Fifteen years ago the field of oxygen free radicals was just beginning to launch into a new area of importance in pathology. Since then, oxygen free radicals have been implicated in a number of disease processes, including atherosclerosis and chronic inflammation. Measuring in vivo Oxidative Damage brings together methods by leading experts in the field of oxidative damage and by scientists from clinical biochemistry laboratories who have had much experience with the practical problems of measuring oxidative damage in vivo. Many of the authors are involved in national and international quality assurance programmes and routinely establish these assays in clinical research laboratories. The book is divided into 5 parts:
* Chromatographic procedures
* Measurement of 8-oxo deoxyguanosine
* Cellular-based methods
* Molecular-based assays
* Antioxidant activity.
Each part is designed to help the clinical scientist evaluate the best method for their particular problem in measuring oxidative damage in vivo. The methods represented are the ones most commonly used and are deemed robust and simple enough to apply to clinical material. Measuring in vivo Oxidative Damage is an ideal practical reference work for the clinical scientist and is a must for all laboratories in hospitals and research institutions which are actively involved in analytical work.
Recenzijas
"This book is an ideal practical reference work for the clinical scientist and is recommended for all laboratories in hospitals and research institutions which are actively involved in analytical work." (Clinical Laboratory International, June 2000)
List of Contributors xiii Preface xv Part I Chromatographic Procedures 1(50) Lipid Peroxidation Determination by HPLC 3(12) Ruth J. Bevan Introduction 3(1) HPLC determination of lipid hydroperoxides 4(8) Determination of lipid hydroperoxides by HPLC with chemiluminescence detection 4(5) Measurement of lipid hydroperoxides using an HPLC-based thiobarbituric acid assay 9(3) Conclusions 12(1) References 13(2) Measurement of Oxidized Bases in DNA and Biological Fluids by Gas Chromatography Coupled to Mass Spectrometry 15(12) Thierry Douki Jean-Luc Ravanat Jean Cadet Introduction 15(1) Reagents 16(1) DNA extraction from liver or cell cultures 17(2) Homogenization of the liver 17(1) Extraction of the nucleic acids 17(1) DNA isolation 18(1) DNA hydrolysis 19(1) Formic acid treatment for the analysis of 5-OHCyt, 5-OHUra, 5-HMUra, 5-ForUra, 8-oxoGua and 8-oxoAde 19(1) HF/pyridine-mediated release of FapyGua from DNA 19(1) Prepurification 20(3) 5-OHCyt, 5-OHUra, 5-HMUra, 5-ForUra and FapyGua 21(1) 8-OxoAde 21(1) 8-OxoGua 21(2) Isolation of modified bases and nucleosides from urine 23(1) GCMS Analysis 23(2) Silylation 23(1) GCMS analyses 24(1) Calibration curve 24(1) Interpretation of the results 25(1) References 25(2) The Measurement of Protein Oxidation by HPLC 27(24) Helen R. Griffiths Ruth Bevan Joe Lunec Introduction 27(1) Markers of protein oxidation in intact proteins 28(5) Induction of novel fluorescence 28(2) Carbonyl measurement 30(3) Oxidized amino acid analysis 33(13) Isolation of oxidized proteins from mixed protein samples 34(1) Protein hydrolysis 35(2) Tryptophan oxidation 37(2) Tyrosine oxidation 39(3) Nitrotyrosine formation 42(1) Valine hydroperoxide analysis 43(3) Conclusion 46(1) Acknowledgements 47(1) References 47(4) Part II Measurement of 8-Oxo Deoxyguanosine 51(30) Measurement of 8-Oxo-2-deoxyguanosine in Cellular DNA by High Performance Liquid Chromatography-Electrochemical Detection 53(10) Mark D. Evans Introduction 53(1) DNA extraction 54(1) Quantitation of DNA 55(1) Calculations 55(1) DNA digestion and analysis of 8oxodG 56(4) Reagents 56(1) HPLC standards 57(1) Calibration standards 58(1) Procedure 59(1) HPLC-EC 60(1) Calculations 60(1) Acknowledgements 61(1) References 61(2) Immunochemical Detection of 8-Oxodeoxyguanosine in DNA 63(6) Marcus Cooke Karl Herbert Introduction 63(1) Immunochemical detection of 8-oxodG in DNA 64(1) Assay principle 64(1) ELISA dynamic range and limit of detection 64(1) General problems in the measurement of 8-oxodeoxyguanosine 64(1) Assay procedure 65(3) Enzymatic hydrolysis of DNA 65(1) Quantitation of deoxyguanosine in DNA digests 65(1) Elisa analysis of 8-oxodG in DNA 66(1) Analysis of results 67(1) Comparison with an established HPLC technique 67(1) Limitations of the assay 67(1) References 68(1) Urinary Measurement of 8-OxodG (8-Oxo-2-deoxyguanosine) 69(12) Henrik E. Poulsen Steffen Loft Allan Weimann Introduction 69(1) Methods of analysis 70(1) HPLC-EC analysis 71(5) Apparatus and system set-up 71(3) Preparation of urine samples 74(1) Alternative HPLC-EC procedures 75(1) Quality control 75(1) Final calculation 75(1) LCMS-MS analysis of 8-oxodG in urine 76(4) Introduction 76(1) Instrumentation set-up 77(1) Experience 78(1) Interpretation of urinary measurements 79(1) Conclusion 80(1) Acknowledgements 80(1) References 80(1) Part III Cellular-based Methods 81(24) Measurement of Oxidative DNA Damage Using the Comet Assay 83(12) Andrew R. Collins Introduction 83(1) Reagents and materials 84(1) Equipment 85(1) Procedure 85(5) Slide preparation 85(1) First agarose layer 85(1) Preparation of cells 86(1) Embedding cells in agarose 86(1) Lysis 87(1) Enzyme treatment (endonuclease III, formamidopyrimidine glycosylase) 87(1) Alkaline treatment 87(1) Electrophoresis 87(1) Neutralization 87(1) Staining 88(1) Quantitation 88(2) Storage and re-examination 90(1) Notes 90(3) Lymphocytes: bulk preparation and storage 90(1) Enzymes 91(1) Treatment with H2O2 92(1) Measuring antioxidant resistance 93(1) Monitoring recovery from oxidative damage in vitro 93(1) References 93(2) Measuring Oxidative DNA Damage by Alkaline Elution 95(10) Michael Pflaum Bernd Epe Introduction 95(1) Principle of the assay 96(1) Scope and limitations 97(1) Reagents 97(1) Instruments 98(1) Repair endonucleases 99(1) Problems and pitfalls 100(1) Procedure 101(4) Calculation of elution rates (for each filter) 102(1) Calculation of single-stand breaks (SSB) and enconuclease-sensitive sites (ESS) 103(1) Detection range 103(1) References 104(1) Part IV Molecular-based Assays 105(38) A 32P-Postlabelling Protocol to Measure Oxidative DNA Damage 107(18) George D.D. Jones Michael Weinfeld Introduction 107(4) Methods 111(8) Materials 111(1) Preparation of polyacrylamide gels 112(3) Postabelling assay 115(2) HPLC analysis of radioactive bands and nearest-neighbour analysis 117(2) Marker compounds 119(1) Discussion 119(4) Notes on the protocol (troubleshooting) 119(1) Quantitative aspects of the assay 120(2) General comments on the assay (advantages and disadvantages) 122(1) Acknowledgements 123(1) References 123(2) Mapping Reactive Oxygen-Induced DNA Damage at Nucleotide Resolution 125(18) Henry Rodriguez Steven A. Akman Oxidative DNA damage 125(1) Nucleotide resolution mapping of ROS-induced DNA damage by the ligation-mediated polymerase chain reaction 125(8) Primer extension 127(1) Ligation 128(1) PCR amplification 128(5) Enhancement of LMPCR damage detection sensitivity by genomic gene enrichment 133(7) BamH I digestion of total genomic DNA 135(1) Loading the DNA sample and running the continuous elution electrophoresis apparatus 135(1) Fraction viewing by standard agarose electrophoresis 136(1) Fraction screening for gene of interest by dot-blot analysis 136(1) Cleavage of enriched samples at ROS-induced modified bases 137(1) Cleavage of non-enriched samples at ROS-induced modified bases 138(1) LMPCR analysis 138(2) The future of DNA damage analysis 140(1) References 141(2) Part V Antioxidant Activity 143(26) Measurements of Plasma Antioxidant Activity 145(24) Simon R.J. Maxwell Introduction 145(1) Principles of measuring `total antioxidant activity 145(3) Total antioxidant assays 148(1) Influences upon plasma `total antioxidant activity 149(1) Interpretation of `total antioxidant assays 150(2) The TRAP assay 152(4) Reagents 152(1) Calibration standards 153(1) Apparatus 154(1) Procedure 154(1) Calculations 154(1) Reference range 155(1) Interference 155(1) Comments 155(1) The enhanced chemiluminescent (ECL) assay 156(5) Reagents 157(2) Calibration standard 159(1) Apparatus 159(1) Procedure 159(1) Calculations 160(1) Reference range 160(1) Interference 161(1) Comments 161(1) The ABTS assay 161(4) Reagents 162(1) Standard solution 163(1) Apparatus 163(1) Manual procedure 164(1) Automated procedure 164(1) Calculations 164(1) Reference range 164(1) Interference 164(1) Comments 165(1) Other antioxidant assays 165(1) The FRAP assay 165(1) The OPD assay 165(1) The phycoerythrin assay 165(1) The cis-parinaric acid assay 166(1) The plasma peroxidation potential assay 166(1) References 166(3) Index 169
J. Lunec and H. R. Griffiths are the authors of Measuring in vivo Oxidative Damage: A Practical Approach, published by Wiley.
