9780471818489

Measuring in Vivo Oxidative Damage : A Practical Approach

by ;
  • ISBN13:

    9780471818489

  • ISBN10:

    0471818488

  • Format: Hardcover
  • Copyright: 2000-07-26
  • Publisher: WILEY
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Summary

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.

Table of Contents

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

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