FREE RADICAL DAMAGE AND ITS CONTROL


New Comprehensive Biochemistry

Volume 28

General Editors

A. NEUBERGER
London

L.L.M. van DEENEN
Uti-echt

ELSEVIER
Amsterdam . London . New York . Tokyo


Free Radical Damage and its Control

Editors

Catherine A. Rice-Evans
Free Radical Research Group, United Medical and Dental Schools,
Guy’s & St. Thomas’s Hospital,
St. Thomas’s Street, London, U K SEl9RT

Roy H. Burdon
Department of Bioscience & Biotechnology, The Todd Centre,

University of Strathclyde, Glasgow, Scotland, U K G4 ONR

Amsterdam

.

1994
ELSEVIER
London . New York . Tokyo


Elsevier Science B.V.
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The Netherlands

Library of Congress Cataloging-in-Publication Data
Free radical damage and its control /editors. Catherine A. Rice
-Evans. Roy H. Burdon.
p. cni. -- (New comprehensive biochernislry ; v. 2x1
Includes hihliographical references and index.
ISBN 0 4 4 4 - X Y 7 16-x (alk. paper).-- ISBN 0-444- XO303 -3 (series:
il I
I . Free radicals (Chemistry)-- Pathophysiology. 2. Activeoxypen.
3. Antioxidants. 1. Rice-Evnns,Catherine.
11. Burdon. R. H . (Roy
Hunter) 111. series.
IDNLM: I . Free Radicals. 2. Reactiveoxygen Species.
WI NE372F v. 2X 1994 / Q V 312 F852S 19941
3.Antioxidmts.

QD41S.N48 vol. 28
[ nB I 701
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lhIh.07'I I
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for Library o f Congress
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ISBN 0 4 4 4 80303-3 (series)

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V

Preface
In this volume of New Comprehensive Biochemistry the international authorship
has aimed to provide a comprehensive treatise on the chemical and biochemical
consequences of damaging free radical reactions, the implications for the pathogenesis of disease and how this might be controlled endogenously and by radicalscavenging drugs. The important developments in biochemistry presented here
impinge not only on fundamental biology, but are also of primary concern to
clinical medicine and human nutrition.
Free radicals are essential to a number of normal biochemical and
physiological processes but are kept under control by the primary antioxidants,
the cytoprotective enzymes, and the secondary antioxidants, such as the
transition-metal and haem protein binders and the interceptors of propagating
radical reactions. Oxidative stress is said to arise when “the balance
between oxidants and antioxidants is tipped in favour of the former”.
This may be influenced by exogenous agents of oxidative stress, radiation,
trauma, drug activation, oxygen excess, for example, or by endogenous
oxidative stress which is associated with many pathological states including
chronic inflammatory disorders, cardiovascular disease, injury to the central
nervous system, connective-tissue damage, etc. These are some of the aspects
we have selected to emphasise in this volume. The approach of novel potentially
therapeutic iron-chelating agents and antioxidants, including the lazaroids and
the hydroxypyridinones is also reviewed. The scene is set by comprehensive
in-depth reports on the chemistry of free radical reactions involving iron and
copper, the major transition metals involved in metalloproteins in living cells,
and the biochemical constraints of their participation in oxidative stress. The
potential mode of action of reactive oxygen species in cell proliferation and the
transmission of messages from the extracellular environment to the nucleus are
also highlighted.

Recent research has focussed on the role of antioxidant nutrients in reducing
the risk of developing coronary heart disease and cancer, the major killers in
Western industrialised society. Several epidemiological studies have reported in
particular the relevance of a-tocopherol in the context of coronary heart disease
and p-carotene in cancer, especially that of the lung. The development of
antioxidant drugs for the treatment of diseases associated with free radicals is
a vibrant area of research but depends on the understanding of the mechanisms


vi

underlying the generation of excessive free radicals in vivo, the factors
controlling their release and the site of their action.
In many disease states, the nature and original location of the radical species
that amplify the primary damage are unknown, making the design and targeting
of appropriate antioxidant drugs difficult. Thus a detailed understanding of
the processes leading to the radical-dependent pathology, as well as to the
nature and sources of the toxic species, is crucial for the design of effective
intervention strategies. The individual chapters present up to date accounts of
the current state of knowledge in these areas.
The editors warmly acknowledge all the contributing authors for participating
in the production of this important text.
Catherine A. Rice-Evans
Roy H. Burdon
May 1993


vii

List of contributors

J.E. Baker, 333
Cardiothoracic Surgery, Medical College of Wisconsin, Milwaukee, WI 53226,
USA
M.S. Baker, 301
Senior Lecture6 Department of Biological Sciences, University of Wollongong,
NorthJield j . Ave., Wollongong, NS W 2522, Australia
D.R. Blake, 361
InPammation Research Group, The London Hospital Medical College, University
of London, Turner Street, London, UK E l 2AD
R.H. Burdon, 155
Department of Bioscience & Biotechnology, The Todd Centre, University of
Strathclyde, Glasgow, Scotland, UK G4 ONR
J. Chaudiire, 25
Centre de Recherche BIOmTECH, Z.A. des petits carreaux, 2 avenue des
coquelicots, 94385 Bonneuil-sur-Marne Cedex, France
C.F. Chignell, 319
Laboratory of Molecular Biophysics, National Institute of Environmental Health
Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
C.E. Cooper, 67
Department of Paediatrics, University College London School of Medicine, The
Rayne Institute, 5 University Street, London, UK WCIE 6JJ
A.T. Diplock, 113
Free Radical Research Group, Division of Biochemistry, United Medical and
Dental Schools of Guyk & St. Thomas? Hospital, St. Thomas Street, London,
UK SEI 9RT
P. Duriez, 257
Dipartement d 'Etudes des Lipides et des Lipoprote'ines, SERLIA et U325 Inserm,
1 rue du Prof Calmette, F59019 Lille Cedex, France
J.C. Fruchart, 257
Dipartement d 'Etudes des Lipides et des Lipoprotkines, SERLIA et U325 Inserm,

1 rue du Pro$ Calmette, F59019 Lille Cedex, France
E.D. Hall, 217
Central Nervous System Diseases Research, The Upjohn Company, Kalamazoo,
MI 49001, USA
R.C. Hider, 189
Department of Pharmacy, King j. College London, University of London,
Manresa Road, London, UK SW3 6LX


...

Vlll

J. Joseph, 333
Biophysics Research Institute, Medical College o j Wisconsin, Milwaukee, WI
53226, USA
B. Kalyanaraman, 333
Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, WI
53226, USA
E.A. Konorev, 333
Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, WI
53226, USA
W.H. Koppenol, 3
Departments of Chemistry and Biochemistry, Louisiana State University, Baton
Rouge, LA 70803, USA
R.P. Mason, 3 19
Laboratory of Molecular Biophysics, National Institute of Environmental Health
Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
C.J. Morris, 361
Injammation Research Group, The London Hospital Medical College, University

of London, Turner Street, London, UK E l 2AD
L. Packer, 239
Department of Molecular and Cell Biology, University of Callfornia, Berkeley,
CA 94720, USA
B.J. Parsons, 281
Multidisciplinary Research and Innovation Centre, North East Wales Institute,
Deeside, Clwyd, UK CH5 4BR
C.A. Rice-Evans, 131
Reader in Biochemistry, Free Radical Research Group, United Medical and
Dental Schools, G u y ) & St. Thomas) Hospital, St. Thomas Street, London,
UK SEl 9RT
S. Singh, 189
Department of Pharmacy, King j . College London, University of London,
Manresa Road, London, UK SW3 6LX
VR. Winrow, 36 1
Inflammation Research Group, The London Hospital Medical College, University
of London, Turner Street, London, UK E l 2AD
PG. Winyard, 361
Injammation Research Group, The London Hospital Medical College, University
of London, Turner Street, London, UK E l 2AD
M. Zaidi, 361
Department of Cellular and Molecular Sciences, St. George b Hospital Medical
School, London, UK


IX

Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
List of contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


PART I

V

vii

Chemical and Biochemical Aspects

.

Chapter 1. Chemistry of iron and copper in radical reactions
KH . Koppenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

Abbreviations . . . . . . . . . . . . . . . . .
...........................
1 . Introduction . . . . . . . . . . . . . . . . . . .
.........................
2 . Autoxidation reactions . . . . . . . . . . . . . . . . . . . . .
..
.............
2.1. Oxygen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Thermodynamics . . . .
..................................
2.3. Kinetics and mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . Fenton reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1, Introduction . . . . . . . . . . . . . . . . .
.......................

3.2. Thermodynamics . . . . . . . . . . . . . . . . . . . .
................
3.3. Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Intermediates . . . . . . . . . . . . . . . . . . . . . . .
4 . Speciation and effectiveness in promoting oxyradical d
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . .
...................................

11
12
13
13
18
20
20

Chapter 2. Some chemical and biochemical constraints of oxidative
stress in living cells
Jean ChaudiBre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

25

Abbreviations . . . . . . . . . .
.....................................
I . The birth of the concept . . . . . . . .
............................
2 . The basic properties of oxygen and th
cept of oxygen activation . . . . . . . . . . .
3 . The puzzling toxicity of superoxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4 . Toxicity of hydroperoxides and their radical by-products . . . . . . . . . . . . . . . . . . .
4.1. Hydrogen peroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Organic hydroperoxides . . . . . . . . . . . . . . .
................
............
4.3. Sodium and calcium homeostasis . . . . . . . . . . . . . . .

25
25
27
34
39
39
40
43

3
3

6
6
7
9

II


X

4.4. Signal transduction . . . . . . . . . . . .

.....
.............
5. Iron transport and the iron-transit pool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Protective pathways of mammalian cells and tissues . . . . . . . . . . . . . . . . . . . . . .
6. I . Hydrophobic protective systems . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. Hydrophilic protective systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3. Regulation of antioxidant enzymes . . . . . . . . . . . . .
.............
7. Protein S-thiolation: signal or damage? . . . . . .
....
.............
8. Conclusion . . . . . .
...
....
.............
References . . . . . . . . .
.........
......................

44
45
46
46
48
52
54
57
58

Chapter 3. Ferry1 iron and protein free radicals

C.E. Cooper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67

.....

....................

Introduction .
...................................
2. Chemical structure of Fe’” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Enzyme catalysis by ferryl ion and free radicals . . . . . . . .
....
3. I . Haem proteins . . . . . . . . . . . . . . . .
...........
3.1.1. Peroxidases and catalases . . . .
.....................
3.1.1.1. Plantifungalibacterial peroxidases . . . . . . . . . . . . . . . . . . . . .
3.1 . I .2. Mammalian peroxidases . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.3. Catalases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2. Oxidasesioxygenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. I .2.1. Cytochrome oxidases . . . . . . . . . . . . . . . . . . . .
....
..................
3.1.2.2. Cytochrome P..................
3.2. Non-haem proteins . . . . . . .
3.2.1. Binuclear iron centres
............................
3.2.2. Mononuclear iron centres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Methods for detecting ferryl iron and protein free radicals . . . . . . . . . . . . . . . . . .

4.1. X-ray crystallography . . . . .
..
..
4.2. X-ray absorption techniques .
................
4.3. Magnetic susceptibility . . . .
......................
4.4. EPWENDOR spectroscopy . . . . . . .
..................
4.5. Optical spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6. Magnetic circular dichroism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7. Mossbauer spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.8. Nuclear magnetic resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.9. Resonance Raman spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Radicals and disease - ferryl gone wrong . . . . . . .
.............
5.1. Enzymes utilising ferryl intermediates . . , , ,
.............
5.2. “Accidental” ferryl states in proteins . . . . . .
.............
5.3. Non-protein catalysed ferryl production
..................
Acknowledgements . . . . . . . . .
..
..............................
References . . .
.............................................
1.

Chapter 4. Antioxidants and free radical scavengers

A.7: Diplock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 , I . Definitions of antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67
67
70
72
72
72
75
16
71
78
78
80
80
80
82
83
83
85
86
87
90
93
94
97

98
100
100
101

I03
103
104

113
I13
1 I3


XI

1.2. Mechanisms in vivo . .
.........
2. Enzymatic mechanisms of protection . . . .
.....................
2 . I . Superoxide dismutases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Catalase and glutathione peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1. Catalase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2. Glutathione peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Minerals and nutritional deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Control of secondary radicals . . . . . . . . . . . . . . . . .
3.1. Vitamin E (a-tocopherol)
....
3.2. Vitamin C (ascorbic acid) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.4. Ubiquinone (CoenzymeQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . Removal of lipid hydroperoxides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 . Other antioxidant mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References
.............
.....
.........

..

1 I3

Chapter 5. Formation of free radicals and mechanisms of action in
normal biochemical processes and pathological states
C.A. Rice-Evans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

131

I . Free radical-generating systems in normal processes in vivo . . . . . . . . . . . . . . . . .
2. Free radical-mediated tissue damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Factors controlling the release of free radicals in disease states . . . . . . . . . . .
2.2. Peroxidation of polyunsaturated fatty acid sidechains . . . . . . . . . . . . . . . . .
2.3. Oxidation of protein substituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Carbohydrate oxidation and consequences for protein function . . . . . . .
3. Haem proteins and the potential for the formation of reactive radical species in
pathological states . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Myoglobin-derived free radicals . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Haemoglobin as a promoter of oxidative processes . . . . . . . . . . . . . . . . . . .
e device . . . . . . . . . . . . . . . .

......
Acknowledgements . .
..
.......
............
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 6. Free radicals and cell proiferation
R.H. Burdon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Proliferation of mammalian cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. The cell division cycle and its control . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. The influence of the extracellular environment
...................
3. Oxidative stress and cell proliferation . . . .
.......
3.1, Oxygen toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Lipid peroxidation . . . . . . .
..............
..
......
3.3. Effects of a-tocopherol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Serum deprivation and lipid peroxidation . . . . . . . . . . . . . . . . . . . . . . . . .
3.5. Polyunsaturated fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6. Lipid peroxidation and signal transduction . . . . . . . . . . . . . . . . . . . . . . . .
4 . Oxygen radicals, and related species that stimulate cell proliferation . . . . . . . . . . .

131
133
133

135
137
139
141
141
143
145
148
151
151

155
155
155

155
157
I58
158
159
159

159
160
161
163


xii
Cellular rclease of superoxide and hydrogen peroxide . . . . . . . . . . . . . . . . . . . . .

lntracellular generation of superoxide . . . .
.......
.......
...
Superoxide and hydrogen peroxide as cellul
ssengers ’ . . . . . . . . . . . . . . . . .
Mechanisms whereby superoxide and hydrogen peroxide promote cell growth or growth
responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1. Redox regulatory paradigm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2. Oxidative inactivation of extracellular protease inhibitors . . . . . . . . . . . . . . .
8.3. Redox rncchanisms and the source of active oxygen species . . . . . . . . . . . . .
9. Active oxygen species and normal cell proliferation . . . . . . . . . . . . . . . . . . . . . .
.................
I0 . Active oxygen species and carcinogenesis . . . . . . . .
10.1 . Initiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.........
10.2. Free radicals and promotion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.3, Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.4. Growth promotion and the tumour phenotype . . . . . . . . . . . . . . . . . . . . . .
10.5. Therapeutic intervention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
..................
1 I . Radicals and the role of ribonucleotide reductase . . .
12. An overview . . . . . . . .
.........
........................
References . . . . . . . . . . . . . . . . . . . . . . . .
........................

5.
6.

7.
8.

PART I1

164
i65
166
168
170
171
173
174
175
175
175
177
177
177
179
179
180

Pathological Aspects

.

Chapter 7. Therapeutic iron-chelating agents
S. Singh and R.C. Hider . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.


2.

3.
4.
5.

6.

...................................
..
..............................
1.2. Iron transport . . . . . . . . . . . . . . . .
.........
.............
1.3. Iron storage . . . . . . . . . . . . . . . . . . . . . .
.......
Iron overload . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . I . Transfusional siderosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Hyperabsorption of iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bidentate and hexadentate iron chelators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chelation therapy . . . . . . . . . .
..
....
..................
Requirements for selective iron chelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. I . Absorption and selective distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Minimal redistribution of iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Negative iron balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4. Lack of acute and long-term toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.5. Metabolism and pharmacokinetic properties of chelating agents . . . . . . . . . .
Design of orally active chclating agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1, Aminocarboxylate ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. Hydroxypyridinone ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3. Desferrithiocin ligands
.....
........................

7. Localised and temporary elevation of iron levels . . . . . . . . . . . . . . . . . . . . . . . .
7.1. lschaemic tissue . . . . . . . . . . . . . . . . . . . . . . . . . .
...
.......
7.2. Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.............
8. Selective inhibition of non-hacm-containing cnzymes . . . . . . . . . . . . . . . . . . . . .
8. I . Ribonucleotide reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

189
190
190
191
191
191

193
193
194
195
195
196

197
197
198
199
199
200
20 I
201
201
205
201
208


...

Xlll

8.1.1. Synchronisation of cell cycling . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8 . I .2. Anti-malarial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2. Lipoxygenase enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9 . Treatment of anaemia with iron complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

208
211
211
212
213

213

Chapter 8. Free radicals in central nervous system injury
E.D. Hall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

217

I . Introduction . . . . . . . . . . .
...........................
2. Oxygen radicals in spinal cor
....
......
2.1. Role in post-traumatic hypoperfusion (secondary ischemia) . . . . . . . . . . . . .
2.2. Role in post-traumatic axonal degeneration . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Role in post-traumatic conduction failure in surviving axons . . . . . . . . . . . .
2.4. Similarity of peroxidative and mechanical spinal injuries . . . . . . . . . . . . . . .
2.5. Effects of anti-oxidants on post-traumatic neurological recovery . . . . . . . . . .
3. Oxygen radicals in head injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . I . Role in post-traumatic microvascular damage
................
3.2. Role in post-traumatic edema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Effects of antioxidants on post-traumatic neurological recovery and survival . .
4 . Clinical evidence of the importance of oxygen radicals in CNS injury . . . . . . . . . .
4.1. High-dose methylprednisolone in spinal-cord injury . . . . . . . . . . . . . . . . . .
4.2. High-dose methylprednisolone in severe head injury . . . . . . . . . . . . . . . . . .
4.3. PEG-SOD in severe head injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Summary . . . . . . . .
.................................
References . . . . . . . . . . . . . . . . . . . . . . . .
...

..................

217
218
218
223
224
226
226
228
228
229
23 1
232
232
233
233
233
234

Chapter 9. Ultraviolet radiation (UVA. UVB) and skin antioxidants
L . Packer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.............
2. Studies on excised hairless mouse skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. UVA irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. UVB irradiation
.................................
2.3. Conclusions from studies on exciscd skin . . . . . . . . . . . . . . . . . . . . . . . . .
3. In vivo irradiation of hairless mouse skin .

..................
3.1. Dose-response for lipophilic antioxida
.........
3.2. Lipid hydroperoxides and effects of vitamin E supplementation . . . . . . . . . . .
3.2.1, Experiment I : irradiation without supplementation . . . . . . . . . . . . . . .
3.2.2. Experiment 2: irradiation with supplementation . . . . . . . . . . . . . . . . .
4 . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

239
240
240
240
242
245
246
247
248
251
252
253
253

Chapter 10. Free radicals and atherosclerosis
J C . Fruchart and P Duriez . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

257

1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 . Arterial wall and oxyradicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

257
257


3. Oxidised low-density lipoproteins (Ox-LDL) . . . . . , , , . , . . . , . .
3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . , , . , , . . , ,
3.2. Chemistry of Ox-LDL . . . . . . . . . . . . . . . . .
.................
3.3. Biology of Ox-LDL and development of the
advanced lesions. , . , . . . . . . . . . . . . . . . .
...........
3.3.1, Infiltration of Ox-LDL across vascular endothelium and the formation of
the fatty streak.
.......................
3.3.2. Evidence for the
X-LDL . , , , , . , , . . . . . . . . . .
3.3.3. The fatty streak and transition to more advanced lesion . . . . . , , ,
3.3.4. Lesion progression . . . . . . . . . . . . . . . .
.................
3.3.5. The mature atherosclerotic plaque (fibrous plaque) . . , . . .
3.3.6. The role of mural thrombosis in plaque growth . . . . . . . . . . . . . , . . ,
..............................
3.3.7. Arterial occlusion . . .
3.3.8. Plaque regression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Antioxidants and Ox-LDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5. Effect of monounsaturated and polyunsaturated fatty acids on the susceptibility
of plasma LDL to oxidative modification . . . . . . . . . . . . . . . . . . . . . . . , ,
3.6. Smoking and coronary heart disease . . . . . . . . . . . . . . , . . . . . .

4. The macrophage scavenger receptors (Fig. I ) . . . . . . . . . , , . , , . .
5. Ox-LDL and vasoconstriction . . . . . . . . .
.................
6. Arrhythmogenic effects of Ox-LDL . . . . .
.................
7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter I I . Chemical aspects of free radical reactions in connective tissue
B.J. Parsons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .
.............
2. Reactions of free radicals with hyaluronic acid in si
2.1. Oxygen-free solutions . . .
. . . . . . . . . . . . . . . . .. . . . . . . . . . .
2.2. Oxygen-containing solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . ,
2.3. Depolymerisation of hyaluronic acid induced by Cu(I1) and hydrogen peroxide
in solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , .
2.4. Factors affecting the efficiency of hydroxyl-radical production in s o h
superoxide radicals and transition-metal ions . .
................
References . . . . . . .
..
. ........................

Chapter 12. Free radicals and connective tissue damage
M.S. Baker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I . Introduction . . , .
............................
2. The release of reactive oxygen intermediates (ROls) during inflammation . . , . , . . ,

3. Composition and organization of connective tissues (with special reference to articular
cartilage) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Connective tissue injury by oxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. I . Direct connective tissue macromolecule degradation by oxidants . . . . . . . . . ,
4.1.1. Hyaluronic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.2. Proteoglycan . . . . . , . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.3. Other connective tissue macromolecules . . . . . . . . . . . . . . . . . . . . . ,

259
259
260
260
260
26 1
263
263
265
266
266
266
267
268
268
270
273
275
275
276

28 1

281
285
286
290
295
295
298

30 1
301
301
302
305
305
306
307
308


xv
Cellular oxidative injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1. Decreased biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.2. Altered macromolecular biosynthesis . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Indirect actions of oxidants which affect structure and function of connective
......................................
tissue . . . . . .
teases via the "cysteine switch" . . . . . . . . . . . . . . . .
..................
4.3.2. Inactivation of proteolytic inhibitors . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

...........
4.2.

309
309
311
312
312
314
316

Chapter 13. Free radicals in toxicology with an emphasis on
electron spin resonance investigations
R.P Mason and C.F Chignell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

319

1 . Introduction . . . .
..........................................
2 . Detection and iden
ation of free radicals in biological systems . . . . . . . . . . . . .
3 . Criteria for free radical toxicity .
....
.........................
4 . Formation of free radicals in biol
..............
4.1. One-electron enzymatic oxidation . . . . . . . . . .
.......
4.2. One-electron enzymatic reduction . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Light-dependent radical formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5. Spin trapping . . . . . .
......................................
5.1. ESR spectrum of the radical adduct of 'CC13 . . . . . . . . . . . . . . . . . . . . . .
5.2. Chlorpromazine . . . . . . . . . . . . . . . . .
.....................
6 . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
......
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
......

319
320
32 1
322
322
324
325
327
328
328
330
330

Chapter 14. Radical generation and detection in myocardial injury
B . Kalyanaraman. E.A. Konorev. J Joseph and J E . Baker . . . . . . . . . .

333

1. Free radicals in myocardial injury . . . . . . . . . . . . . . . . . . .
......

2 . Indirect evidence for the role of the oxy radicals . . . . . . . . . . . . . . . . . . . . . . . .
2.1, Cellular sources of oxy radical and isolated heart models . . . . . . . . . . . . . .
2.2. Interaction of calcium and oxy radicals . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Compartmentalization of radical reactions in the heart
................
................
4 . Detection of oxygen-derived free radicals . . . . . . . .
4.1. ESR technology . . . . . . . . . . . . . . . . . . . . . . . . . .
.............
4.2. Artifactual generation of free radical signals in
4.3. An alternative tissue processing technique . . . . . . . . . . . . . . . . . . . . . . . . .
5. Spin traps in myocardial ischemia and reperfusion injury . . . . . . . . . . . . . . . . . . .
5.1. Vasodilatory activity of spin traps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Spin trapping using DMPO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. PBN as the spin trap of choice in ischemia and reperfusion studies . . . . . . . .
5.4. Trapping of free radicals with PBN during myocardial ischemia and reperfusion
5.5. Detection of PBN adduct in coronary effluents during reperfusion . . . . . . . . .
5.6. Detection of PBN-OH adduct formed in a Fenton system . . . . . . . . . . . . . .
5.1. Solvent effects on ESR parameters of PBN/'OH . . . . . . . . . . . . . . . . . . . .
5.8. ESR parameters of PBN adducts formed during myocardial ischemia and
reperfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.9. GC-MS of derivatized PBN adducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.10. Protective effect of PBN on ischemic-reperfused myocardium . . . . . . . . . . . .

333
334
334
335
335
338

338
339
340
342
343
345
347
341
348
348
348
349
352
353


XVI

6 . Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 15. Free radical pathways in the inJlammatory response
PG. Winyard. C.J Morris. l!R . Winrow, M . Zaidi and D.R. Blake'

...

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . The generation of free radicals in inflammatory diseases . . . . . . . . . . . . . . . . . . .
2.1. Activation of NADPH oxidase and myeloperoxidase systems . . . . . . . . . . . .

2.2. Uncoupling of the xanthinc dehydrogenase system . . . . . . . . . . . . . . . . . . .
2.3. Uncoupling of mitochondria1 and endoplasmic reticulum electron-transport chains
2.4. Non-enzymatic reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Inhibition of free radical pathways in inflammation . . . . . . . . . . . . . . . . . . . . . . .
3 . I . Enzymatic removal of oxygen radicals . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Chelation of catalytic iron and free radical scavengers . . . . . . . . . . . . . . . . .
4 . Pathways involving free radicals as second messengers in inflammation - some topical
examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Vasodilation . . . .
................
..
....
4.2. Fibrosis . . . . . . .
..
....
4.3. Gene transcription . .
..................................
4.3.1. Nuclear factor
..................
4.3.2. Activator protein 1 (AP-I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.3. Haem oxygenasc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.4. Tyrosine phosphatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.5. Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 . Free radical pathways of macromolecular damage and tissue destruction - some topical
cxamples . .
....................................
.....
5.1. Inactivation of scrpins and activation of latent metalloproteinases in pulmonary
emphyscrna and rheumatoid arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Bone rcsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.3. Oxidative modification of low-density lipoprotein in atherosclerosis and
rheumatoid arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4. Oxidative DNA damagc as a cause of ageing, cancer and autoimmunity . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rcfercnccs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

354
355
355

36 1
361
361
362
362
364
365
365
365
366
367
367
367
369
369
370
370
371

371
372
372
374
375
376
379
370


PART I

Chemical and Biochemical Aspects


This Page Intentionally Left Blank


C.A. Rice-Evans and R.H. Burdon (Eds.), Free Radical Damage and its Control
6 1994 Elsevier Science B.V All rights reserved

3

CHAPTER 1

Chemistry of iron and copper
in radical reactions
W.H. KOPPENOL
Departments of Chemistry and Biochemistq, and Biodynamics Institute,
Louisiana State University, Baton Rouge, LA 70803, USA


Abbreviations
adp
amp
atp
dtpa
edda
edta

adenosinediphosphate
adenosinemonophosphate
adenosinetriphosphate
diethylenetriamine-N, N, M,M'.MIpentaacetate
ethylenediamine-N, A"-diacetate
ethylenediamine-N, N, M ,M-tetraacetate

gtp
hedta
nta
phen
PQ'+
utp

guanosine triphosphate
(N-hydroxyethy1)ethylenediamineN, M,M-triacetate
nitrilotriacetate
1,lO-phenanthroline
paraquat radical
uridine-5'-triphosphate


I . Introduction
In a comparison of several elements it was shown by George[1] in 1965 that
oxygen is unique because its reduction by organic compounds is favourable and
because the reaction product, water, is not toxic. As such oxygen is the best
element on which to base life. Yet oxygen also plays an important role in free
radical biology in that it is also essential in the initiation of, and greatly amplifies,
damage to biomolecules. Oxyradicals have been implicated in numerous diseases
and disorders.
Iron and copper catalyse the formation of oxyradicals. Three reactions are
relevant in this context: (1) Autoxidation of metal complexes may yield the
superoxide radical which by itself is not very reactive, but is a precursor of more
reactive radical species. (2) The one-electron reduction of hydrogen peroxide the Fenton reaction - results in hydroxyl radicals via a higher oxidation state of
iron [2]. (3) A similar reaction with organic peroxides leads to alkoxyl radicals,
although a recent report alleges that hydroxyl radicals are also formed [3]. There
is a fourth radical, the formation of which does not require mediation by a
metal complex. This is the alkyldioxyl radical, ROO', which is formed at a


A

TABLE 1
Reduction potentials of oxyradicals”
Inorganic
Couple

Organic
E“‘W 7 ) (V)

Couple


EO’(PH7) (V)

One-electron reduction potentials
0210;
-0.33
~

HOiIH202

1.07

ROO’IROOH

1.o

‘OHIH20
H202I’OH, H2O

2.3 1
0.32

RO‘IROH
ROOHIRO’, H20
bis-allylic’lbis-allylicH

1.7
1.9
0.6

Two-electron reduction potential

H20212H20

1.32

ROOHIROH, H20

1.8

”

Data for H- and 0-containing radicals were taken from a recent compilation [4]. The values for the
carbon containing radicals arc estimates derived from bond-dissociation enthalpies [5]. The value
for E”’(ROO’IRO0H) has recently been confirmed experimentally [6].

nearly diffusion-controlled rate from an alkyl radical and dioxygen. As shown
in Table 1, the hydroxyl, the alkoxyl and the alkyldioxyl radical are oxidizing
species. For a quantitative description one needs to know rate constants for
the formation of these radicals and for their subsequent reactions in order to
determine which reaction will dominate under physiological conditions. This
requires inter aha knowledge of the precise chemical composition of the cell;
we are still a long way from this goal.
In principle, there are three mechanism for damage: a single reaction, a chain
reaction, or a branching mechanism. A single reaction is not likely to lead to
extensive damage. However, when a radical like the hydroxyl radical reacts with
a biomolecule, another radical is created. A chain reaction ensues that stops
only when a radical reacts with another radical or with a transition-metal ion.
For extensive damage to occur it might be necessary that branching occurs.
For instance, superoxide production may lead to lipid peroxidation; alkenals
formed as products of that process are substrates for xanthine oxidase [7]; more
superoxide is produced, and a new chain reaction is started. Similarly, during

ischaemia atp is converted to hypoxanthine [8]. Any iron that was tightly bound
to atp is now bound elsewhere, possibly in a more “open” complex. Since
rate constants increase with a decrease in coordination number (see below),
such an iron complex is likely to be more reactive.
Given the concentration of “reaction sites” in vivo and the magnitude
of the relevant rate constants it is not possible to intercept effectively the
hydroxyl, alkoxyl or alkyldioxyl radicals [9]. For less reactive compounds,


5

such as hydrogen peroxide and superoxide, nature has developed enzymes to
dispose of them. The strategy adopted by nature is threefold: (1) interception
with superoxide dismutase and proteins such as catalase and glutathione
peroxidase that react with hydrogen peroxide and small alkyl hydroperoxides;
(2) repair of water-soluble biomolecules with glutathione, and ( 3 ) inhibition
of lipid peroxidation with vitaminE, which might [lo], or might not [ l l ] , be
regenerated by vitamin C. Part of the defence mechanism may be that radicals
are interconverted to superoxide via the glutathione radical; superoxide, acting
as a radical sink, is subsequently scavenged by superoxide dismutase [12]. The
overall energetics of these reactions are extremely favourable [ 131.
Excess amounts of transition metals, in particular iron and copper, are toxic.
For instance, it has recently been suggested that excess iron plays a role in
heart disease [ 14,151. Transition-metal ions are sequestered by proteins: iron in
ferritin and transferrins [ 161, and copper in caeruloplasmin. However, a small
concentration of low-molecular-weight complexes is likely to be present at all
times because of transfer of metals from storage proteins to metalloproteins,
and from the turnover of these proteins. Under oxidative stress this pool of iron
is increased due to reductive mobilisation and destruction of ferritin [ 17-25].
Desferrioxamine, being a stronger complexing agent than the naturally occurring

ligands, chelates iron and prevents oxidative injury in hepatocytes [26]. The
precise nature of the low-molecular-weight complexes present in viva is not
known with certainty. Evidence has been presented that iron bound to atp,
amp [27], gtp [28] and possibly citrate [29] may be present in tissues in the
micromolar range [21]. No information is available on copper.
Some metal- (especially copper) complexes catalyse the dismutation of superoxide at rates that compare favourably with catalysis by superoxide dismutase.
One could therefore argue that the presence of such complexes in viva might be
beneficial. There are, however, additional considerations: (1) such metal complexes may also reduce hydrogen peroxide, which could result in the formation
of hydroxyl radicals, and (2) it is extremely likely that the metal will be displaced
from its ligands (even when those ligands are present in excess), and becomes
bound to a biomolecule, thereby becoming less active as a superoxide dismutase
mimic. As an example, copper binds well to DNA and catalyses the formation
of hydroxyl radicals in the presence of hydrogen peroxide and ascorbate [30].
Both the reduction of superoxide and that of hydrogen peroxide appear to be
inner-sphere reactions; that is, a ligand of the metal ion has to be replaced by
superoxide or hydrogen peroxide for the reaction to take place. For superoxide
this involves overlap between a metal d-orbital and its own accessible T * orbital.
Reduction of hydrogen peroxide involves electron transfer to an empty 0 * orbital
which is not very accessible [31]. Thus, reductions of hydrogen peroxide are
generally slower than those of superoxide. The reductions of alkylhydroperoxides
are even slower, due to steric hindrance [32,33].


6

This review is concerned with the quantitative aspects of metal-catalysed
oxyradical reactions. As such one will find discussions of structures of metal
complexes, rate constants and reduction potentials, not unlike our review of
1985 [34]. Two areas related to the role of transition metals in radical chemistry
and biology have been reviewed recently; these are the metal-ion-catalysed

oxidation of proteins [35] and the role of iron in oxygen-mediated toxicities [36].
These topics will not be discussed in detail in this review. Related to this
work is a review on the role of transition metals in autoxidation reactions [37].
Additional information can be obtained from Afanas'ev's two volumes on
superoxide [38,39]. This subject is also treated in a more general and less
quantitative manner by Halliwell and Gutteridge [40].

2. Autoxidation reactions
2.1. Oxygen

It is well known that oxygen does not react directly with organic molecules
because of spin restrictions: ground-state oxygen is a triplet molecule, and
most organic molecules are in the singlet state (see ref. [37]). In the past we
have explained this phenomenon qualitatively in the following fashion: Prior
to a reaction, overlap is necessary between orbitals of the reactants; this can
only occur rapidly between half-filled orbitals (of the proper symmetry) which
organic molecules generally do not have [3 11. Similarly, triplet oxygen and
organic radicals react at near difhsion-limited rates because both have half-filled
orbitals. Often, authors of reviews try to explain the singlet and triplet states of
oxygen with a diagram as depicted in the middle column of Fig. 1 [36,40,41].
Such primitive representations have been criticised [42] because, for one, it does
not show that there are 6 ways in which 2 electrons can be distributed over
two orbitals. There are 6 different microstates that belong to three different
energy levels: ground state (3C,) oxygen is three-fold degenerate, the 'A,state
is two-fold degenerate, and the 'CH state is not degenerate. A correct orbital
occupation-energy diagram, taken from refs. [42,43], is depicted in the righthand
column of Fig. 1. In the lowest energy state, 'Xi,the two electrons move in
mutually perpendicular planes, minimizing repulsion, with parallel spin. In the
highest state, 'Xi,which because of its extremely short lifetime is biologically
not relevant, the electrons move in the same plane with paired spins, while in

the 'A, state both electron orientations occur. Thus, this state can undergo both
two-point additions and single-point attachments [42,43]. A well-known twopoint reaction is the addition of A, to a double bond. Single point attachments
do not lead to a reaction with singlet organic molecules, but would allow 'A,
to react like 3X; oxygen with radical species.

'


State

Orbital Assiament
Primitive

’cg+ Ox@,
1

A,

oxoy

7

Correct

oxoy oxoy
+

oxoy
- 0,
oy

@,ay
- ox0,

Fig. 1. Spin-orbital diagram of the different states of oxygen. The x and y refer to the two perpendicular
antibonding orbitals of oxygen. On the right this diagram depicts the real wave-functions for the
lowest electronic states. From ref. [43].

-A

2.2. Thermodynamics

At low pH the iron(I1) ion is stable with respect to oxidation, due to the high
value of the reduction potential of the Fe3’/Fe2+ couple, 0.77V versus the
normal hydrogen electrode. Above pH 2.1, Fe(III), but not Fe(II), hydrolyses,
which results in a reduction potential that decreases with 59 mV per
pH unit to a value of 0.48V at pH7. This applies only to very dilute
solutions, since iron(II1) hydroxide precipitates above pH 3. Complexation
by aminopolycarboxylates, such as edta, which provide mainly oxygen as
donor atoms, also reduces the reduction potential, generally to a value
near 0.1 V [34]. The standard reduction potential of the oxygen/superoxide
couple is -0.33V (see Fig. 2), independent of pH[44,45]. Although in such
an instance the one-electron reduction of oxygen by such metal complexes
is thermodynamically unfavourable by approximately 10kcal/mol, the reaction
proceeds because the product, superoxide, disappears by disproportionation. In
contrast, reduction by two electrons to hydrogen peroxide is favourable: the
Gibbs energy change is -8.1 kcal per two moles of Fe(I1) edta, as calculated
from E0’(02/H202) = 0.305 V at pH 7 [44] and the reduction potential of the
Fe(II1)-/Fe(II)-edta couple of 0.12 V [46].
It has been suggested that a (dioxygen)iron(II), or “perferryl” I complex,
a likely intermediate in the autoxidation of iron(II), could abstract an

allylic hydrogen and initiate lipid peroxidation [48]. Such complexes are weak
oxidants at best, as has been shown before[49] and, with the exception of
iron(I1) edta [50], have not been observed. Constraints on the reduction potential

’

The name “perferryl”, indicating an oxidation state beyond that of ferry], iron(IV), is not
recommended by the current IUPAC guidelines for the nomenclature of inorganic chemistry [47].
This name would only be defensible if both oxygen were attached to the iron, which they are not.
The use of this misleading name should be discontinued.


8

!

pH 7
po,= 1 atm

HO’

t

03.

0
0

w
c

-1

-2
-2

0

-1

n

Fig. 2. Oxidation state diagram of oxygen at pH 7 at otherwise standard conditions ( 1 molal
concentrations, 1 atm for gases). The x-axis gives the oxidation state, the y-axis the product
of reduction potential and oxidation state. As such the slope represents the reduction potential.
Adapted from ref. [4]. A compound that lies above a line joining its neighbours is unstable with
respect to disproportionation, as is the case for superoxide and hydrogen peroxide. The line from
hydrogen peroxide to the middle of the water-hydroxyl line represents the one-electron reduction
potential of the couple H202I’OH, H 2 0 .

Eo’(HLFe1102/HLFe111,
H202) come from the following thermodynamic cycle and
considerations. If such a complex were to be an initiator of oxyradical damage,
one might expect that approximately 1YOof the low-molecular-weight iron(I1) be
complexed to oxygen at a cellular oxygen tension of, say, 0.01 atm. This requires
a standard Gibbs energy change of Okcal/mol. In the following sequence of
reactions HL represents a ligand with a covalently bound hydrogen:
HLFe(II)02
0 2

+ HLFe(I1) + 0 2


+ 2H’ + 2e-

-+

H202

HLFe(I1) -+ HLFe(II1) + eHLFe(II)02 + 2Hf + e-

-+

(AGO’ = 0 kcaVmol),

(1)

(Lo’= 0.305 V),

(2)

-0.1 V),

(3)

(EO’

=

HLFe(II1) + H202

(EO’


=

0.2 V).

(4)

The reduction potential of 0.2V for Reaction (4) at pH7 depends very
much on the reduction potential of the Fe(III)/Fe(II) couple, Reaction ( 3 ) .
The 0.1 V assumed here for that half-reaction is close to that of various ironaminopolycarboxylate complexes. The uncertainty in our reduction potential
for Reaction (4) is estimated at 0.2V The abstraction of a doubly allylic
hydrogen is estimated to require a reduction potential of 0.6V (see Table l),