Measuring in vivo Oxidative Damage A Practical Approach

by Lunec, J.; Griffiths, H. R.

• ISBN13: 9780471818489
• ISBN10: 0471818488
• Edition: 1st
• Format: Hardcover
• Copyright: 2000-07-26
• Publisher: WILEY

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.

Author Biography

J. Lunec and H. R. Griffiths are the authors of Measuring in vivo Oxidative Damage: A Practical Approach, published by Wiley.

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