Free Radicals A Practical Approach

by Punchard, Neville; Kelly, Frank

• ISBN13: 9780199635603
• ISBN10: 0199635609
• Format: Hardcover
• Copyright: 1996-10-10
• Publisher: Oxford University Press

Summary

With the recognition that oxygen and nitrogen radicals are involved in normal cell metabolism, free radical research has begun to feature in most disciplines in the life sciences. Increasingly, their implication in a number of human disease processes is being accepted, altough conclusiveevidence is lacking in many instances, partly due to the difficulties in detecting and measuring free radicals that have lifetimes measured in microseconds. This book outlines the latest techniques for quantifying radicals and their effects, and features detailed protocols, hints, and tips forsuccess, troubleshooting comments, sample data, and key literature citations.

Table of Contents

List of contributors	xix	(4)
List of abbreviations	xxiii
1. Introduction	1	(10)
Neville A. Punchard
Frank J. Kelly
1. Free radicals	1	(1)
2. Sources of free radicals	2	(3)
3. Antioxidants	5	(1)
4. Measurement of free radicals	6	(1)
5. Conclusion	7	(4)
Part I. Free radical detection: physico-chemical methods	11	(54)
2. In vitro and in vivo detection of free radical metabolites with electron spin resonance	11	(14)
Ronald P. Mason
1. Introduction	11	(1)
2. Direct free radical detection with electron spin resonance	12	(5)
Freeze-quench technique	12	(1)
Quartz flat cells	12	(1)
Steady-state condition	13	(1)
Fast-flow technique	13	(1)
In situ technique	13	(3)
Rapid sampling	16	(1)
3. Spin trapping detection of free radicals	17	(6)
In vivo spin trapping	17	(1)
Folch extraction of radical adducts	18	(2)
Detection of radical adducts in biological fluids	20	(1)
Ex vivo radical adduct formation	21	(1)
Bioreduction of radical adducts	22	(1)
References	23	(2)
3. Detection of free radical reaction products by high-field nuclear magnetic resonance spectroscopy	25	(22)
D. P. Naughton
E. Lynch
G. E. Hawkes
J. Hawkes
D. R. Blake
M. Grootveld
1. Introduction	25	(1)
2. NMR analysis of chemical model systems	26	(2)
Keto acid anions as antioxidants	26	(1)
Lipid peroxidation products	27	(1)
Heparin degradation	28	(1)
DNA-protein crosslinks	28	(1)
3. NMR analysis of biofluids and tissues	28	(8)
H2O2 scavengers of healthy and diseased human biofluids	28	(3)
y-radiolysis of human biofluids	31	(1)
Redox-active transition metal ion speciation	32	(3)
Detection of lipid peroxidation products in biological samples	35	(1)
Therapeutic applications of free radical species	36	(1)
4. Applications to cell culture systems	36	(3)
Applications of methionine as a reactive oxygen species 'target' molecule	36	(1)
Assessment of free radical-based drug resistance in leukaemic cell lines	36	(3)
Protection against paraquat toxicity via thiourea antioxidant activity	39	(1)
Investigation of the mechanism of reperfusion-induced myocardial injury	39	(1)
5. NMR analysis of food	39	(5)
1H NMR analysis of y-irradiated food	39	(1)
1H NMR analysis of the thermally-induced oxidative deterioration of glycerol-bound polyunsaturates in culinary oils and fat	40	(4)
Acknowledgements	44	(1)
References	44	(3)
4. Pulse radiolysis	47	(18)
J. Butler
E. J. Land
1. Introduction	47	(1)
2. Design of experiments	48	(3)
Polar solvents	49	(1)
Non-polar solvents	50	(1)
3. Physico-chemical parameters	51	(6)
Handling of raw data from oscilloscopes/digitizers	51	(1)
Dosimetry	52	(1)
Spectra	53	(1)
Kinetics	53	(1)
Reduction potentials	55	(2)
4. Examples	57	(3)
Oxygen radicals	57	(1)
Radical transfer in proteins	58	(1)
Carotenoid radical ions	59	(1)
Acknowledgement	60	(1)
References	60	(1)
Further reading	60	(5)
Part II. Free radical detection: biochemical methods	65	(54)
5. Intrinsic (low-level) chemiluminescence	65	(18)
Yury A. Vladimirov
1. Introduction	65	(1)
2. Instruments	65	(1)
3. Experimental material	66	(6)
Whole organs	66	(2)
Tissue homogenates, mitochondria, and microsomes	68	(2)
Blood plasma	70	(2)
Phospholipid vesicles	72	(1)
4. Reaction kinetics	72	(3)
Analysis of the kinetic curves	74	(1)
5. Main factors affecting low-level chemiluminescence	75	(2)
Iron ions	75	(1)
Aeration	76	(1)
Temperature	77	(1)
6. Chemiluminescence spectra	77	(2)
7. The sensitization phenomenon	79	(1)
8. Conclusion	80	(1)
References	81	(2)
6. Visual assessment of oxidative stress by multifunctional digital microfluorography	83	(18)
Makoto Suematsu
Geert W. Schmid-Schonbein
Yuzuru Isimura
Masaharu Tsuchiya
1. Introduction	83	(1)
2. Intravital fluorescence video microscopy	84	(1)
3. Fluorescent probes for assessment of oxidative stress	85	(10)
Autofluorescence	85	(1)
Dichloroflurorescein and its derivatives	86	(9)
4. Other applications of digital microfluorography: leucocyte-dependent tissue injury in vivo	95	(3)
5. Conclusion	98	(1)
6. Acknowledgements	98	(1)
7. References	98	(3)
7. Salicylic acid and phenylalanine as probes to detect hydroxyl radicals	101	(18)
Harparkash Kaur
Barry Halliwell
1. Introduction	101	(2)
Detection of free radicals	101	(2)
The hydroxyl radical	101	(1)
Detection of hydroxyl radicals: 'trapping assays'	102	(1)
2. Aromatic hydroxylation	103	(12)
Salicylate	106	(3)
Phenylalanine	109	(6)
3. Conclusions	115	(1)
Acknowledgements	115	(1)
References	115	(4)
Part III. Measurement of free radical products	119	(94)
8. Peroxides and other products	119	(14)
R. K. Brown
F. J. Kelly
1. Introduction	119	(1)
Initiation of lipid peroxidation	119	(1)
Detection and measurement of lipid peroxidation	120	(1)
2. HPLC determination of lipid hydroperoxides	120	(5)
Peak identification and quantification	123	(2)
Plasma concentrations of lipid hydroperoxides	125	(1)
3. Determination of malondialdehyde	125	(5)
Introduction	125	(1)
Determination of malondialdehyde	126	(2)
Detection of malondialdehyde by HPLC	128	(2)
4. Conclusions and future directions	130	(1)
Acknowledgements	130	(1)
References	131	(1)
Further reading	131	(2)
9. Quantitative analysis of 4-hydroxy-2-nonenal	133	(14)
Michael Kinter
1. Introduction	133	(1)
2. Determination by UV spectroscopy	134	(3)
Principle of analysis	134	(3)
Adaptability to other aldehydes of interest	137	(1)
3. Determination by HPLC	137	(2)
Principle of analysis	137	(2)
Adaptability to other aldehydes of interest	139	(1)
4. Determination by gas chromatography-mass spectrometry (GC-MS)	139	(4)
Principle of analysis	139	(4)
Adaptability to other aldehydes of interest	143	(1)
5. Summary	143	(1)
References	144	(1)
Further reading	144	(3)
10. F2-Isoprostanes: Prostaglandin-like products of lipid peroxidation	147	(12)
Jason D. Morrow
L. Jackson Roberts
1. Introduction	147	(2)
2. Method of assay	149	(6)
Handling and storage of biological fluids and tissues for quantification of F2-isoprostanes	149	(1)
Extraction and hydrolysis of F2-isoprostane-containing phospholipids	149	(2)
Hydrolysis of lipid extracts	151	(2)
Purification and derivatization of F2-isporistanes	153	(2)
Quantification of F2-isoprostanes using GC-MS	155	(1)
3. Application of the assay to biological samples	155	(1)
Acknowledgements	156	(1)
References	156	(1)
11. Determination of carbonyl groups in oxidized proteins	159	(12)
Emily Shacter
Joy A. Williams
Earl R. Stadtman
Rodney L. Levine
1. Introduction	159	(1)
2. Reaction with 2,4-dinitrophenylhydrazine in guanidine	160	(5)
Calculations	163	(1)
Comments	164	(1)
3. Reaction with DNPH in sodium dodecyl sylphate	165	(5)
Comments	168	(2)
Acknowledgement	170	(1)
References	170	(1)
12. Protein hydroperoxides, protein hydroxides, and protein-bound DOPA	171	(14)
Roger T. Dean
Shanlin Fu
Steven Gieseg
Sharyn G. Armstrong
1. General background	171	(1)
2. General methods involved in determination of protein hydroperoxides, hydroxides, and protein-bound DOPA	172	(2)
3. Protein hydroperoxides	174	(5)
Introduction	174	(1)
Determination of stable derivatives of protein hydroperoxides	174	(1)
Preparation of B-hydroxyvaline standard	175	(1)
Detection of B-hydroxyvaline on oxidized protein molecules	175	(4)
4. Protein-bound DOPA	179	(3)
Introduction	179	(2)
DOPA: general comments	181	(1)
References	182	(3)
13. Investigating the effects of oxygen free radicals on carbohydrates in biological systems	185	(16)
H. R. Griffiths
J. Lunec
1. Introduction	185	(2)
Methods of choice	186	(1)
2. In vitro analyses	187	(9)
Methods of carbohydrate damage by in vitro OFR generation	187	(2)
Analysis of OFR damage to Monosaccharides	189	(3)
Complex sugar analysis using a lectin-based ELISA	192	(2)
Deoxyribose degradation by OFRs in situ	194	(2)
3. Ex vivo techniques	196	(2)
OFR effects on mucopolysaccharides	196	(1)
Hyaluronic acid degradation by OFRs	196	(2)
4. Conclusions	198	(1)
Acknowledgement	198	(1)
References	198	(1)
Further reading	199	(2)
14. Measurement of products of free radical attack on nucleic acids	201	(12)
M. Lindsay Maidt
Robert A. Floyd
1. Introduction	201	(4)
Biological consequences	201	(1)
HPLC detection of DNA/RNA products	202	(3)
2. DNA isolation method	205	(1)
3. DNA digestion	206	(1)
4. HPLC analysis	207	(1)
HPLC	207	(1)
Analysis of results	208	(1)
5. Applications	208	(1)
8-OHdG detection in human urine	208	(1)
Future developments	209	(1)
Acknowledgements	209	(1)
References	209	(4)
Part IV. Mesurement of antioxidants	213	(88)
15. Glutathione	213	(14)
Mary E. Anderson
1. Introduction	213	(1)
2. Sample preparation	214	(3)
3. Assays for the determination of GSH	217	(3)
DTNB-GSSG reductase recycling assay for GSH	217	(3)
DTNB-GSSG reductase recycling assay for GSSG	220	(1)
4. Column methods for determination of GSH	221	(4)
Ion-exchange chromatography of GSH and other thiols	221	(1)
Ion-exchange chromatogrphy of GSH and GSSG	222	(1)
Determination of GSH using monobromobimane derivatization and HPLC	223	(2)
5. Conclusions	225	(1)
Acknowledgement	225	(1)
References	226	(1)
16. Glutathione peroxidase: activity and steady-state level of mRNA	227	(14)
Daret K. St Clair
Ching K. Chow
1. Introduction	227	(1)
2. Measurement of GSH perioxidase activity	228	(3)
Calculation of GSH peroxidase activity	230	(1)
3. Detection of GSH peroxidase expression	231	(4)
4. Analysis of steady-state mRNA by Northern hybridization	235	(4)
References	239	(2)
17. Superoxide dismutase	241	(16)
Sara Goldstein
Gidon Czapski
1. Introduction	241	(1)
2. Mechanism of the catalysis of superoxide dismutation by SOD and SOD mimics	241	(2)
3. Methods of determining SOD activity	243	(10)
Direct methods	243	(5)
The indirect method	248	(5)
4. Properties required for SOD mimics operating in vitro to work also in vivo	253	(1)
References	254	(3)
18. Deuterated vitamin E: measurement in tissues and body fluids	257	(14)
Graham W. Burton
Malgorzata Daroszewska
Introduction	257	(1)
2. Synthesis and administration of deuterium-labelled tocopherols	258	(1)
Syntheses of deuterated tocopherols	258	(1)
Administration of deuterated tocopherols to humans and animals	259	(1)
3. Extraction of vitamin E from tissues, cell suspensions, and fluids	259	(4)
4. Purification and derivatization	263	(1)
Purification	263	(1)
Derviatization	264	(1)
5. GC-MS analysis of tocopherols	264	(3)
Calculation of tocopherol concentrations	266	(1)
6. Sensitivity and limitations	267	(1)
7. Applications	268	(1)
References	269	(1)
Further reading	270	(1)
19. Selective and sensitive measurement of vitamin C, ubiquinol-10, and other low-molecular-weight antioxidants	271	(16)
Detlef Mohr
Roland Stocker
1. Introduction	271	(2)
2. Properties of selected natural antioxidants	273	(10)
Water-soluble, non-proteinanceous antioxidants	273	(4)
Lipid-soluble, non-proteinaceous antioxidants	277	(6)
Acknowledgement	283	(1)
References	283	(4)
20. Measurement of antioxidant gene expression	14	(287)
Colin D. Bingle
1. Introduction	287	(1)
2. Isolation of RNA	287	(3)
Precautions to reduce potential RNase contamination	287	(1)
Isolation of total RNA	288	(2)
Quantification of RNA	290	(1)
3. Analysis of RNA	290	(5)
Choice of analysis method	290	(3)
Choice and labelling of probes	293	(2)
Removal of unincorporated NTPs	295	(1)
4. Hybridization and washing conditions	295	(3)
Hybridization of RNA blots with Synthetic oligonucleotide probes	297	(1)
5. Analysis of results	298	(1)
References	299	(1)
Further reading	300	(1)
A1. Addresses of suppliers	301	(4)
Index	305