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Preface

Through the ages man has sought to harness the knowledge of exercise; interest
in exercise science dates back at least to ancient Greece. Today, exercise is seen
as not merely a leisure activity, but as an effective preventive and therapeutic
tool in medicine. The study of exercise physiology and biochemistry has had a
revolutionary overall impact on biomedical research, and exercise has been used
as a model for studying the response of physiological regulatory mechanisms to
stress. At the same time, advances in oxygen free radical biochemistry have lured
exercise biochemists to study the effects of the increased oxygen consumption
that accompanies exercise. The first reports in this field, published in the early
1970s, indicated that strenuous physical exercise might cause oxidative lipid
damage in various tissues. Since then, a considerable body of research has accu-
mulated concerning the effects of exercise, nutrition and training on indices of
oxidative stress and antioxidant responses in various tissues. Most studies sup-
port the contention that during strenuous exercise, generation of reactive oxygen
species (highly reactive, partially reduced metabolites of oxygen) is elevated to a
level that overwhelms tissue antioxidant defence systems. The result is oxidative
stress. The magnitude of the stress depends on the ability of the tissues to detoxify
reactive oxygen species; i.e., antioxidant defences. Endurance training enhances
such defence in various tissues, especially in skeletal muscle and heart. Anti-
oxidants produced by the body act in concert with their exogenous (mainly diet-
ary) counterparts to provide protection against the ravages of reactive oxygen as
well as nitrogen species.
Exercise and Oxygen Toxicity was first published in 1994. The purpose of this
multiauthor volume — the first of its kind — was to examine different aspects
of exercise-induced oxidative stress, its management, and how reactive oxygen
may affect the functional capacity of various vital organs and tissues. Remarkable
interest of the readers and favourable critical reviews published in leading jour-
nals provided the impetus to put together an enlarged second edition. We started
to accomplish that goal. After two years of tireless effort of many, the current
volume was ready to go the press. This volume was over double in size of the ori-
ginal edition. Key related issues such as analytical methods, environmental fac-
tors, nutrition, aging, organ function and several pathophysiological processes
were thoroughly addressed. Leading experts provided unprecedented insight into
the understanding of the role of reactive species and antioxidants. The combina-
tion of these properties makes this volume an authoritative treatise.
During the course of review of the finalized manuscripts at the publishing
house it was brought to our attention that the structure and contents of this
volume more closely resembled a Handbook than just a second edition of Exer-
vi Preface

cise and Oxygen Toxicity. This view was shared by many of our colleagues and
peers, and thus we decided to name this volume as the Handbook of Oxidants
and Antioxidants in Exercise.
Since Exercise and Oxygen Toxicity was pubhshed, interest in exercise, reactive
species and the possible role of endogenous and supplemented antioxidants has
soared. Search of the PubMed database of the National Library of Medicine
using a combination of the keywords "exercise" and "antioxidant" show that the
number of research reports in 1997 is more than double of that in 1994. This
handbook is therefore a timely publication. It is relevant to all those who have
interest in biomedical sciences and is designed to be intelligible to a general
scientific audience.
We are dehghted to be involved in this project. The excellent editorial assistance
of Dr. Savita Khanna and the outstanding contribution of authors are gratefully
acknowledged. We hope that this volume will contribute to the further develop-
ment of this important late-breaking field of research.

Chandan K. Sen
Lester Packer
Osmo Hdnninen
 Vll

Contents

Preface v

Part I: Introduction to free radicals

1. Free radical chemistry
K.-D. Asmus and M. Bonifacic 3

Part II: Reactive species in tissues

2. Exercise and oxygen radical production by muscle

MJ. Jackson 57
3. Exercise and xanthine oxidase in the vasculature:
superoxide and nitric oxide interactions
C.R. White, IE. Shelton, D. Moellering, H. Jo, R.R Patel and
V Darley-Usmar 69

Part III: Oxidative stress: Mechanisms and manifestations

4. Chemical bases and biological relevance of protein oxidation

O. Tirosh and A.Z. Reznick 89
5. Lipid peroxidation in healthy and diseased models:
influence of different types of exercise
KM. Alessio 115
6. Metal binding agents: possible role in exercise
R.R. Jenkins and J. Beard 129
7. The role of xanthine oxidase in exercise
Y. Hellsten 153
8. Acute phase immune responses in exercise
JG. Cannon and JB. Blumberg 177
9. Oxidative DNA damage in exercise
A. Hartmann and A.M. Mess 195
viii Contents

Part IV: Antioxidant defenses

10. Physiological antioxidants and exercise training

S.K. Powers and C.K. Sen 221
11. Superoxide dismutases in exercise and disease
K. Suzuki, H. Ohno, S. Oh-ishi, T. Kizaki, T. Ookawara, J. Fujii,
Z. Raddk and N. Taniguchi 243
12. Antioxidants and physical exercise
C.K. Sen and AH. Goldfarb 297

Part V: Nutrition

13. Dietary sources and bioavailability of essential and nonessential

antioxidants
E.A. Decker and P.M. Clarkson 323
14. Vitamin E
M.G. Traber 359

Part VI: Cellular and molecular mechanisms

15. Biological thiols and redox regulation of cellular signal transduction

pathways
C.K. Sen 375
16. Regulation and deregulation of vascular smooth muscle cells by
reactive oxygen species and by a-tocopherol
A. Azzi, D. Boscoboinik, N.K Ozer, R. Ricciarelli and E. Aratri . . . . 403

Part VII: Analytical methods

17. Oxidative stress indices: analytical aspects and significance

D. Han, S. Loukianoff, and L. McLaughlin 433
18. Noninvasive measures of muscle metaboHsm
T. Hamaoka, KK McCully, T. Katsumura, T. Shimomitsu and
B. Chance 485

Part VIII: Environmental factors

19. Air pollution and oxidative stress

D.M. MeacherandD.B. Menzel 513
Contents ix

20. Risk of oxidative stress at high altitude and possible benefit of

antioxidant supplementation
I. M. Simon-Schnass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555
2 1. Oxidants in skin pathophysiology
S.Weber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579

Part IX: Organ functions

22. Muscle fatigue: mechanisms and regulation

M.B. Reid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
23. Oxidative stress in muscular atrophy
H.Kondo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 1
24. Protection against free radical injury in the heart and cardiac
performance
D. K. Das and N Maulik . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 655
25. Exercise-induced oxidative stress in the heart
L.L.Ji . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
26. Influence of exercise-induced oxidative stress on the
central nervous system
S. M. Somani and K. Husain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713

Part X: Aging

27. Oxidants and aging

K. B. Beckman and B. N Ames . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755
28. Caloric restriction, exercise and aging
R.JM. McCarter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
29. Oxidative stress and the pathogenesis of sarcopenia
M.E. Lopez, T A . Zainal, S.S. Chung, J M . Aiken and
R.Weindruch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 1
30. Molecular mechanisms of oxidative stress in aging: free radicals,
aging, antioxidants and disease
M. Pollack and C. Leeuwenburgh . . . . . . . . . . . . . . . . . . . . . . . . . . 88 1

Part XI: Disease processes

31. Oxidative stress, antioxidants and cancer

M.Gerber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 927
X Contents

32. Alcohol and oxidative stress

as. Lieber 951
33. Cigarette smoking as an inducer of oxidative stress in relation to
disease pathogenesis
G.G. Duthie, J.R. Arthur and S.J. Duthie 977
34. Regulation of inflammation and wound healing
J.J. Maguire 995
35. Oxidative stress induced by the metabohsm of medical and
nonmedical drugs
M Paolini and G. Cantelli-Forti 1021
36. The paradoxical relationship of aerobic exercise and the oxidative
theory of atherosclerosis
R. Shern-Brewer, N. Santanam, C. Wetzstein, JE. White-Welkley,
L. Price and S. Parthasarathy 1053
37. Claudication, exercise and antioxidants
P.V.Tisi and C.P Shearman 1069
38. Exercise and oxidative stress in diabetes mellitus
D.E. Laaksonen and C.K. Sen 1105
39. Exercise induces oxidative stress in healthy subjects and in chronic
obstructive pulmonary disease patients
J. Vina, E. Servera, M. Asensi, J. Sastre, F.V. Pallardo, A. Gimeno,
L. Heunks, P.N.R. Dekhuijzen and J. A. Ferrero 1137
40. Hypoxia, oxidative stress and exercise in rheumatoid arthritis
S. Jawed, S.E. Edmonds, V. Gilston and D.R. Blake 1147

Index of authors 1189

Subject index 1191

 Parti

Introduction to free radicals

This Page Intentionally Left Blank
 ©2000 Elsevier Science B.V. All rights reserved.
Handbook of Oxidants and Antioxidants in Exercise.
C.K. Sen, L. Packer and O. Hanninen, editors.

Part I • Chapter 1

Free radical chemistry

Klaus-Dieter Asmus^ and Marija Bonifacic^
^Radiation Laboratory, University of Notre Dame, Notre Dame, IN 46556, USA
'^Ruder Bockovic Institute, Department of Physical Chemistry, RO. Box 1016, 10001 Zagreb, Croatia

1 INTRODUCTION
2 PROPERTIES AND DETECTION OF FREE RADICALS
2.1 What is a free radical?
2.2 How fast are free radical reactions?
2.3 Detection of free radicals by time-resolved optical spectroscopy
2.4 Detection of free radicals by electron spin resonance
2.5 Spin trapping
2.6 Product analysis
2.7 Reduction potentials
2.8 Where to find rate constants, ESR splittings, reduction potentials, etc.
3 OXYGEN RADICALS
3.1 The hydroxyl radical
3.2 Peroxyl radicals
3.3 Reversible oxygen addition
3.4 Alkoxyl radicals
3.5 Phenoxyl radicals
3.6 Semiquinone radicals
3.7 Superoxide (02*" / HOs*)
4 SULFUR-CENTERED FREE RADICALS
5 CONCLUSION
6 SUMMARY
7 ACKNOWLEDGEMENTS
8 ABBREVIATIONS
9 NOTES
10 REFERENCES

1 INTRODUCTION

It is well accepted these days in the scientific community that free radicals par-
ticipate in many biological processes, often in a quite decisive manner [1—8].
This clearly emerges from the still rapidly increasing number of studies on this
subject and the very important fact that such investigations are not restricted to
the views of just one particular group of researchers. Free radical studies in bio-
logical systems embrace practically all fields from fundamental chemistry to
applied clinical studies these days and have proved to be particularly successful
when scientists of the various fields engage in collaborative work and extensive
interdisciplinary discussions.
In living systems, including humans, the presence of free radicals is something
very natural. The decision of nature to provide the enzyme superoxide dismutase
4 Part I: Introduction to free radicals

(SOD), for example, clearly hints on the existence of the superoxide radical, O2* ,
- considered to be one of the most prominent radical species in oxygenated bio-
logical systems - and it also shows the obvious need of cellular systems to regu-
late its concentration [9—15]. While this serves as an example that a "too much"
of a particular radical species is not in the interest of a biological system there
are, on the other hand, also observations where a "too little" of radicals seems
equally unwanted. For example, healthy cervix tissue has been reported to con-
tain quite a measurable radical concentration whereas a cancerous tissue is
almost completely devoid of radical species [16]. In general, however, it seems
that the majority of malignant disturbances is associated with an increase in free
radical concentration [17]. Free radicals are undoubtedly involved in many
degenerative processes including aspects of aging, cancer, cardiovascular dis-
eases, arteriosclerosis, neural disorders, skin irritations and inflammations.
Reperfusion injuries, e.g., in the aftermath of surgery, are also typically character-
ized by an enhanced formation of free radicals [18]. Actually, any metabolism
involving redox active centers is, per se, a possible source of continuous free radi-
cal production. This certainly allows the extrapolative conclusion that any stress
situation, including exercise, carries the potential of excess free radical produc-
tion associated with all the chemical consequences arising thereof
Most of the radicals relevant in biological systems are either derived from or
associated with the presence of molecular oxygen, in particular, the already men-
tioned superoxide anion (O2*) and peroxyl radicals (ROO*). Other oxygen-
centered and highly reactive radical species are hydroxyl (*OH) and oxyl radicals
(RO*). Their biological significance is not in doubt but somewhat more in the
debate than that of 02*^ and ROO*. Of course, it is not only oxygen-centered
free radicals which are of importance and interest. Many others, generated both
from endogenous as well as exogenous substrates are also of significance. A par-
ticularly relevant example is the thiyl radical, RS*, which is the one-electron
redox intermediate between thiols and disulfides, two vital classes among the
biologically abundant substrates [19,20].
The action of free radicals is generally determined by their chemical reactivity
and the availability of a suitable reaction partner in the vicinity of their produc-
tion site. In some cases such a radical-molecule interaction may directly lead to
a biological damage in a few or even just one reaction step [7]. Thus, an •OH
reaction with the sugar moiety of DNA can result in a strand break due to a radi-
cal specific phosphate cleavage reaction. Such a singular event does, however,
not necessarily imply cell death as there are enzymatic and chemical, possibly
even radical associated repair mechanisms. Accumulation of hazardous com-
pounds as a result of free radical reactions is often the reason for long-term
effects. In this case it may even be difficult to prove the free radical's responsibil-
ity It should also be pointed out that the appearance of free radicals does not
automatically imply their direct participation in the disturbance of vital biologi-
cal functions. In other words, free radical reactions are not necessarily the cause
of disorders but may equally well just be a consequence thereof In this case free
Chapter 1: Free radical chemistry 5

radicals and their reactions may, however, serve as useful markers.

Nature and modern medicine related science have provided us with mecha-
nisms and substrates to cope with free radicals by "deactivation". Enzymes such
as the above already mentioned SOD serve this function. Another important
group of compounds in this respect are the so-called antioxidants [22,23]. Most
prominent representatives in this respect are vitamin E (a-tocopherol) and vita-
min C (ascorbate) which are most effective free radical scavengers in the lipid
cell membrane and adjacent, more aqueous compartments, respectively. The
same function is assigned to many other redox-active compounds such as, for
example, thiols, carotenoids, quinones, etc.
Whenever a free radical reacts with a molecular compound it loses its identity
but at the same time it proliferates its general radical properties to a new radical
formed in this reaction. In order to assess the action of free radicals it is, there-
fore, necessary to know not only the properties of the initiating but also of all
subsequently generated species. The aim of the present article is, therefore, to elu-
cidate on all these aspects and to provide an understanding for the chemical basis
of free radical processes. In the first part of our essay we shall focus on the gener-
al properties of free radicals, their generation in chemical model systems, and
provide some basic information on the most common methods of their detection.
Later we shall focus on the identification and chemical properties of specific
groups or individual free radical species which are considered to be of potential
significance in biological processes. To find out whether the latter actually applies
is then a challenge for the biochemist, biologist and medical researcher. It must,
of course, always be remembered that a chemical "test tube" or in vitro experi-
ment can only provide the basic information what a free radical can and will do
chemically when meeting a suitable reaction partner. Such knowledge is an abso-
lute prerequisite to understand a free radical's potential action. By no means
can an in vitro experiment, however, substitute for the in vivo situation where
many more parameters need to be taken into the consideration.

2 PROPERTIES AND DETECTION OF FREE RADICALS

2.1 What is a free radical?

A free radical is, per definition, a molecule or just a single atom with an unpaired
electron, conventionally symbolized by a radical dot "*". Examples, extremes
from the molecular size point of view, would be a DNA radical (4' in sugar moi-
ety) and a bromine atom.
phosphate - O - CH2 base

7
O— phosphate ^^^'^
6 Part I: Introduction to free radicals

Both are species of interest in certain biological processes. The DNA radical is
generated, for example, by reaction of an •QH radical with this vital macro-
molecule in any cellular system exposed to ionizing radiation and is a direct pre-
cursor for a potential strand lesion [7]. The bromine atom, on the other hand, is
formed en route of reductive degradation of 1,2-dibromoethane, a well-known
toxin [24-30].
What means "unpaired" electron? Typically, any electron seeks the association
with another of its kind, so that they can pair up their spins in a system of lower
energy A spin is the rotational motion of an electron which can attain two direc-
tions and, viewed in a simplified picture, sort of represent a right handed (or
"up") and a left handed (or "down") version. Two radicals "shaking hands", i.e.,
teaming up two individually provided unpaired electrons of opposite spin, form
a new bond with this process:

T + i ^ n (1)

A chemical example would be the combination of two bromine atoms which

results in the formation of the Br-Br a-bond:
Br*^ + Br*^ -^ Br - Br (with the spins t i in the bond) (2)

Generally, any even number of electrons in a molecule arranges in "up"-"down"

spin coupled pairs, both in bonds or as nonbonding electron pairs. Usually this
is an energetically quite favorable process. Sometimes, however, it may be more
economic from the energy point of view that two electrons remain uncoupled,
even within the same molecule. In this case the two unpaired electrons attain
the same spin direction and the chemist talks about a "triplet state" or, if they
are located at different atoms, a biradical. A most important example for this
situation is the molecular oxygen which we generally view as a molecule with a
double bond (a). The two electrons of its p-bond seem, however, to some extent
separated and may thus act as if there were two centers providing one unpaired
electron each (b):

0 = 0 (a) ^•O-O*^ (b)

The presence of unpaired electrons or unpaired spins has a most important con-
sequence, namely the generally very high reactivity of such species.^ This, of
course, reflects the genuine desire of the radical species to attain the most favor-
able energetic state by coupling its unpaired spin with that of an opposing sign
in a new bond between two spin carrying atoms. Accordingly, most radical-
radical and also many radical-molecule reactions occur as soon as the two react-
ing species meet each other. Such a reaction is, as we say, a diffusion controlled
process. The time it actually takes for completion of any chemical reaction
depends on two parameters, a reaction specific rate constant (which attains its
Chapter 1: Free radical chemistry 7

highest value for a diffusion controlled reaction) and the concentration of the
reacting species. In biological systems the free radical concentration is usually
very low and rarely acquires values high enough so that a radical-radical reaction
could beat a reaction of a radical with a suitable molecular partner. The latter
are typically present at much higher concentration in the near-by vicinity
In order to illustrate this, let us discuss a simple example. Carbontretrachlo-
ride, CCI4, known as a potent liver toxin, is readily reduced by the enzymatic
P-450 system of the endoplasmic reticulum [31]. In the presence of oxygen this
results in the formation of CCI3OO*, i.e., trichloromethylperoxyl radicals as an
important intermediate in the CCI4 degradation process (further mechanistic
details on peroxyl radical decay will be presented later in this article). Its steady
state concentration is, however, extremely small and only few research groups
with highly sensitive equipment and great experience have been able to actually
detect this radical in vivo [32]. It is probably even lower or, at most, comparable
to the steady state radical concentration of ^ 1 pM (^10"^^ M) prevailing in sys-
tems which are subjected to y-irradiation in an average research ^^Co-source. In
the absence of any suitable reaction partner the above peroxyl radicals have,
indeed, no other chance than to react with each other:

CClsOO* + CClsOO* -> products (2k = 2 x 10^ M ^ s"^) [33] (3)

The half-life for such a so-called second order process is mathematically given by
an equation which includes the rate constant (for this kind of second order pro-
cess defined as "2k") and the radical concentration (in molar units) [34]:

ti/2 = 1 / {(2k) X [CCIBOOT } = 1 / {2 X 10^ X 10"^^} = 5 000 s (I)

On the other hand, if a scavenger such as vitamin E (a-tocopherol) was around in

just nanomolar (10^ M) concentration the following reaction would successfully
take over:

CClsOO* + Vit E ^ products (k = 5 x 10^ M~^ s^) [35] (4)

When the concentration of the molecular reaction partner (Vit E) significantly

exceeds that of the radical (CCI3OO*) the reaction becomes of pseudo-first order
(reflecting the fact, that the concentration of the excess component hardly
changes during the course of the reaction). For such a condition, the mathemati-
cal equation attains a slightly different form, the most important difference being
that it contains the concentration of the molecular reaction partner and not that
of the radical:

ti/2 = In 2/ {k X [Vit E] } = 1 / {5 x 10^ x 10 ^} = 1.4 s (II)

By comparison, these two half-lives show that the scavenging of the CCI3OO*
8 Part I: Introduction to free radicals

radical by Vit E is several orders of magnitude faster, i.e., more probable to occur
than the mutual radical-radical deactivation.
While this example, from the biological point of view, pertains more to the lipid
phase (where Vit E is preferentially located) a similar system may be considered
for the aqueous compartments. Thiyl radicals, RS*, although not oxygen-centered
but nonetheless equally frequent and important as any oxygen-centered free radi-
cal in cellular systems, readily react with the water-soluble ascorbate (Vit C), as
formulated in the following equation for the thiyl radical, GS*, from the probably
most abundant thiol, glutathione (GSH):

OS* + ascorbate (AH) -^ GS" + A*" (5)

With a rate constant of k = 6 x 10^ M^^ s"^ [36] and an estimated Vit C concen-
tration (e.g., in liver tissue) in the low millimolar range (10~^ M) the half-life of
this process, calculated according to eq.(II), assumes a value of about 1 ms. For
a second order thiyl radical recombination process (2k6 ^ 10^ M ^ s~^) [37] to
compete, i.e., to occur with a comparable half-life, a GS* concentration of about
1 mM would be necessary (calculated with eq. (I)). Such a high steady-state radi-
cal concentration is, however, pretty unrealistic in any biological system.

GS* + GS* -> GSSG (6)

Therefore, any radical-radical reaction is extremely unlikely to occur and the dis-
cussion of radical reactions in biological systems should accordingly always be
checked first with respect to possible radical-molecule processes. Even if the lat-
ter are slow they may still benefit from the usually considerably higher concentra-
tion of the molecular reaction partner as compared to the free radical concentra-
tion.

2.2 How fast are free radical reactions?

As mentioned above most radical reactions are essentially controlled just by the
diffusion of the reacting species. In liquid environment this typically results in
rate constants within the order of 10^ M ^ s~^ with the individual value depend-
ing on the size and structure of the respective species, on solvation effects, and
also on the viscosity of the environment. One intrinsic parameter lowering this
value could be an internal stabilization of the free radical, meaning delocaliza-
tion of spin and electron density into the existing regular bonds or to atoms of
particularly high electron affinity. Often it is feasible to characterize such radicals
by resonance structures, especially when aromatic TT-systems are involved. For
example, phenoxyl type radicals (structurally similar to the chromanoxyl radical
obtained upon oxidation of Vit E) mostly behave as oxygen-centered radicals (a)
but certain reactions such as di- and oligomerizations can only be understood
in terms of their carbon-centered resonance forms (b) and (c) [38]:
Chapter 1: Free radical chemistry

°^ °=0
(a) (b) (c)

Sometimes such resonance structures are so stable that the radical becomes more
and more persistent and may, in extreme cases, even be bottled. At the same
time the reactivity towards other radicals and molecular substrates becomes low-
er and lower, and rate constants accordingly diminish, possibly by many orders
of magnitude.
A second parameter which influences the rate of a radical reaction pertains to
radical-molecule reactions:

R* + A- B -^ R - B + A* (7)

In this case, the initial radical R* succeeds in pairing up its lonesome electron, a
process certainly beneficial for a high rate constant. At the same time, however,
it is necessary to break the molecular A - B bond, and this is a process which
requires energy and is, therefore, of adverse influence on the overall rate of reac-
tion. In fact, the complete assessment of the kinetics of such processes must
also take into account the energy contents of the product species and, in particu-
lar, of the transition state in going from the educt to the product system. This
transition state, in which (with reference to the above example) R* has associated
already with A - B but A* has not left yet (i.e., [R...B...A]*, has, per definition,
always the highest energy content along the reaction trajectory. The energy differ-
ence between the educts and the transition state is the so-called activation energy,
and this is essentially the parameter which decides on the overall rate constant
of a reaction. For a typical diffusion controlled process the activation energy is
so small that it is essentially provided by the Brownian motion of the reacting
partners. However, if this activation energy becomes higher statistically fewer
and fewer of the reacting species provide the necessary internal energy to, lit-
erally spoken, push the reaction over the transition state mountain. Such reac-
tions are not any more diffusion but activation energy controlled. An example,
quite relevant and important from the biological point of view, would be an oxi-
dative cleavage of a C - H bond by, e.g., a trichloromethyl peroxyl radical:
I i
-C-H + CC1300* - ^ -C* + CCI3OOH (8)

Abstraction of a bisallylic hydrogen atom from linoleic acid, for example, has
been reported to occur with a rate constant on the order of 10^ M"^ s^ [39],
i.e., three orders of magnitude below the diffusion limit. This means that, despite
the strong activation these hydrogen atoms receive from the allylic double bonds
of this PUFA, statistically only one in about a thousand encounters actually leads