NEUROMETHODS 0 11

Carbohydrates and Energy Metabolism

NEUROMETHODS Procrram Editors: Alan A. Boulton and Glen B. Baker

1 General Neurochemical Techniques Edlted by Alan A. Boulton and Glen B. Baker, 1985

2 Amines and Their Metabolites Edited by AIan A. Boufton, Glen B. Baker, and Judith M. Baker, 1985

3 Amino Acids Edited by Alan A, Boulton, Glen B. Baker, and James D. Woo4 1985

4 Receptor Binding Edlted by Alan A. Boulton, Glen B. Baker, and Pave1 D. Hrdlna, 1986

5 Neurotransmltter Enzymes Edited by Alan A. Boulton, Glen B. Baker, and Peter H. Vu, 1986

6. Peptides Edlted by Alan A. Boulton, Glen B. Baker, and Quentln Plttman, 1987

7 Lipids and Related Compounds Edlted by Alan A. Boulton, Glen B. Baker, and Llogd A. Horrocks, 1988

8 Imaging and Correlathre Physicochemlcal Techniques

Edited by Alan A. Boulton, Glen B. Baker, and Donald P. Boisvert, 1988 9. The Neuronal Microenvironment

Edited by Alan A. Boulton, Glen B. Baker, and Wolfgang Walz, 1988 10. Analysis of Psychiatric Drugs

Edlted by Alan A Boulton, Glen B. Baker, and Ronald T. Coutts, 1988 11 Carbohydrates and Energy Metabolism

Edlted by Alan A. Boulton, Glen B. Baker, and Roger F. Butterworth, 1989 12 Drugs as Tools in Neurotransmitter Research

Edited by Alan A. Boulton, Glen B. Baker, and August0 V. Juorio, 1989 13 Psychopharmacology

Edlted by Alan A. Boulton, Glen B. Baker, and Andrew J. Greenshaw, 1989 14. Neurophysiology

Edked by Alan A. Boulton, Glen B. Baker, and Case H. Vanderwolf 15 Neuropsychology

Edited by Alan A. Boulton, Glen B. Baker, and Merrll H&cock 16 Molecular Neurobiological Techniques

Edited by Alan A. Boutton, Glen 8. Baker, and Anthony T. Campagnonl

NEUROMETHODS Program Editors: Alan A. Boulton and Glen B. Baker

NEUROMETHODS I 11

Carbohydrates and Energy Metabolism

Edited by

Alan A. Boulton University of Saskatchewan, Saskatoon, Canada

Glen B. Baker University of Alberta, Edmonton, Canada

and

Roger F. Butterworth University of Montreal, Montreal, Canada

Humana Press l Clifton, New Jersey

Library of Congress Cataloging in Publication Data

Mam entry under title

Carbohydrates and energy metabolism

(Neuromethods , 11) Includes blbhographles and index 1 Bram-Metabohsm-Research-Methodology

2 Carbohydrates-Metabohsm-Research-Methodology 3 Energy metabolism-Research-Methodology I Boulton, A A (Alan A ) II Baker, Glen B ,

Date III Butterworth, Roger F IV Series. [DNLM 1 Carbohydrates 2 Energy Metabohsm

Wl NE337G v 11 / QU 75 C2642] QP376 C35 1988 599' 0188 88-26609

ISBN o-89603-143-8

Q 1989 The Humana Press lnc

Crescent Manor PO Box 2148 Cl&on, NJ 07015

All nghts reserved

No part of this book may be reproduced, stored m a retneval system, or transmltted m any form or by any means, electromc, mechamcal, photocopymg, microhlmmg, recordmg, or otherwise without wntten permlsslon from the Pubhsher

Printed m the United States of America

Preface to the Series

When the President of Humana Press first suggested that a series on methods in the neurosciences might be useful, one of us (A. A. B.) was quite skeptical; only after discussions with G. B. B. and some searching both of memory and library shelves did it seem that perhaps the publisher was right. Although some excellent methods books have recently appeared, notably in neuroanatomy, it is a fact that there is a dearth in this particular field, a fact attested to by the alacrity and enthusiasm with which most of the con- tributors to this series accepted our invitations and suggested additional topics and areas. After a somewhat hesitant start, es- sentially in the neurochemistry section, the series has grown and will encompass neurochemistry, neuropsychiatry, neurology, neuropathology, neurogenetics, neuroethology, molecular neurobiology, animal models of nervous disease, and no doubt many more “neuros.” Although we have tried to include adequate methodological detail and in many cases detailed protocols, we have also tried to include wherever possible a short introductory review of the methods and/or related substances, comparisons with other methods, and the relationship of the substances being analyzed to neurological and psychiatric disorders. Recognizing our own limitations, we have invited guest editors to join with us on most volumes in order to ensure complete coverage of the field and to add their specialized knowledge and competencies. We anticipate that this series will fill a gap; we can only hope that it will be filled appropriately and with the right amount of expertise with respect to each method, substance or group of substances, and area treated.

Alan A. Boulton Glen B. Baker

V

Preface

The mammalian central nervous system depends almost ex- clusively on glucose as its major energy source. In addition, glu- cose participates in other cerebral metabolic functions including the biosynthesis of neurotransmitters, such as acetylcholine and the amino acids. This volume of Neuromethods assembles currently available methods for the study of cerebral glucose and energy metabolism in vitro and in mm.

In the first chapter, Lust et al. describe the various methods available for the appropriate fixation of brain tissue necessary for the study of cerebral energy metabolism. Different fixation methods are compared, and some concerns raised by the USDHHS in their guidelines for the care and use of laboratory animals are addressed. Specific fixation methods pertinent to the various measurements are also covered in other chapters. In vitro pre- parations have, despite certain limitations, been found to be useful in the study of brain metabolism, since the biochemical environ- ment is amenable to rapid, controlled manipulation. The chapter by Lai and Clark describes methods for the isolation and characterization of metabolically active preparations of synaptic and non-synaptic mitochondria from brain, and studies of en- zymes involved in glucose metabolism and glucose-derived neurotransmitter synthesis in these preparations are summarized. The chapter by Whittingham discusses methods of preparations of hippocampal slices for use in the study of energy metabolism. Measurement of glucose and of glycolytic and dicarboxylic acid cycle intermediates in neural tissues are described in the chapter by Bachelard.

Increasing attention has been focused in recent years on the use of quantitative autoradiography for the study of local cerebral glucose utilization (LCGU). Such methods allow the comparison of the relative rates of glucose utilization in various structures of the brain. Two chapters are devoted to the subject of LCGU using

vii

. . . Vlll Preface

either “C-deoxyglucose (Sokoloff et al.) or 14C-glucose (Hawkins and Mans). In addition to providing information on rates of glu- cose utilization in different brain structures, the autoradiographs afford pictorial representations of relative cerebral function in these structures. Details of experimental procedures, as well as methods of calculation of rates of LCGU and recent advances in computerized image processing are provided in these chapters.

Three chapters deal with the measurement of enzymes in- volved in cerebral glucose metabolism. The chapter by Clark and Lai focuses on spectrophotometric methods for the study of glycolytic, tricarboxylic acid cycle, and ketone body-metabolizing enzymes. Some phylogenetic and ontogenic aspects are discussed. In view of the difficulties frequently encountered in their assay, a single chapter is devoted to the measurement of the five enzymic components of the pyruvate dehydrogenase complex (Butter- worth); both radiometric and spectrophotometric methods are in- cluded. The chapter by Pate1 describes methods available for the study of CO*-fixing enzymes in brain and discusses their signifi- cance in brain metabolism.

No volume on methods for the study of brain energy metabo- lism would be complete without a chapter on the measurement of ATP and other high-energy phosphates. In the chapter by Dwor- sky et al., methods for the fluorimetric measurement of high- energy phosphates in microdissected, microgram, and submicro- gram samples of brain tissue are described.

Roger F. Buttenoorth

Contents

Preface to the Series ............................................................. v Preface ................................................................................ vii List of Contributors ............................................................ xix

METHODS OF FIXATION OF NERVOUS TISSUE FOR USE IN THE STUDY OF CEREBRAL ENERGY METABOLISM W. David Lust, Anthony J. Ricci, Warren R. Selman, and Robert A.

Ratcheson

1. Introduction .................................................... 1 1.1. Rationale for Rapid Brain Fixation .................. 2 1.2. Criteria for Use of Rapid Fixation ................... 3 1.3. Methods of Rapid Fixation in the Brain ........... 3 1.4. Evolution of Brain Fixation Methods ............... 4

2. Fixation Techniques by Rapid Freezing ................ 5 2.1. Coolants ..................................................... 2.2. Rates of Freezing ......................................... 2 2.3. Comparison of Brain Metabolite

Concentrations ........................................... 11 2.4. Animal Welfare Concerns on Euthanasia ........ 16 2.5. Effects of Neurotropic Agents on the

Fixation Process .......................................... 16 2.6. Protocols for Freezing Methods.. ................... 17

3. Microwave Irradiation ...................................... 26 3.1. Animal Welfare Concerns and Microwave

Irradiation ................................................. 26 3.2. Microwave Radiation ................................... 26 3.3. Microwave Apparatus ................................. 28 3.4. Microwave Irradiation Protocol ..................... 29

ix

X Contents

3.5. Discussion of Microwave Irradiation for Studies on Cerebral Metabolism .................... 29

4. Tissue Extraction ............................................. 30 4.1. Preparation of Tissue ................................... 33 4.2. Tissue Extraction ........................................ 33

5. Summary ....................................................... 35 References ....................................................... 36

ISOLATION AND CHARACTERlZATlON OF SYNAPTIC AND NONSYNAPTIC MlTOCHONDRlA FROM MAMMALIAN BRAlN James C. K. Lai and John B. Clark

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 1.1. Historical Aspects of Brain Mitochondrial

Isolation and Metabolism .,.....*..................... 43 1.2. Methodological Problems Associated

with Early Studies of Brain Mitochondrial Metabolism . . . . ..I............................... . . . . . . . . *. 44

1.3. Heterogeneity of Brain Mitochondria and Metabolic Compartmentation.. . . . . . . . . . . . . . . . , . . . . . . 44

1.4. Anatomical and Functional Classification of Heterogeneous Populations of Brain Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . t . . . . . 46

1.5. The Scope of This Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 2. Methods for the Isolation of Relatively Pure

and Metabolically Active Fractions of Synaptic and Nonsynaptic Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . 47

2.1. Isolation of Nonsynaptic Mitochondria from Whole Brain . . . ..a................................. 47

2.2. Isolation of Synaptic Mitochondria from Whole Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

2.3. Isolation of Synaptic and Nonsynaptic Mitochondria from Discrete Brain Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*.*............... 62

2.4. Isolation of Mitochondria from the Developing and Aging Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

3. Characterization of Preparations of Synaptic and Nonsynaptic Brain Mitochondria . . . . . . . . . . . . . . . . . 62

3.1. The Purity of Various Preparations . . . . . . . . . . . . . . . . 62

Contents xi

3.2. The Structural and Metabolic Integrity of Different Mitochondrial Preparations ............. 63

3.3 Respiratory Properties of Isolated Mitochondria ............................................. 65

3.4. The Cytochromes in Synaptic and Nonsynaptic Mitochondria ........................... 67

3.5. Enzymatic Properties of Different Mitochondrial Populations from Whole Brain and from Discrete Regions ................... 68

3.6. The Relations Between the Heterogeneity of Brain Mitochondria and Metabolic Compartmentation ...................................... 75

4. Integrated Metabolic Studies with Various Intact Mitochondrial Populations ....................... 78

4.1. Metabolism of Pyruvate and Ketone Bodies .... 78 4.2. Metabolism of Glutamate and GABA ............. 81 4.3. Mitochondrial-Cytosolic Transport and

Shuttle Systems .......................................... 83 5. Pathophysiological and Toxicological Studies

with Brain Mitochondria ................................... 84 5.1. Pathophysiological Studies ........................... 85 5.2. Toxicological Studies ................................... 87

6. Concluding Remarks and Prospects for Future Studies .......................................................... 88

6.1. Concluding Remarks ................................... 88 6.2. Prospects for Future Studies ......................... 88

7. Summary ....................................................... References .......................................................

THE USE OF HIPPOCAMPAL SLICES FOR THE STUDY OF ENERGY METABOLISM Tim S. Whittingham

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Basic Considerations ..,,.,.................**.,.*.**.,. Liz

2. Theoretical Considerations . . . ..*........................ 101 2.1. Coupled Enzymatic Fluorescent Measure-

ments of Metabolite Levels.. . . . . . . . . . . . . . . . . . . . . . . . 102

xii Contents

2.2. Luminescence Measurements of Adenylate Phosphates and Phosphocreatine ................ 103

2.3. Comparison of Metabolite Values.. .............. 105 3. Methodology ................................................ 107

3.1. Brain Slice Preparation and Incubation.. ....... 107 3.2. Preparation of Samples for Analysis ............ 110 3.3. Analysis of ATP, Phosphocreatine, and

Creatine from Brain Slices .......................... 116 4. Summary ..................................................... 127

References ..................................................... 128

MEASUREMENTOFCARBOHYDRATESANDTHEIR DERIVATIVESIN NEURALTISSUES Herman S. Bachelard

1. Introduction ................................................. 133 2. Preparation of Tissue Extracts ......................... 134

2.1. Rapid-Freezing Techniques ......................... 134 2.2. Freeze-Blowing ......................................... 137 2.3. Microwave Fixation ................................... 137 2.4. Metabolic Inhibitors .................................. 138 2.5. Tissue Extraction ...................................... 138

3. Estimation of Glucose .................................... 138 3.1. Radioactive Glucose .................................. 139 3.2. Nuclear Magnetic Resonance ...................... 140

4. Hexose Phosphates ........................................ 141 4.1. Separation Methods .................................. 141 4.2. Enzymic Methods ..................................... 142 4.3. Deoxyglucose and Its 6-Phosphate ............... 142

5. Intermediates of the Pentose Phosphate Pathway ...................................................... 142

6. Intermediates of the Tricarboxylic Acid Cycle .... 146 7. Glycogen ..................................................... 146

7.1. Extraction ................................................ 146

7.2. Estimation ............................................... 146 7.3. Specific Activity of r4C-labeled Glycogen ...... 148

Contents I.,

xi11

8. Complex Carbohydrates-Glycosaminoglycans . . 148 8.1. Extraction of Glycosaminoglycans ................ 149 8.2. Enzymic Analysis ..................................... 150 References ..................................................... 151

THE['4C]DEOXYGLUCOSEMETHODFORMEASUREMENT OF LOCAL CEREBRAL GLUCOSE UTILIZATION Louis Sokoloff, Charles Kennedy, and Carolyn B. Smith

1. Introduction ................................................. 155 2. Theoretical Basis of Radioactive Deoxyglucose

Method ........................................................ 156 3. Procedure .................................................... 160

3.1. Preparation of Animals .............................. 161 3.2. Administration of [14C]Deoxyglucose and

the Sampling of Arterial Blood .................... 146 3.3. Analysis of Arterial Plasma for [14C]Deoxy-

glucose and Glucose Concentrations.. .......... 167 3.4. Processing of Brain Tissue .......................... 168 3.5. Preparation of Autoradiographs .................. 170 3.6. Densitometric Analysis of Autoradiographs . . 172 3.7. Calculation of Rate of Glucose Utilization ..... 173 3.8. Normal Rates of Local Cerebral Glucose

Utilization in Conscious Animals ................. 174 4. Theoretical and Practical Considerations ........... 175

4.1. Rate Constants ......................................... 177 4.2. Lumped Constant ..................................... 179 4.3. Role of Glucose-6-phosphatase .................... 180 4.4. Influence of Varying Plasma Glucose

Concentration .......................................... 182 4.5. Animal Behavior During the Experimental

Period ..................................................... 183 5. The Use of [14C]Deoxyglucose in Metabolic

Mapping and Other Nonquantitative Studies ..... 183 6. Recent Technological Advances ....................... 185

6.1. Computerized Image-Processing ................. 185

xiv Con tents

6.2. Microscopic Resolution .............................. 186 6.3. [18F]Fluorodeoxyglucose Technique .............. 187

7. Summary ..................................................... 188 References ..................................................... 189

DETERMINATIONOFCEREBRALGLUCOSEUSE IN RATS USING ['4C]GLUCOSE Richard A. Hawkins and Anke M. Mans

1. Introduction ................................................. 195 2. Biochemical Theory ....................................... 196 3. Mathematical Theory ..................................... 200

3.1. Model and Assumptions.. .......................... 201 3.2. Behavior of Circulating [6-14C]Glucose ......... 202 3.3. Behavior of Tissue [6- 4C]Glucose ................ 203

4. Preparation of the Rats ................................... 204 4.1. Acute Preparation ..................................... 204 4.2. Anesthesia ............................................... 205 4.3. Chronic Preparation .................................. 205

5. Experimental Procedure ................................. 205 5.1. Preparation of Label .................................. 205 5.2. Blood Samples., ........................................ 206 5.3. Brain Removal and Treatment for

Quantitative Autoradiography .................... 5.4. Brain Sections ..........................................

zii

5.5. Tissue Thickness and Attenuation ............... 208 5.6. Films and Exposure Time.. ......................... 209 5.7. Standards ................................................ 210 5.8. Brain Sampling by Dissection ..................... 211 5.9. Treatment of Blood and Plasma .................. 211

6. Collection of Data and Calculation of Rates ....... 212 6.1. Fitting the Plasma Specific Radioactivity

Curve ..................................................... 215 7. Experimental Considerations ........................... 217

7.1. Experimental Time .................................... 217 7.2. Accuracy of the Kinetic Constants ............... 223 7.3. Preliminary Glucose Use Measurement ........ 227

8. Caveat ......................................................... 228 8.1. Injury ..................................................... 228

Contents ml

8.2. Anaerobic Glycolysis ................................. 228 8.3. Breakdown of the Blood-Brain Barrier., ........ 228 8.4. Compartmentation .................................... 229 8.5. Steady-state ............................................. 229 8.6. Autoradiographs ....................................... 229 References ..................................................... 230

GLYCOLWIC, TRICARBOXYLIC ACID CYCLE AND RELATED ENZYMES IN BlWN John B. Clark and James C. K. Lai

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 1.1. Literature Survey and the Scope of this

Review . . . . . . . . . . . . . . . . . . . . . . . ..*.a........................ 233 1.2. The Importance of Glucose Utilization in

Brain Energy Metabolism and Production . . . . . . . . . . . . . . . . . . . . ..*......*............**... 233

1.3. Regulatory Aspects of Brain Glycolysis and Citric Acid Cycle . ..*..................*......... 234

1.4. Regulatory Aspects of the Citric Acid Cycle in Brain . . . . . ..~~~...~........~~.~~.~........~.~.~.....~..~ 236

1.5. The Concept of Metabolic Compartment- ation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

1.6. Relations between the Compartmentation of Brain Citric Acid Cycle Metabolism and Neurotransmitter Metabolism . . . . . . . . . . . . . . . . . . . . . 239

2. Assays of the Enzymes of the Glycolytic, Citric Acid Cycle and Related Pathways.. . . . . . . . . . . 240

2.1. Glycolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 2.2, Citric Acid Cycle Enzymes . . . . . . , . . . . . . . . . . . . . . . . . . 242 2.3. Enzymes Related to the Citric Acid Cycle . . . . . 245

3. Brain Regional Variations of Glycolytic, Citric Acid Cycle and Related Enzymes.. . . . . . . . . . . . . . . . . . . . 250

3.1. Phylogenetic Aspects and Sex Differences . . . . 250 3.2. Changes during Development . . . . . . . . . . . . . . . . . . . . 254 3.3. Changes during Aging . . . . . . . ..*..........*......... 258

4. Cellular and Subcellular Compartment- ation of Glycolytic, Citric Acid Cycle and Related Enzymes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

xvi Contents

4.1. Neuronal and Glial Localization . . . . . . . . . . . . . . . . . . 260 4.2. Subcellular Localization . . . . . . . . . . ...*.........*..*.. 265

5. Pathophysiological States and Changes in Glycolytic, Citric Acid Cycle and Related Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

5.1. Deficiency States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 5.2. Toxicological States q....................,............. 269

6. Conclusions and Prospects for Future Studies.. . . 270 6.1. Concluding Remarks . . . . . . . . . . . . . . . ..*............... 270 6.2. Prospects for Future Studies.. . . . . . . . . . . . . . . . . . . . . . 271

7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

ENZYMESOFTHEPYRUVATEDEHYDROGENASECOMPLEX OFMAMMALIANBRAIN Roger F. Butteworth

1. Introduction ................................................. 283 2. Regulation of PDHC in Mammalian Brain ......... 284 3. Methods of PDHC Assay in Brain.. .................. 285

3.1. Measurement of PDHC (Total Complex). ...... 285 3.2. Measurement of PDHC (Constituent

Enzymes) ................................................ 292 4. Regional and Subcellular Localization of

PDHC in Brain .............................................. 296 4.1. Regional Distribution of PDHC Enzymes.. .... 296 4.2. Subcellular Localization of PDHC in Brain .... 298

5. PDHC and Brain Development ........................ 298 6. PDHC Deficiency Disorders ............................ 299

6.1. Inherited Disorders of PDHC.. .................... 299 6.2. Acquired Disorders of PDHC.. .................... 301 6.3. Pathophysiology of PDHC Deficiency

Disorders ................................................. 301 7. Summary ..................................................... 303

References ..................................................... 304

C02-FWNGENZYMES Mulchand S. Pate1

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

Contents xvii

2. Biotin-Dependent CO2 Fixation ....................... 311 2.1. Pyruvate Carboxylase ................................ 311 2.2. Acetyl-CoA Carboxylase ............................ 318 2.3. Propionyl-CoA Carboxylase ........................ 321 2.4. 3-Methylcrotonyl-CoA Carboxylase .............. 322 2.5. Mechanism of Action of Biotin-

Dependent Carboxylation ........................... 323 3. Biotin-Independent CO2 Fixation ..................... 325

3.1. Phosphoenolpyruvate Carboxykinase ........... 325 3.2. NADP-Malate Dehydrogenase (Malic

Enzyme) .................................................. 327 3.3. NADP-Isocitrate Dehydrogenase ................. 329

4. Significance of COz-Fixing Enzymes in Brain ..... 330 References ..................................................... 333

HIGH-ENERGYPHOSPHATES IN BRAIN FROM DISSECTED FREEZE-DRIED REGIONS TO SINGLE CELLS Suzanne Dworsky, Marian Namovic, and David W. McCandless

1. Introduction ................................................. 343 2. Adequate Tissue Fixation ................................ 344

2.1. Rapid Submersion in Liquid N2 .................. 344 2.2. Funnel Freezing ........................................ 345 2.3. Freeze Blowing ......................................... 345 2.4. Microwave Irradiation ............................... 345

3. Preparation, Dissection, and Weighing of Samples for Analysis ..................................... 346

3.1. Removal of Brain from Cranial Vault ........... 346 3.2. Sectioning the Brain .................................. 347 3.3. Dissection of the Samples .......................... 348 3.4. Weighing Microsamples ............................. 352

4. Fluorometric Enzymatic Analysis of Samples for High-Energy Phosphates ........................... 355

4.1. ATP and PCr ........................................... 355 4.2. Fluorometer Direct Assay, 0.1-S X 10m9

mol. ADP ................................................ 365 4.3. Cycling l-10 x lo-l2 mol. Tissues, AMP ...... 365 References ..................................................... 369

Index . . . . . . . . . ..~.........................................................~.......... 371

Contributors

HERMAN S. BACHELARD l Division of Biochemistry, St. Thomas’s Hospital, London, U. K. GLEN B. BAKER l Neurochemzcal Research Unit, Department of Psy- chiatry, Unfversity of Alberta, Edmonton, Alberta, Canada ALAN A. BOULTON l Neuropsychlatric Research Unit, University of Saskatchewan, Saskatoon, Saskatchewan, Canada ROGER F. BUTTERWORTH l Andr&VzaZlet Clmzcal Research Center, Hdpital Sam t-Luc, Universe ty of Montreal, Montreal, Quebec, Canada JOHN B. CLARK l Medrcal College of St. Bartholomew’s Hospztal, University of London, London, U. K. SUZANNE DWORSKY l Unzversity of Health Sczences, The Chxago Medical School, North Chicago, lllinols RICHARD A. HAWKINS l College of Medrcine, The Pennsylvania State Unzversity, Hershey, Pennsylvanza CHARLES KENNEDY l Department of Pediatrics, Georgetown Umver- sity School of Medicine, Washmgton, D. C. JAMES C. K. LAI l Laboratory of Cerebral Metabolism, Cornell Uni- versrty Medical College, New York, New York W. DAVID LUST l Case Western Reserve Universzty School of Medl- cme, Cleveland, Ohio ANKE M. MANS l College of Medicine, The Pennsylvania State Um- verszty, Hershey, Pennsylvanza DAVID W. MCCANDLESS l Umversity of Health Sciences, The Chxa- go Medical School, North Chrcago, lllmols MARIAN NAMOVIC l Unzversity of Health Sciences, The Chzcago Medical School, North Chzcago, lllmois MULCHAND S. PATEL l Case Western Reserve University School of Medicine, Cleveland, Ohlo ROBERT A. RATCHESON l Case Western Reserve University School of Medicine, Cleveland, Ohio

XiX

.x.x Contributors

ANTHONY J.RICCI l Case Western Reserve Unzversity School ofMedz- tine, Cleveland, Ohio WARREN R. SELMAN l Case Western Reserve Universtty School of Medzcine, Cleveland, Ohlo CAROLYN B. SMITH l National Institute of Mental Health, U. S. Department of Health and Human Services, Bethesda, MD LOUIS SOKOLOFF l National ZnstituteofMental Health, U. S. Depart- ment of Health and Human Services, Bethesda, MD TIM S. WHITTINGHAM l Case Western Reserve University School of Medicine, Cleveland, Ohio