

8.0

i

7.0 m

: (II L

6.0

; ‘;

5.0

;

4.0

s 3.0 2.0

1

1

1

1

1

t

L

t

1

1

1

1 2 3 4 5 6 7 8 9 1011 Fraction number

1

12

from stnatal prisms by F% 1 Release of ‘H-DA (&)-amphetamine (10 pM) The control fractions are represented by open circles Amphetamine was added to superfusmg medium at fractlon 4, and fractions collected m the presence of this drug are represented by the open triangles

caudate-putamen sected

so

as

complex (hereafter called the stnatum) IS dlsto

exclude

the

globus

palhdus

and

nucleus

accumbens, and extends from the frontal plane of the anterior commissure to the mldportlon of the body of the caudate-putamen complex Tissue weights (10-15 mg) are determined prior to sllcmg A Sorvall mechanical chopper IS used to slice (0 2 mm thickness) the strlatum m a dlrectlon parallel to the internal capsule fibers 6.3 3. Release

Procedure

The sliced strlatum 1s placed in a tube contammg 4 mL of oxygenated (95% 02, 5% COz) standard medium (37°C) wlthm 5 mm of

Amine

Transport

3 30

501

In vitro

1

..I..

14 C-DA

H-g-TA 1 UM Ver --

3

4

5676910

Ca*+ present No Ca*+

345670910

T

T *

“5

T

10 IJM Ver

...._T.,. .

;*.

‘.., : ‘.A I.,a \..... ‘..

10

345676910

;

fraction

345678910

T -.:-

T

number

Fig. 2. Release of 3H-p-TA and 14C-DA from brain slices by veratridine. Values represent means +SEM. Using Student’s f-test for unequal variance, *p ~0.05, comparing release into fraction 6 in the presence 1 ure reproduced from Dyck, 1984a, with perand absence of Ca2+. (F’g mission from Humana Press.)

Baker and Dyck

502

killing the rat, and the tube IS gently mixed to separate and suspend the mdrvidual slices. This suspensron 1s poured into a transfer holder assembly (free-floatmg type) and contmually oxygenated at 37°C. The slices are premcubated for 20 min. The transfer holder IS then lifted out of the tube to dram off the fluid, and the shces transferred mto the next tube and incubated for 5 mm m 4 mL of the standard medmm containing the nitrated and/or 14C amme (0.5 PM). The incubation medium is dramed off, and the slices are transferred every 5 mm through a sequence of 10 tubes, each containmg 4 mL of substrate-free medium maintained at 37°C and contmually oxygenated to agitate the mdivrdual slices The first five tubes contam the Krebs buffer, the remaining five tubes contam a “releasing” drug (e.g., amphetamine, methylphenidate) drssolved in the buffer After the slices have been transferred through the last tube of the sequence, they are trapped onto a paper disk, transferred mto a scmtrllatron vial, with 1.5 mL NCS tissue solubrlrzer added, and the sample left overnight. Then glacial acetic acrd (70 PL) IS added to neutralize the tissue solubrhzer and 10 mL of a toluene-based scintrllation cocktail added The radioactrvrty IS then assessed m a liquid scmtillation counter. The medium in each of the tubes (now called a fraction) is poured into a scintillatron vial and 15 mL of Aquasol is added The radioactrvrty spontaneously “washed” mto each fraction or released by the drugs is then assessed by lrquid scintillation spectrometry A typical release curve IS shown m Fig. 2 The radioactivity in the ten “release” fractions plus the radioactrvrty left m the tissue are added together, and the amount of radroactivrty released into each fraction 1s expressed as a percentage of this total.

Acknowledgments The authors gratefully acknowledge support from the Mental Health Advisory Councrl, the Alberta Heritage tion for Medical Research, the Medical Research Councrl ada, the University of Alberta Hosprtal Special Servrces search Committee, and Saskatchewan Health.

Alberta Foundaof Canand Re-

References Aceves J, and Cue110 A C (1981) Dopamme

release induced

cal stlmulatlon of mlcrodlssected caudate-putamen mgra of the rat brain Neurosczence 6, 2069-2075

by electn-

and substantla

Amine

Transport

In Vitro

503

Ahluwaha I’. and Smghal R. L (1982) Effect of lithium treatment and withdrawal on uptake of noradrenalme into rat brain synaptosomes A kmetic study Pro8 Neuropsychopharmacol & Blol Psychratry

6, 339-342

Almgren 0 and Jonason J, (1976) Extraneuronal amme transport m glandular tissue, m The Mechantsm of Neuronal and Exfraneuronal Transporf of Catecholamznes (Paton D M., ed ) pp. 299-311 Raven Press, New York Anderson S D. and Roberts P J (1978) Ammo acid-mduced stimulation of 3H-dopamme release from rat striatum m vitro Brat ] Pharmaco/ 64, 429P. Andrews D W., Patrick R L , and Barchas J D (1978) The effects of 5-hydroxytryptophan and 5-hydroxytryptamme on dopamme synthesis and release m rat brain stnatal synaptosomes. 1 Neurockem. 30, 465-470 Annunziato L., Leblanc P , Kordon C., and Weiner R. I (1980) Differences m the kmetics of dopamme uptake m synaptosome preparations of the median eminence relative to other dopammergically mnervated brain regions Neuroendocrmology 31, 316-320 Arbllla S , Briley M S , Dubocovich M L., and Langer S Z. (1978) Neuroleptic binding and their effects on the spontaneous and otassmm-evoked release of “H-dopamme from the stnatum and of !?H-norepmephrme from the cerebral cortex Life Scl 23, 1775-1780 Arbilla S , Kamal L A , and Langer S. Z (1981) Amphetamme mhibits the electrical strmulation-evoked release of [7H]-dopamme from the rabbit caudate nucleus Brll J Pkarmacol. 72, 499P-500P Arbilla S , Kamal L. A , and Langer S Z (1980) Presynaptic dopamme receptors modulate electrical stimulation but not amphetammeevoked release of trmated dopamme from the rabbit caudate nucleus Bnt 1. Pkarmacol. 70, 45P46P. Arbilla S and Langer S. Z (1978) Effects of GABA on the K+- and TAinduced release of “H-NA from rat occrpital cortex slices. Brlf J Pkarmacol 63, 389P-390P.

Arbilla S. and Langer S. Z (1980) Influence of monoamine oxidase mhibition on the release of 3H-dopamme elicited by potassium and by amphetamine from the rat substantia nigra and corpus striatum Naunyn Sckmredeber;y’sArch Pharmacol 311, 45-52

Arrang J M , Garbarg M , and Schwartz J. C (1983) Automhibitton of brain histamine release mediated by a novel class (H3) of histamine receptor. Nature (London) 302, 832-837. Ary T E. and Komiskey H L (1982) Phencyclidme-induced release of [7H]dopamme from chopped striatal tissue. NeuropkarmacoloXy 21, 639-645 Atack C and Carlsson A. (1972) In vitro release of endogenous histamine, together with noradrenaline and 5-hydroxytryptamme, from slices of mouse cerebral hemispheres 1 Pkarm Pkarmacol 24, 990-992

504

BakerandDyck

Aurlella S , Langer S Z ‘3 and Lehmann J (1981) Dopamme autoreceptors mhlbltmg [ HI-dopamme release m the caudate nucleus of the cat Evidence for a role of endogenously released dopamme But ] Pharmncol 74, 226P Azzaro A J and Demarest K T (1982) Inhibitory effects of type A and type B monoamme oxldase mhlbltors on synaptosomal accumulation of [3H]dopamme A reflectlon of antidepressant potency. B~ochem

Pharmacol

31, 2195-2197

Baker G B , Hlob L E , and Dewhurst W G (1980) Effects of monoamme oxldase mhlbltors on release of dopamme and 5-hydroxytryptamme from rat strlatum m vitro Cell Mel Blol 26, 182-186 Baker G B , Martin I L , and Mitchell I’ R (1977) The effects of some mdolalkylammes on the uptake and release of 5-hydroxytryptamme m rat strlatum Brlf ] Pharmcol , 61, 151P-152P Baker G B and Yasensky D L (1981) Interactions of trace ammes with dopamme m rat strlatum Progr Netlro-Psydlopharlnacol 5, 577-580 Baldessarml R J and Vogt M (1971) The uptake and subcellular dlstnbutlon of aromatic ammes m the brain of the rat I Ne~?ochcnl 18, 2519-2533 Baldessarml R J and Vogt M (1972) Regional release of aromatic ammes from tissues of the rat bram m vitro j Neurochem 19, 755761 Balfour D J. K (1980) Effects of GABA and dlazepam on 3H-serotonm release from hlppocampal synaptosomes Eur ] Pharmcol 68, 11 Balfour D J K (1973) Effects of mcotme on the uptake and retention of “C-noradrenalme and “C-5-hydroxytryptamme by rat brain homogenates. Eur J Phamacol 23, 19-26 Baneqee S P , Snyder S H , and Mechoulam R (1975) Cannabmolds Influence on neurotransmltter uptake m rat brain synaptosomes 1 Pharmacol Exp Ther 194, 74-81 Barbaccla M. L , Gandolfl 0 , Chuang D M , and Costa E. (1983) Modulation of neuronal serotonm uptake by a putative endogenous 11gand of lmlpramme recogmtlon sites Pruc NnfI Acad Scl (USA) 80, 5134-5138

Barker L A. (1979) Choline avallablhty-choline high-affinity transport and the regulation of acetylcholme synthesis, m Brmn Acefylcholzne and Neuru-psychzatnc Dlseuse(Davis K L and Berger P. A , eds ), pp 515-531, Plenum, New York Barker L A and Mlttag T W (1975) Comparative studies of substrates and mhlbltors of choline transport and choline acetyltransferase 1 Pharmacul. Exp Ther 192, 86-94 Bartholml G , Stadler H , Carla M G , and Lloyd K G (1976) The use of the push-pull cannula to estimate the dynamics of ACh and catecholammes wlthm various brain areas Ncuropharmacolo~y 15, 515-519.

Amine Transport In titro

505

Baumann P A. and Maitre L. (1976) Is drug mhibition of dopamme uptake a mismterpretation of m vitro experiments? Nature (London) 264, 789-790. Baumann I’. A and Waldmeier I’ C. (1981) Further evidence for negative feedback control of serotonm release m the central nervous system. Nuunyrz Schmledekrg’s Arch. Pharmacol 317, 36-43 L , Blorklund A , Baumgarten H G , Klemm H. I’ , Lachenmeyer Lovenberg W , and Schlossberger H G. (1978) Mode and mechanism of action of neurotoxic mdoleammes A review and a progress report Ann N Y Acad Scr 305, 3-24 Belm M. F , Kouyaumdlian J C., Bardakdlian J , Duhault J , and Gonnard I’ (1976) Effects of fenfluramme on accumulation of 5-HT and other neurotransmitters into synaptosomes of rat brain Neuroyharmacologq 15, 613-617 Benharari R R and Youdim M B H (1981) Ontogenesis of uptake and deammatron of 5-hydroxytryptamme, dopamine, and betaphenylethylamme m isolated perfused lung and lung homogenates from rats But 1 Pharnracol 72, 731-737 Blanc] C , Tanganelli S , Marzola G , and Beam L (1982) GABA-induced changes m acetylcholme release from slices of guinea-pig brain Naunyn Schmledeberg’s Arch Pharmacol 318, 253-258 Blanch1 L , Stella L , Dagnmo G , de Gaetano G and Ross1 G (1981) The uptake of tyramme by rat platelets Blochem Pharnzacol 30, 709-713 Biggs M J and Johnson E S (1980) Electrically-evoked release of [3H]-histamme from the guinea-pig hypothalamus. Brat J Pharmacol 70, 555-560. Bomsch H (1980) Extraneuronal transport of catecholammes Pharmacology 21, 93-108 Borawska M and Wismewski K (1981) The influence of bradykmin m and release of dopamme by rat striatal vitro on uptake synaptosomes. Pal. ] Pharmacol Pharrn 33, 585592 Born G. V R., Juenglaroen K , and Michal F (1972) Relative activities on and uptake by human blood platelets of 5-hydroxytryptamine and several analogues But 1 Pharmacol. 44, 117-139. Borrom E , Ceci A , Garattmi S , and Menmm T. (1983) Differences between d-fenfluramme and d-norfenfluramme m serotonm presynaptic mechanisms J Neurochem 40, 891-893 Bosse A and Kuschmsky K (1978) Potassium-Induced release of “C-dopamme from synaptosomes of corpus striatum of rats Effects of morphine Arznem Forsch 28, 2100 -2102. Boulton A A (1979) Trace ammes m the central nervous system, m Iuf Rev. Blochem Physlol Pharmacol Blochem , vol 26 (Tipton K F , ed ) pp. 179-206 Umverslty Park Press, New York Boulton A A. and Juorio A V (1982) Brain trace ammes, m Handbook of Neurochemzstry, vol 1 (Laltha A., ed ) pp 189-222 Plenum Press, New York

506

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increases 3H-GABA release from slices of rat substanha mgra m vitro. Nature (London) 268, 652-654. Reubi J.-C , Iversen L L , and Jesse11 T M. (1978) Regulation of CABA release by dopamme m the rat substantia nigra, m Advances zn Blochemzcal Psychopharmacology, vol. 19 (Roberts I’ J , Woodruff G N , and Iversen L L , eds ) pp 401404 Raven Press, New York. Richter J A (1976) Characteristics of acetycholme release by superfused slices of rat brain 1 Neurochem. 26, 791-797 Roberts P J and Anderson S D (1979) Stimulatory effect of L-glutamate and related ammo acids on [3H] dopamme release from rat striatum an m vitro model for glutamate actions 1 Neurochem 32, 1539-1545 Roberts I’. J and Shanf N. A (1978) Effects of L-glutamate and related ammo acids upon the release of [3H] dopamme from rat striatal slices Bram Res 157, 391-395 Robmson C. M and Marsden C. A (1984) Tryptamme-induced changes m endogenous 5-hydroxytryptamme and [“HI-5-HT release from mouse hypothalamic slices, m Neurobiology of the Trace Ammes (Boulton A A., Baker G B , Dewhurst W G , and Sandler M , eds ) pp. 265270 Humana Press, Clifton, N J Rommelspacher H., Strauss S M., and Rehse K (1978) Beta-carbolmes A tool for mvestigatmg structure-activity relationships of the highaffinity uptake of serotonm, noradrenalme, dopamme, GABA and choline mto a synaptosome-rich fraction of various regions from rat brain 1. Neurochem 30, 1573-1578 Rommelspacher H and Subramaman N (1979) Tetrahydronorharmane modulates the depolarisation-induced efflux of 5-hydroxytryptamme and dopamme and is released by high potassium concentration from rat brain slices Eur J Pharmacol 56, 81-86 Roskoskr R Jr (1978) Acceleration of choline uptake after depolarization-induced acetylcholme release m rat cortical synaptosomes I Neurochem 30, 1357-1361 Ross S B (1980) Neuronal transport of 5-hydroxytryptamme. Pharmacology 21, 123-131 Ross S B (1977) Potentiation by reserpme of the mhibition by amphetamme of 3H-DA accumulation m the rat striatum ] Pharm Pharmacol 29, 433434. Ross S B and Ask A -L (1980) Structural requirements for uptake mto serotonmergic neurones Acta Pharmacol et Towcol 46, 270-277 Ross S B and Kelder D (1977) Efflux of 5-hydroxytryptamme from synaptosomes of rat cerebral cortex Acta Physlol Stand 99, 27-36 Ross S B. and Renyi A. L (1967) Accumulation of tntiated 5-hydroxytryptamme m brain slrces. Lrfe Scr 6, 1407-1415 Ross S. B and Renyi A L (1971) Uptake and metabolism of B-phenylethylamme and tyramme m mouse brain and heart slices. 1 Pharm Pharmacol 23, 276-279. Ross S B and Renyi A L (1978) Effect of (+)-amphetamme on the retention of 3H-catecholammes m slices of normal and reserpmlzed rat brain and heart Acta Pharmacol. et Toxtcol 42, 328-336

Amine Transport In Wtro

527

Ross S B and Renyi A. L (1975a) Inhibition of the uptake of 3H-dopamme and ‘“C-5-hydroxytryptamme m mouse striatum slices Acta Pkarmacol et Toxtcol. 36, 56-66 Ross S B and Renyi A. L. (1975b) Tricychc antidepressant afents I. Comparison of the mhibmon of the uptake of 3H-NA and ’ C-5-HT m slices and crude synaptosome preparations of the midbramhypothalamus region of the rat brain. Acta Pkarmacol et Tox~ol. 36, 382-394

Ross S. B., Renyi A L , and Brunfelter uptake of sympathomimetic ammes Pkarmacol

B. (1968) Cocaine-sensitive m nerve tissue 1 Pkavm

20, 283-288.

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eds.) pp 159-177 Marcel Dekker, New York Salzman P M and Roth R. H (1980) Poststimulation catecholamme synthesis and tyrosme hydroxylase activation m central noradrenergic neurons. II Depolarized hippocampal slices 1 Pkarmacol.

Exp

Tker. 212, 74-84

Salzman S. K. and Stepita-Klauco M. (1981) Cadaverme m the rat brain: regional distribution and acylahon of [ i4C] cadaver-me m vlvo and uptake m vitro I, Neurockem 37, 1308-1315. Sanders-Bush E. (1982) Regulation of serotonm storage and release Adv

Blockem

Psyckopkarmacol

34, 17-34

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528 desrpramme 318,

pretreatment.

Baker

and Dyck

Nuunyr~ Schmtedeberg’s Arch

Pharmncol

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Amrne Transport In V&o

529

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Baker

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and Dyck

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196, 276-281 Stoof J C., Llem A. L., and Mulder A M (1976) Release and receptor strmulahng properties of p-tyramme m rat brain Arch Int Pharmacodyn 220, 62-71 Stromblad B C R (1959) Uptake of mlected ‘%Z-adrenaline m denervated and in normally innervated submaxillary glands of the cat. Brtt ] Pharmacol Chemother. 14, 273-276 Subramaman N. and Mulder A H. (1977) Modulatron by hrstamme of the efflux of radtolabeled catecholammes from rat brain shces Eur 1 Pharmacol 43, 14%152 Suszklw J B and Polar G R (1976) Selective localrzahon of a high affinity cholme uptake system and its role m ACh formation m cholmerglc nerve terminals 1, Neurochem 26, 1133-1138 Suszkrw J B. and Whittaker V. I’ (1979) Role of vesicle recycling m vesicular storage and release of acetylcholme m Torpedo electroplaque synapses. Progr Bram Res 49, 153-162 Suter A H and Collard K J (1983) The regulatron of 5-hydroxytryptamme release from superfused synaptosomes by 5-hydroxytryptamme and Its lmmedrate precursors. Neurochem. Res 8, 723-730. Taklmoto G S , Amm B A , and Cho A K. (1981) Sympathomlmetlc amme-induced release of norepmephrme-3H from different mtraneuronal storage compartments Pharmacology 23, 31&325 Takrmoto G S., Strttsworth J. D Jr., and Stephens J. K. (1983) [3H]-Dopamme depletion from osmotically defined storage sites Ef-

AmIne

TranSpOJT

In

VJtrO

531

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Sot 21, 7-9

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Amine

Transport

In Vitro

533

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534

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Wu

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49, 141-151 Zimmermann H. (1984) False transmitters or false concepts7 Neurosczence 12, 343-344

Index A, see Adrenaline Acetylcholme, 64, 326, 377, 384, 387, 389, 395, 407, 408, 433-441, 457, 472, 474-476, 489, 491 N-Acetylserotonm, 88, 89, 95-103, 139, 140, 147, 270-272, 277, 278, 281, 282, 288-290 N-Acetyltransferase, 95-98, 102, 121 S-Adenosyl-L-methronme (SAME), 87-89, 98, 99, 101-103, 105, 106, 109, 110, 113, 115, 116, 119, 120, 122 Adrenaline (A, epmephrme), 4-11, 19, 20, 53-55, 87-89, 92, 93, 103-106, 130-136, 149-154, 169-174, 273, 275, 283, 284, 289, 294, 308, 326, 339, 351, 352, 354, 409, 429433, 478 Adrenergrc receptors, 376, 389, 396 Adrenochrome, 7 Adrenolutine, 7, 8 Aldehyde dehydrogenase, 419 Alkalr flame detectors, See Nitrogen-phosphorus detectors L-ammo acid decarboxylase, 97, 103, 121, 412, 413, 419, 420 y-Ammobutyrrc acid, see GABA p-Ammoclomdme, 389, 395 4-Ammoqumolme, 476 Amphetamine, 60, 230-232, 234236, 242, 252, 253, 255, 308, 313, 458, 473 Antiserum affinity, 276 535

Antiserum binding capacity, 277 Ascorbic acid, 7, 8, 202, 203, 210, 219, 220-225, 227, 229, 230-240, 243, 245, 249, 250, 253, 254 Autoradrography, 373403, 460 exposure, 390-394 mcubatlon, 387-390 llgands, 383-387 qualitatwe assessment, 394-397 quantrtatron, 397-402 techniques, 377-379 tissue preparation, 377-379 BANSYL derrvatrves, 16, 343, 344 Benztropme, 242, 473 Blcuculline, 387 rw-Bungarotoxm, 389 t-Butyl drmethylsrlyl derrvatrves, 344, 346 y-Butyrolactone, 251 Cadaverme, 63, 476 Caprllary columns, 46, 48, 51, 57, 59, 63, 65-67, 70, 72, 306, 307, 321, 327, 329, 331, 334, 346, 357 Carbodllmlde couplmg, 268, 272, 273, 282, 284 Catechol 0-methyltransferase (COMT), 95, 103-106, 108, 121, 283, 421, 489 Chemllummescence, 461 Chemography, 390 Chlonmlpramine, 474 p-Chlorophenylalanme, 28, 238, 245, 288, 289, 411

536

Index

Chlorpromazme, 240 Choline, 64, 326, 408, 409, 433-441, 457, 472, 474476 Choline acetyltransferase, 433, 434, 475, Cholmesterase, 433, 435, 474 Citalopram, 474 Clorgylme, 239 Cocaine, 473, 497 Colchlcme, 289, 462 COMT, see Catechol 0-methyltransferase Curare, 377

3,4-Dlhydroxyphenylethanol (DHPE), 327 3,4-Dlhydroxyphenylethylamme, see Dopamme 3,4-Dlhydroxyphenylethylene glycol (DHPG, DOPEG),

55,

57, 108, 136, 141, 225, 327, 423, 427, 429

5,6-Dlhydroxytryptophan, 239 3,4-Dlmethoxyphenylethylamme, 273, 275, 284

p-Dnn;;hylammobenzaldehyde, p-Dl~;thylammocmnamaldehyde,

D-600, 462 DA, see Dopamme Dansyl denvatwes, 6, 12, 16, 17, 19-21, 120, 158, 167, 171, 338, 343, 344, 347-352,

359

Densltometry, 19, 379, 398 Desmethyhmlpramme, 427, 497 DHPE, see 3,4-Dlhydroxyphenylethanol DHPG, see 3,4-Dlhydroxyphenylethylene glycol 1,2-Dlammoethane, see Ethylenedlamme derlvatlves Dlazotlzatlon, 268, 272, 275 Dlchlorvos, 435 Dlethyldlthlocarbamlde, 426 Dlhydroalprenolol, 389 Dlhydroxymdole derwatlve, 5, 9 Dihydroxymandehc acid (DOMA), 108 3,4-Dlhydroxyphenylacetlc acid (DOPAC), 5, 12, 13, 21-24, 57, 106, 108, 136, 138-140, 150, 158, 162, 163, 174-176, 203, 210, 248-256, 319, 326, 358, 419,

221-225, 227, 243, 308, 311, 312, 317, 327, 339, 346, 356, 421

3,4-Dlhydroxyphenylalanme (DOPA), 9, 10, 12, 106, 108, 109, 143, 144, 210, 275, 284, 419, 420, 422, 423, 426, 428

Dmltrophenyl derivatives, 341 DOMA, see Dlhydroxymandehc acid DOPA, see 3,4-Dlhydroxyphenylalanme DOPAC, see 3,4-Dlhydroxyphenylacetlc acid DOPA decarboxylase, 426 Dopamme (DA), 5, 6, 9, 10, 12, 18-21, 25-30, 53-55, 67-69, 87-89,

92, 93, 95, 103-106, 138, 149-154, 169-174, 202-204, 219-225, 227, 229, 230-238, 240-245, 248-256, 275, 283, 284, 289, 292, 308, 312, 316, 318, 325, 339, 341, 351, 352, 354, 377, 389, 407, 409, 419-422, 423, 426-428, 457, 472, 473, 476, 478, 488-492, 497-502

108, 119, 130-136,

Dopamme-p-hydroxylase, 112, 113, 115-120,

95, 426

DOPEG, see 3,4-Dlhydroxyphenylethylene glycol EC, see Electrochemical detectors ECD, see Electron-capture detectors Electrochemical detectors (EC),

Index

537

21, 70, 71, 329, 131, 132, 13.5-138, 144, 145, 147, 149-152, 155, 157-159, 162-164, 168, 169, 171, 174, 178-181, 216, 230, 235, 253, 460 Electron-capture detectors (ECD), 48-50, 55, 57-60, 62, 63, 66-68, 70 Enkephalms, 248, 251 Epmephrme, see Adrenaline Ethylenedramme derwatwes, 10-13, 21-24 Flame romzation detectors, 48, 49, 54, 55, 57, 59, 62-64 Flophemsyl derwatrves, 341 Fluorescamme, 17, 133, 167, 171, 343 Fluorescem, 287, 291 Fluorescence, l-30 advantages of, 21, 22 derrvatrves for, 4-18, 19-30 detection, 2, 5, 6, 18, 19, 129, 133-138, 144, 145, 147, 149, 152-154, 158, 159, 162, 164, 166, 171, 174, 178, 180, 181 instrumentation, 1, 2 methodological problems, 3-5 mrcromethods, 19, 20 native, 10, 13, 14, 18, 19, 131, 133-136, 144, 167, 171, 174, 178, 180, 181 quantrtatron, 6-20, 287, 288 sample methods, 22-30 theory of, 1, 2 a-Fluoromethylhlstldme, 442, 443 Formaldehyde condensatron, 268, 270-272, 274, 275, 283, 289, 290, 293, 294 Formaldehyde derrvatrves, 6, 11, 13 Fourier transform mass spectrometer, 328, 331

GABA,

12, 18, 20, 275, 295, 384,

387, 389, 395, 409, 476, 491, 492 Gas chromatography (GC), 22, 45-72, 3b5 advantages of, 70-72 capillary columns, 46, 48, 51, 57, 59, 63, 65-67, 70, 72 columns, 46 derrvatwes, 52, 53 heptafluorobutyrlc anhydrrde, 53, 59, 62, 63 pentafluorobenzene sulfonyl chlorrde, 60 pentafluorobenzoyl chlorrde, 54, 60, 63 pentafluoropropionic anhydrrde, 53, 57-59, 67, 69 trlchloroacetlc anhydrrde, 60 trrfluoroacetrc anhydrrde, 53-55, 57-59, 61-63, 66-69 trrmethylsllylatmg reagents, 54, 55, 57, 59 detectors, 48-51 electron capture, 48-50, 55, 57-60, 62, 63, 66-68, 70 flame lomzatron, 48, 49, 54, 55, 57, 59, 62-64 mass spectrometry, 49, 50, 51, 62, 149, 157, 164, 166, 180, 280, 305-323, 325 nitrogen-phosphorus, 48, 50, 60, 63, 64, 70 thermal conductivity, 48, 49 injection systems, 51 sample methods, 64-70 statronary phases, 47 support materials, 46 Gas chromatography-mass spectrometry (GC-MS), 305-323, 325-327, 329, 331, 333, 334, 341, 346, 352, 356, 357, 360 (see also Gas chromatography detectors) analysis, 311-321 of catecholamines and

538 metabolites, 311-313 of mdoleammes and metabohtes, 313 of phenylacetic acid, 314, 315 of phenylalanme, 313, 314 of phenylethylamme, 313 of tyrosine, 313, 314 derwatization, 307-309 extraction, 311 quantification, 309-311 derrvatrzatron, 307-309 extraction, 311 quantification, 309-311 Gas-liquid chromatography, see Gas chromatography GC, see Gas chromatography GC-MS, see Gas chromatography-mass spectrometry Glutamate, 275, 295, 491 Glutamic acid decarboxylase, 295 Glutaraldehyde condensation, 268, 273, 275, 290, 293, 295 Glycme, 389, 476, 492 HA, see Histamine Haloperidol, 232, 236, 240, 243, 255, 292 Halothane, 234 Hapten, 268, 274, 287, 291 Hemichohnmm-3, 435 Heptafluorobutyric anhydride (HFBA), 53, 59, 62, 63, 341, 344, 346 Hexafluororsopropyl derivatives, 344, 346 HFBA, see Heptafluorobutyric anhydride 5-HIAA, see 5-Hydroxymdole-3acetic acid High-performance liquid chromatography, see Highpressure liquid chromatography High-pressure liquid chromatography (HPLC), 18, 19, 21, 70, 71, 122, 129-183,

Index 216, 230, 235, 255, 281, 305, 329, 335, 338 for catecholammes, 130-136, 149-154, 169-174 for catecholamme metabolites, 136-144, 154-158, 162, 163, 174-180 for dihydroxyphenylacetic acid, 143, 144 for mdoles, 144-149, 158, 162, 164, 180-182 for phenohc trace ammes, 168, 169 for tryptamme and metabolites, 164-168 Histamine (HA), 6, 12, 14-16, 18, 19, 22, 60, 62, 63, 87-89, 92, 93, 95, 109-111, 275, 295, 326, 338, 389, 442, 443, 472, 476 Histamine-N-methyltransferase, 95, 109, 110, 121, 442 Histidme, 14, 16, 62, 111, 443, 476 Histidme decarboxylase, 121, 442 Histochemical fluorescence, 289 6-HMEL, see 6-Hydroxymelatonm Homovamllic acid (HVA), 5, 12, 13, 19, 21-24, 57, 108, 132, 136, 138-140, 145, 155, 157, 162-164, 174-176, 222, 227, 230, 236, 245, 249, 254, 308, 311, 312, 317, 319, 326, 327, 339, 346, 356, 358, 419, 421 HPE, see Hydroxyphenylethanol HPG, see Hydroxyphenylethylene glycol HPLC, see High-pressure liquid chromatography 5-HT, see 5-Hydroxytryptamme 5-HTOL, see Hydroxytryptophol 5-HTP, see 5-Hydroxytryptophan ol-Hydrazme histidme, 442 6-Hydroxydopamme, 29, 229, 231, 234, 250, 253, 289 5-Hydroxymdole-3-acetic acid (5-HIAA), 5, 13, 14, 18, 19, 21, 57, 59, 100, 101, 132, 144,

539

lndex 145, 180, 236, 254, 326, 412,

147, 181, 238, 270, 339, 413,

158, 161-164, 222, 225, 227, 239, 245-248, 282, 283, 308, 346, 356, 358, 416-418

167, 230, 252, 313 410,

Hydroxymdole-O-methyltransferase, 95-102, 121 Hydroxymandehc acid, 5, 62, 308, 316, 326, 356, 358

6-Hydroxymelatonm 149, 270, 280 Hydroxyphenylacetic

(6-HMEL), acid, 5, 62,

catecholammes, mdolealkylammes, radioimmunoassay, 277-286,

292-294 287, 288 267,

288

catecholammes, 283-286 mdolealkylammes, 277-283 Indole-3-acetic acid (IAA), 18, 19, 62, 147, 164, 165, 182, 308,

313, 326, 339, 346, 356, 358, 441 Iontophoresis, 221, 248 Isoprenalme, 488 Isothiocyanate derivatives, 341

69, 70, 308, 312, 320, 326, 327, 339, 346, 356-360

Hydroxyphenylethanol

(HPE),

327

Hydroxyphenylethylene glycol (HPG), 327 5-Hydroxytryptamme (5-HT, serotonm), 5, 6, 13, 15, 18-21, 25-30, 58, 59, 66-69, 87-89, 92, 93, 95-97, 100-103, 119, 132, 144, 145, 147, 158-161, 180, 181, 202, 222, 224, 225, 229, 238, 239, 245-248, 250, 252, 254, 270-272, 282, 288-290, 308, 313, 326, 339, 389, 409-418, 421 457, 472-474, 490, 491, 497-499

5-Hydroxytryptophan (5-HTP), 13, 14, 18, 97, 103, 144, 147, 158, 159, 161, 238, 410, 411, 413, 416, 418

5-Hydroxytryptophol (5-HTOL), 144, 147, 327, 413 IAA, see Indole3-acetic acid Imrpramine, 497 Immunological techniques, 267-296

antigens, 267-277 catecholammes, 273-275 mdolealkylammes, 268-272 immunohistochemistry, 267, 287-292

Kau-uc acid, 492 LSD, see Lysergic acid diethylamide Lysergic acid diethylamide

(LSD),

389

Magnetic sector mass spectrometer, 328, 329, 330, 331 Manmch reaction, see Formaldehyde condensation MAO, see Monoamine oxidase Mass fragmentography, see Gas chromatography-mass spectrometry Mass spectrometry, 21, 70, 112, 325-360, 460 (see also Gas chromatography-mass spectrometry and Gas chromatography detectors) derivatives, 340-347 experimental protocols, 347-359 acids and alcohols, 356-359 catecholammes, 351, 352 trace amines, 347-351 mstrumentation, 327-338 high resolution, 329-332 ion formation, 335-338 low resolution, 328, 329

540 quantitation, 338-340 sample mtroductlon, 333-335 selected ion momtormg (SIM), 328, 331, 332, 347, 352-356 Mazmdol, 497 Melatonm, 18, 19, 96, 98-103, 147, 149, 270-272, 278-282, 287, 289 Metanephrme (MN), 9-10, 11, 54, 55, 57, 103, 105, 106, 109, 115, 136, 142, 178-181, 273, 275, 283, 285, 294, 308 Metastable Ions, 329, 331-333, 359 Methamphetamme, 308, 313 3-Methoxy-4-hydroxyphenylethylene glycol (MOPEG, MHPG), 55, 57, 108, 136, 141, 155, 162, 163, 178, 273, 285, 308, 311, 312, 317, 327, 339, 356, 358, 423, 426, 427, 429 5-Methoxymdoleacetlc acid (5-MIAA), 147 3-Methoxysynephrme, see Metanephrme 5-Methoxytryptamme (5-MT) 13, 14, 18, 282, 290, 421 5-Methoxytryptophol (5-MTOL), 147, 283, 288 3-Methoxytyramme (3-MTA), 5, 9, 10, 12, 54, 55, 60, 64-66, 103, 105, 106, 136, 142, 203, 222, 273, 275, 284, 292, 308, 316, 419, 422, 488 3-Methyldopamme, see 3-Methoxytyramme Methylhlstamme (MHA), 16, 60, 62, 63, 110, 111, 326, 443 Methylphemdate, 473, 478 N-Methylscopolamme, 389 a-Methyl-p-tyrosme (wMPT), 9, 28, 29, 231, 249, 251, 292, 420, 426, 429 MHA, see Methylhlstamme 5-MIAA, see 5-Methoxymdoleacetlc acid MID, see Multiple Ion detectlon Monoamine oxldase (MAO), 10,

Index 121, 412, 413, 419421, 441, 443, 490 cu-MPT, see a-Methyl-p-tyrosme 5-MT, see 5-Methoxytryptamme 3-MTA, see 3-Methoxytyramme 5-MTOL, see 5-Methoxytryptophol Multiple ion detection (MID), 310 Musclmol, 384, 387, 389, 395, 396 NA, see Noradrenalme Nlalamlde, 289 Nmhydrm denvatlves, 14, 17, 27, 158 Nitrogen-phosphorous (NP) detectors, 48, 50, 60, 63, 64, 70 NMN, see Normetanephrme Nomlfensme, 473 Noradrenalme (NA, norepmephrme), 4, 5, 6-12, 18-20, 25-30, 53-55, 67-69, 87-89, 92, 93, 103-106, 108, 119, 130-136, 138, 149-154, 169-174, 202-204, 219, 225, 229-238, 248, 275, 283, 289, 292, 293, 308, 312, 316, 318, 326, 339, 351, 352, 354, 409, 423-429, 457, 472, 473, 478, 489, 491, 497-499 Norepmephrme, SW Noradrenalme Norharman denvatwes, 13 Normetanephrme (NMN), 9-12, 18, 54, 55, 60, 64-66, 103, 105, 106, 109, 119, 136, 142, 178-181, 203, 273, 292, 308, 312, 316, 318, 423, 426, 427 NP, see Nitrogen-phosphorous detectors NSD-1015, 239, 412 NSD-1055, 442 OA, see Octopamme Octopamme (OA), 6, 12, 18, 20, 62, 87-89, 95, 112, 113, 115,

Index 117-120, 122, 168, 169, 273, 292, 308, 325, 339, 350, 354, 443, 472, 477 OPT dertvatlves, 6, 13-19, 21, 22, 25-30, 133, 152, 158, 167, 181 Ormthme, 476 PAA, see Phenylacetlc acid Paper chromatography, 5, 10, 12 Paraoxon, 435 Pargylme, 232, 240, 242, 243, 289, 412, 413, 427, 441, 442 PBC, SW Propylbenzllyl cholme mustard PE, set Phenylethylamme PEG, see Phenylethyl glycol Pentafluorobenzene sulfonyl chloride (PFBS), 60 Pentafluorobenzoyl chloride (PFBC), 54, 60, 63, 344, 346 Pentafluorobenzyllmmo derrvatlves, 341 Pentafluoro-I?-propyl derrvatlves, 344, 345, 356 Pentafluoroproplomc anhydride (PFPA), 53, 57-59, 67, 69, 307, 311, 313, 314, 315, 341, 345-347, 356, 357, 359 Pergollde, 234, 249 Peroxldase, 287, 289 PFBC see Pentafluorobenzoyl chloride PFBS, see Pentafluorobenzene sulfonyl chloride PFPA, see Pentafluoroproplomc anhydride Phemprazme, 412 Phenylacetlc acid (PAA), 62, 273, 306-309, 314, 316, 321, 326, 327, 339, 345, 346, 356-358, 360 Phenylalanme, 308, 309, 313, 316, 326 Phenylethanolamme, 6, 12, 87-89, 95, 112-115, 117-120, 122, 308, 316, 319, 325, 341, 350, 354, 443, 472, 477

541 Phenylethanolamme-N-methyltransferase, 95, 109, 112, 115-121, 283, 429 Phenylethylamme (PE), 6, 12, 17, 52, 55, 59, 60, 62, 64-66, 87-89, 95, 112, 115-117, 119, 122, 292, 305, 306, 308, 313, 316, 319, 321, 325, 338, 339, 341, 342, 344, 347-350, 352, 354, 443, 472, 477 Phenylethyl glycol (PEG), 327 o-Phthalaldehyde, see OPT Polyammes, 6, 15-17, 20, 62, 63, 326, 472, 476, see also mdlvldual compounds Probenecid, 239, 413, 421, 426, 427, 429, 441 Propansyl derrvatlves, 343, 344 Propylbenzllyl choline mustard (PBC), 389 Propyldopacetamlde, 411 PUT, see Putrescme Putrescme (PUT), 6, 16, 17, 20, 63, 476 Pyrogallol, 292 QNB, see Qumuclldmyl benzllate Quadrupole mass spectrometer, 328, 329 Qumuchdmyl benzllate (QNB), 384, 389, 395

Radloenzymatrc assays, 22, 70, 71, 87-122, 150-152, 460 for catecholammes and derrvatlves, 103-106 acid metabohtes, 106-108 adrenaline, 104-106 L-DOPA, 108 dopamme, 104-106 noradrenalme, 104-106 normetanephrme, 109 enzyme purrficatlon, 94-96 general procedure, 89-91 for hlstamme, 109-111 for mdoleammes, 96-103

542 N-acetylserotonin, 98-101 serotonm, 101-103 for octopamine, 117, 118 for phenylethanolamme, 113-115 for phenylethylamme, 115-117 and “punch” dlssectmg technique, 91-94 senwtlvity, 88, 89 for tyramine, 118-120 Radlolmmunoassay, see Immunological techmques Release of neurotransmltters, 411, 457-502 Ro 4-4602, 412 SAME, see S-adenosylmethlonme Serotonin, see 5-Hydroxytryptamme SIM, see Mass spectrometry, selected ion momtormg SPD, see Spermldme Spermldme (SPD), 6, 15, 16, 20, 63, 476 Spermme (CPM), 6, 16, 20, 63, 476 Splropendol, 389 SPM, see Spermme Synephrme, 117, 118, 120, 273, 283-285, 294, 325, 350, 354 T, see Tryptamme TA, see Tyramme TFAA, see Tnfluoroacetlc anhydride Thermal conductwty detectors, 48, 49 Thin-layer chromatography, 2, 5, 6, 17, 19, 20, 21, 99, 100, 102, 103, 105, 106, 108, 109, 113, 117-120, 176, 178, 325, 327, 333, 341, 342, 347-352, 356 Time of flight mass spectrometer, 328 TP, see Tryptophan Transport, extraneuronal, 478, 488, 489

Index Transport, neuronal, 457-502, see also Release of neurotransmitters of acetylcholme, 474-476 antidepressant drug bmdmg, 492, 497 of catecholammes, 472, 473 of choline, 474476 defmltlons, 457, 458 drug effects, 479488 experimental condltlons, 459-472 experlmental protocols, 498-502 of histamine, 476 of 5-hydroxytryptamme, 473, 474 mteractions among neurotransmitters, 491-497 of polyammes, 476 presynaptic receptor effects, 489-491, 493497 tissue preparation, 458, 459 of trace ammes, 477, 478 Tranylcypromme, 61 Trazodone, 473 Trlchloroacetlc anhydride, 60 Trlcychc antldepressants, 473, 492, 497, see also mdlvldual compounds Trlfluoroacetlc anhydride (TFAA), 53-55, 57-59, 61, 62, 63, 66-69, 341, 346 Trlfluoroethyl denvatlves, 344-346, 356, 359, 360 Trlhydroxymdole denvatlves, 4, 7, 8, 10, 25-30, 133, 152, 171 Trlmethylsllyl derwatwes, 54, 55, 57, 59, 341, 344, 346, 347 Tropolone, 242, 421 Tryptamme (T), 5, 6, 12, 13, 18, 20, 52, 58-60, 66, 67, 97, 119, 147, 164, 282, 290, 308, 313, 325, 339, 344, 347-350, 354, 441, 442, 472, 477 Tryptophan (TP), 13, 144, 145, 147, 158, 161, 164, 167, 180, 181, 289, 326, 410, 411-413, 417, 419, 441

Index Tryptophan hydroxylase, 97, 121, 238, 291, 411 Tryptophan pyrollase, 411 Turnover rates, 407-444 of acetylcholme, 433-441 of adrenaline, 429-433 of dopamme, 419-422 of histamine, 442, 443 of 5-hydroxytryptamme, 410-418 of noradrenalme, 423-429 theory of, 407-410 of tryptamme, 441, 442 TYR, see Tyrosme Tyramme (TA), 5, 6, 12, 17, 18, 20, 52, 55, 59, 60, 62, 64-66, 71, 87, 88, 89, 95, 112,, 115, 118-120, 122, 168, 169, 273, 286, 305, 306, 308, 316, 319, 320, 325, 339, 341, 347-350, 354, 359, 443, 472, 477, 478, 488, 499-502 Tyrosme (TYR) 158, 164, 308, 309, 313, 316, 326, 417, 419, 422, 423, 426, 428, 429 Tyrosme hydroxylase, 121, 419, 420, 426, 478

Ultraviolet (UV) detectors, 158, 174, 180, 181 Uptake of neurotransmltters, 457-502 Uric acid, 237, 238 UV, see Ultraviolet detectors

Valine, 411 Vamllylmandelrc acid (VMA), 19, 57, 136, 155, 1744176, 308, 311, 312, 316, 319, 326, 327, 346, 356, 358, 423 Verapamll, 462 Veratndme, 462, 478 VMA, set Vanlllylmandellc acid Voltammetry, m vrvo, 197-256 appllcatlons, 241-256

543 of catecholammes,

202-204, 230-238, 241-245, 248-256 electrodes, 216-221 calibration, 227-229 carbon epoxy, 206, 218, 219, 221, 222, 225, 236, 253, 254 carbon fiber, 206, 209, 214, 219-221, 222, 227, 231, 234, 239, 240, 241, 248, 252 carbon paste, 202, 203, 206, 210, 218, 219, 221, 224, 225, 229, 230, 235, 237, 238, 245, 249, 250, 254, 255 modtfrcatron, 221-225 response, 225-227 electronics, 214-216 mterpretatron, 229-240 dopamme, DOPAC, and ascorbic acid, 230-238 mdoles, 238, 239 neurotransmitter release, 239, 240 techniques, 204-213 chronoamperometry, 205-208, 214, 220, 225, 227, 230, 231, 235-237, 250-253, 255 cychc, 203, 211-213, 229 differential double pulse, 211 differential pulse, 209-211, 214, 234, 239, 240, 248, 249, 252 linear sweep, 214, 235, 249 with semldifferentlatlon, 213, 214, 229, 248 normal pulse, 208, 209, 211, 234, 241 theory of, 198-202

WB4101,

Zlmelldme,

389

473, 474