Mitochondrial Dysfunction (eBook)

Front Cover 1
Mitochondrial Dysfunction 4
Copyright Page 5
Dedication 6
Table of Contents 8
Contributors 14
Preface 20
Introduction: Criteria for Assessing Normal and Abnormal Mitochondrial Function 22
Mitochondrial Heterogeneity 22
Mitochondrial Plasticity 24
Causes of Mitochondrial Dysfunction 24
Programmed Cell Death 27
REFERENCES 28
Chapter 
29 
INTRODUCTION 29
ISOLATION OF MITOCHONDRIA FROM LIVER AND KIDNEY HOMOGENATES 29
SUBFRACTIONATION OF ISOLATED MITOCHONDRIA FROM LIVER AND KIDNEY 37
ISOLATION OF MITOCHONDRIA FROM INTACT HEPATOCYTES AND KIDNEY CELLS: DIGITONIN FRACTIONATION 42
ISOLATION OF MITOCHONDRIA FROM INTACT HEPATOCYTES: 
 
44 
COMMENTS 47
REFERENCES 48
Chapter 
50 
INTRODUCTION 50
MATERIALS AND REAGENTS 51
PROCEDURES 52
COMMENTS 55
ACKNOWLEDGMENTS 61
REFERENCES 61
Chapter 
62 
INTRODUCTION 62
ISOLATION OF INTACT RAT FOREBRAIN MITOCHONDRIA 62
PROPERTIES OF RAT BRAIN MITOCHONDRIAL PREPARATION 64
COMMENTS 69
REFERENCES 70
Chapter 4. Study of Skeletal Muscle Mitochondrial Dysfunction 72
INTRODUCTION 72
PREPARATION OF MITOCHONDRIAL FRACTIONS 73
MEASUREMENT OF MITOCHONDRIAL OXIDATIONS 74
MEASUREMENT OF ACTIVITY OF RESPIRATORY CHAIN COMPLEXES I-IV 76
MEASUREMENT OF MITOCHONDRIAL ß-OXIDATION FLUX 80
ENZYMES OF MITOCHONDRIAL FATTY ACID OXIDATION 82
MEASUREMENT OF MITOCHONDRIAL FATTY ACID OXIDATION ENZYME ACTIVITIES IN FIBROBLAST HOMOGENATES 88
ACKNOWLEDGMENTS 89
REFERENCES 90
Chapter 
91 
INTRODUCTION 91
PREPARATION OF INTACT MITOCHONDRIA AND MITOCHONDRIAL FRAGMENTS 92
PROPERTIES 95
COMMENTS 101
REFERENCES 103
Chapter 
105 
INTRODUCTION 105
CONCEPTS OF METABOLIC CONTROL ANALYSIS 108
EXPERIMENTAL METHODS EMPLOYED IN CONTROL ANALYSIS 112
CONTROL ANALYSIS AS A TOOL IN DIAGNOSIS 113
CONTROL ANALYSIS OF POTENTIAL THERAPIES 114
CONTROL BY MITOCHONDRIAL FUNCTION 116
ACKNOWLEDGMENTS 117
REFERENCES 117
Chapter 
119 
ASSESSMENT OF MITOCHONDRIAL FUNCTION in Vivo 119
THERAPEUTIC EVALUATIONS 121
SUMMARY 122
ACKNOWLEDGMENTS 122
REFERENCES 122
Chapter 8. Noninvasive Assessment of Mitochondrial Function by Breath Analysis Using Ketoisocaproic Acid 123
INTRODUCTION 123
MATERIAL AND REAGENTS 123
PROCEDURES 124
COMMENTS 125
ACKNOWLEDGMENTS 126
REFERENCES 126
Chapter 
128 
INTRODUCTION 128
OPTICAL BEHAVIOR OF THE MITOCHONDRIAL ELECTRON TRANSPORT SYSTEM 128
METHODS FOR OPTICAL SPECTROSCOPY OF INTACT TISSUES 130
APPLICATIONS OF OPTICAL SPECTROSCOPY in Vivo 138
SUMMARY 144
REFERENCES 144
Chapter 
148 
INTRODUCTION 148
MITOCHONDRIAL FUNCTION AND CARBON TRACERS 149
DETECTION OF 13C-ENRICHED COMPOUNDS BY NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 151
SOURCES OF ACETYL-COENZYME A 157
CITRIC ACID CYCLE KINETICS 161
ANAPLEROSIS: ANALYSIS UNDER STEADY-STATE CONDITIONS 162
SUBSTRATE SELECTION IN HEART TISSUE 163
LIMITATIONS OF ISOTOPOMER ANALYSIS BY 13C NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 
164 
ACKNOWLEDGMENTS 164
REFERENCES 164
Chapter 11. Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy in the Study of Mitochondrial Metabolism 167
INTRODUCTION 167
REGULATION OF OXYGEN CONSUMPTION IN SKELETAL MUSCLE 167
REFERENCES 179
Chapter 
183 
INTRODUCTION 183
MITOCHONDRIAL DYSFUNCTION IN ISCHEMIC LIVER 183
DYSFUNCTION OF ISOLATED MITOCHONDRIA DURING ANOXIC INCUBATION 186
LEAKAGE OF MITOCHONDRIAL ENZYMES DURING REOXYGENATION 189
REFERENCES 190
Chapter 
192 
INTRODUCTION 192
METHODS 193
COMMENTS 195
ACKNOWLEDGMENTS 198
REFERENCES 198
Chapter 
200 
INTRODUCTION 200
MITOCHONDRIAL VOLUME DENSITY AND SURFACE DENSITY 200
MITOCHONDRIAL INNER MEMBRANE SURFACE DENSITY 203
MITOCHONDRIAL NUMBER, SIZE, AND SHAPE 205
THREE-DIMENSIONAL RECONSTRUCTION 206
MITOCHONDRIAL DISTRIBUTION 208
CONCLUSIONS 210
ACKNOWLEDGMENT 211
REFERENCES 211
Chapter 15. Megamitochondria 
212 
INTRODUCTION 212
EXPERIMENTAL INDUCTION OF MEGAMITOCHONDRIA 
215 
ANALYSIS OF FUNCTION OF MEGAMITOCHONDRIA 218
ANALYSIS OF MECHANISMS OF MEGAMITOCHONDRIA FORMATION 222
CONCLUSION 226
ACKNOWLEDGMENTS 226
REFERENCES 226
Chapter 16. Redox Status and Mitochondrial Inner Membrane Permeability 
228 
INTRODUCTION 228
MATERIALS 229
PROCEDURES 229
COMMENTS 231
REFERENCES 238
Chapter 
240 
INTRODUCTION 240
MEASUREMENT OF MITOCHONDRIAL GLUTATHIONE IN HEPATOCYTES AND OTHER CELLS 241
SAMPLE PREPARATION 242
MITOCHONDRIAL GLUTATHIONE 244
ACKNOWLEDGMENTS 247
REFERENCES 247
Chapter 
248 
INTRODUCTION 248
MATERIALS 249
PROCEDURES 249
COMMENTS 253
REFERENCES 254
Chapter 19. Determination of Flux, Activity, and Activity State of Mitochondrial a-Keto Acid Dehydrogenase Complexes 
256 
INTRODUCTION 256
MATERIALS AND REAGENTS 257
PROCEDURES 258
REFERENCES 265
Chapter 
267 
INTRODUCTION 267
METHODS 267
RESULTS 271
COMMENTS 274
ACKNOWLEDGMENTS 275
REFERENCES 275
Chapter 
277 
INTRODUCTION 277
EXPERIMENTAL PROCEDURES 278
CHEMICALS 279
RESULTS 279
CONCLUSIONS 285
REFERENCES 285
Chapter 
287 
INTRODUCTION 287
METHODS 289
COMMENTS 294
REFERENCES 297
Chapter 
298 
INTRODUCTION 298
COMPARISON OF UBIQUINOL AND TOCOPHEROL ANTIOXIDANT ACTIVITIES IN MEMBRANES AND IN LIPOSOMES 299
REDUCTION OF VITAMIN . RADICALS BY UBIQUINOLS IN LIPOSOMES AND IN MEMBRANES 301
REFERENCES 305
Chapter 
307 
INTRODUCTION 307
ROLE OF NADPH OF MITOCHONDRIAL ORIGIN IN DRUG METABOLISM AND TOXICITY 307
DIRECT MEASUREMENT OF NADPH AND NADH WITH ENZYMATIC CYCLING 313
ROLE OF MITOCHONDRIA IN ZONE-SPECIFIC TOXICITY TO THE LIVER LOBULE 317
REFERENCES 319
Chapter 
322 
INTRODUCTION 322
MATERIALS AND REAGENTS 323
PROCEDURES 324
COMMENTS 327
ACKNOWLEDGMENT 330
REFERENCES 330
Chapter 26. Identification of Mitochondrial Dysfunction at Coupling Site I: Loss of Activity of NADH-Ubiquinone Oxidoreductase during Myocardial Ischemia 
331 
INTRODUCTION 331
MATERIALS AND REAGENTS 333
PROCEDURES 333
COMMENTS 336
ACKNOWLEDGMENTS 343
REFERENCES 343
Chapter 27. Identification of Mitochondrial Dysfunction at Coupling Site II 
345 
INTRODUCTION 345
SPECTROPHOTOMETWC ASSESSMENT OF COMPLEX III ACTIVITY 347
POLAROGRAPHY 353
MEASUREMENT OF MITOCHONDRIAL CYTOCHROME CONCENTRATIONS BY LOW TEMPERATURE DIFFERENCE SPECTROSCOPY 353
WESTERN BLOTTING 354
APPLICATION OF TECHNIQUES TO DIAGNOSTIC INVESTIGATIONS 356
ACKNOWLEDGMENTS 356
REFERENCES 357
Chapter 
358 
INTRODUCTION 358
MATERIALS AND REAGENTS 360
PROCEDURES 361
COMMENT 374
ACKNOWLEDGMENTS 375
REFERENCES 375
Chapter 
377 
INTRODUCTION 377
MATERIALS AND REAGENTS 379
PROCEDURES 379
REFERENCES 387
Chapter 
388 
INTRODUCTION 388
MATERIALS AND REAGENTS 390
PROCEDURES 391
COMMENTS 393
ACKNOWLEDGMENTS 399
REFERENCES 399
Chapter 
401 
INTRODUCTION 401
TECHNIQUES FOR in Situ IMAGING OF MITOCHONDRIAL MEMBRANE POTENTIAL 402
PROBING MITOCHONDRIAL CALCIUM CYCLING WITH CYCLOSPORIN A 406
USE OF SAPONIN-PERMEABILIZED CELLS TO STUDY MITOCHONDRIAL CALCIUM HOMEOSTASIS 408
REFERENCES 411
Chapter 
413 
INTRODUCTION 413
PREPARATION OF INTACT AND PERMEABILIZED MITOCHONDRIA AND MITOCHONDRIAL EXTRACTS 414
CALCIUM BUFFERS AND THEIR USE 416
ASSAY OF PYRUVATE DEHYDROGENASE ACTIVITY WITHIN INTACT AND PERMEABILIZED MITOCHONDRIA 418
ASSAY OF 2-0X0GLUTARATE DEHYDROGENASE AND NAD+-ISOCITRATE DEHYDROGENASE ACTIVITIES IN MITOCHONDRIAL EXTRACTS, PURIFIED PREPARATIONS AND PERMEABILIZED MITOCHONDRIA 421
ASSAY OF 2-OXOGLUTARATE DEHYDROGENASE AND NAD+-ISOCITRATEDE HYDROGENASE ACTIVITIES WITHIN INTACT MITOCHONDRIA 423
FINAL COMMENTS 425
ACKNOWLEDGMENTS 425
REFERENCES 426
Chapter 
427 
INTRODUCTION 427
MEMBRANE POTENTIAL IN ISOLATED MITOCHONDRIA 429
MITOCHONDRIAL MEMBRANE POTENTIAL IN CELL SUSPENSIONS 430
FLUORESCENCE MICROSCOPY OF SINGLE CELLS 432
QUANTITATIVE IMAGING OF MEMBRANE POTENTIAL BY CONFOCAL MICROSCOPY 434
CONCLUSION 437
ACKNOWLEDGMENTS 438
REFERENCES 438
Chapter 
439 
INTRODUCTION 439
OXIDATIVE PHOSPHORYLATION 439
UNIQUE CHARACTERISTICS OF MITOCHONDRIAL GENETICS 440
MITOCHONDRIAL DNA MUTATIONS AND DISEASE 441
GENETIC APPROACHES TO MITOCHONDRIAL DNA DISEASE 443
CONCLUSION 448
REFERENCES 449
Chapter 
451 
INTRODUCTION 451
MATERIALS AND PROCEDURES 452
SUMMARY COMMENTS 458
ACKNOWLEDGMENTS 459
REFERENCES 459
Chapter 
461 
INTRODUCTION 461
METHODS BASED ON A CELLULAR APPROACH TO MITOCHONDRIAL DNA SYNTHESIS 462
MOLECULAR APPROACH TO MITOCHONDRIAL DNA SYNTHESIS 468
CONCLUSION 474
REFERENCES 475
Chapter 37. Covalent Binding of Reactive Intermediates of Xenobiotics to Mitochondrial DNA 
476 
INTRODUCTION 476
PREPARATION OF MITOCHONDRIA 477
ISOLATION OF MITOCHONDRIAL DNA 478
ACKNOWLEDGMENTS 482
REFERENCES 482
Chapter 
484 
INTRODUCTION 484
MATERIALS AND REAGENTS 486
PROCEDURES 487
COMMENTS 494
REFERENCES 499
Index 500