FREE RADICAL FORMATION
Atoms are most stable in the ground state.
An atom is considered to be "ground" when every electron
in the outermost shell has a complimentary electron that spins
in the opposite direction. By definition, a free radical is any
atom (e.g. oxygen, nitrogen) with at least one unpaired electron
in the outermost shell, and is capable of independent existence
(13). A free radical is easily formed when a covalent bond between
entities is broken and one electron remains with each newly formed
Free radicals are highly reactive due to
the presence of unpaired electron(s). The following literature
review addresses only radicals with an oxygen center. Any free
radical involving oxygen can be referred to as reactive oxygen
species (ROS). Oxygen centered free radicals contain two unpaired
electrons in the outer shell. When free radicals steal an electron
from a surrounding compound or molecule, a new free radical is
formed in its place. In turn, the newly formed radical then looks
to return to its ground state by stealing electrons with antiparallel
spins from cellular structures or molecules. Thus the chain reaction
continues and can be "thousand of events long." (7).
The electron transport chain (ETC), which is found in the inner
mitochondrial membrane, utilizes oxygen to generate energy in
the form of adenosine triphosphate (ATP). Oxygen acts as the
terminal electron acceptor within the ETC. The literature suggests
that anywhere from 2 to 5% (14) of the total oxygen intake during
both rest and exercise have the ability to form the highly damaging
superoxide radical via electron escape. During exercise, oxygen
consumption increases 10 to 20 fold to 35-70 ml/kg/min. In turn,
electron escape from the ETC is further enhanced. Thus, when
calculated, .6 to 3.5 ml/kg/min of the total oxygen intake during
exercise has the ability to form free radicals (4). Electrons
appear to escape from the ETS at the ubiqunone-cytochrome c level
Polyunsaturated fatty acids (PUFAs) are
abundant in cellular membranes and in low-density lipoproteins
(LDL) (4). The PUFAs allow for fluidity of cellular membranes.
A free radical prefers to steal electrons from the lipid membrane
of a cell, initiating a free radical attack on the cell known
as lipid peroxidation. Reactive oxygen species target the carbon-carbon
double bond of polyunsaturated fatty acids. The double bond on
the carbon weakens the carbon-hydrogen bond allowing for easy
dissociation of the hydrogen by a free radical. A free radical
will steal the single electron from the hydrogen associated with
the carbon at the double bond. In turn, this leaves the carbon
with an unpaired electron and hence, becomes a free radical.
In an effort to stabilize the carbon-centered free radical, molecular
rearrangement occurs. The newly arranged molecule is called a
conjugated diene (CD). The CD then very easily reacts with oxygen
to form a proxy radical. The proxy radical steals an electron
from another lipid molecule in a process called propagation.
This process then continues in a chain reaction (9)
TYPES OF FREE
There are numerous types of free radicals
that can be formed within the body. This web site is only concerned
with the oxygen centered free radicals or ROS. The most common
ROS include: the superoxide anion (O2-), the hydroxyl radical
(OH ·), singlet oxygen (1O2 ), and hydrogen peroxide (H2O2)
Superoxide anions are formed when oxygen (O2) acquires an additional
electron, leaving the molecule with only one unpaired electron.
Within the mitochondria, O2- · is continuously being formed.
The rate of formation depends on the amount of oxygen flowing
through the mitochondria at any given time. Hydroxyl radicals
are short-lived, but the most damaging radicals within the body.
This type of free radical can be formed from O2- and H2O2 via
the Harber-Weiss reaction. The interaction of copper or iron
and H2O2 also produce OH · as first observed by Fenton.
These reactions are significant as the substrates are found within
the body and could easily interact (9). Hydrogen peroxide is
produced in vivo by many reactions. Hydrogen peroxide is unique
in that, it can be converted to the highly damaging hydroxyl
radical or be catalyzed and excreted harmlessly as water. Glutathione
peroxidase is essential for the conversion of glutathione to
oxidized glutathione, during which H2O2 is converted to water
(2). If H2O2 is not converted into water, 1O2 is formed. Singlet
oxygen is not a free radical, but can be formed during radical
reactions and also cause further reactions. Singlet oxygen violates
Hund's rule of electron filling in that, it has eight outer electrons
existing in pairs, leaving one orbital of the same energy level
empty. When oxygen is energetically excited one of the electrons
can jump to empty orbital creating unpaired electrons (13). Singlet
oxygen can then transfer the energy to a new molecule and act
as a catalyst for free radical formation. The molecule can also
interact with other molecules leading to the formation of a new
All transition metals, with the exception
of copper, contain one electron in their outermost shell and
can be considered free radicals. Copper has a full outer shell,
but loses and gains electrons very easily making itself a free
radical (9). In addition, iron has the ability to gain and lose
electrons (i.e. (Fe2+«Fe3+) very easily. This property
makes iron and copper two common catalysts of oxidation reactions.
Iron is major component of red blood cells (RBC). A possible
hypothesis is that, the stress encountered during this process
may break down RBC releasing free iron. The release of iron can
be detrimental to cellular membranes because of the pro-oxidation
effects it can have. Zinc only exists in one valence (Zn2+) and
does not catalyze free radical formation. Zinc may actually act
to stop radical formation by displacing those metals that do
have more than one valence.
Free radicals have a very short half-life,
which makes them very hard to measure in the laboratory. Multiple
methods of measurement are available today, each with their own
benefits and limits. Radicals can be measured using electron
spin resonance and spin trapping methods. The methods are both
very sophisticated and can trap even the shortest-lived free
radical. Exogenous compounds with a high affinity for free radicals
(i.e. xenobiotics) are utilized in the spin techniques. The compound
and radical together, form a stable entity that can be easily
measured. This indirect approach has been termed "fingerprinting."
(12). However, this method is not 100% accurate. Spin-trapping
collection techniques have poor sensitivity, which can skew results
(1) A commonly used alternate approach measures markers of free
radicals rather than the actual radical. These markers of oxidative
stress are measured using a variety of different assays. These
assays are described below. When a fatty acid is peroxidized,
it is broken down into aldehydes, which are excreted. Aldehydes
such as thiobarbituric acid reacting substances (TBARS) have
been widely accepted as a general marker of free radical production
(3). The most commonly measured TBARS is malondialdehyde (MDA)
(13). The TBA test has been challenged because of its lack of
specificity, sensitivity, and reproducibility. The use of liquid
chromatography instead spectrophotometer techniques help reduce
these errors (15). In addition, the test seems to work best when
applied to membrane systems such as microsomes (8). Gases such
as pentane and ethane are also created as lipid peroxidation
occurs. These gases are expired and commonly measured during
free radical research (13). Dillard et al. (6) was one of the
first to determine that expired pentane increased as VO2 max
increased. Kanter et al. (11) has reported that serum MDA levels
correlated closely with blood levels of creatine kinase, an indicator
of muscle damage. Lastly, conjugated dienes (CD) are often measured
as indicators of free radical production. Oxidation of unsaturated
fatty acids results in the formation of CD. The CD formed are
measured and provide a marker of the early stages of lipid peroxidation
(9). A newly developed technique for measuring free radical production
shows promise in producing more valid results. The technique
uses monoclonal antibodies and may prove to be the most accurate
measurement of free radicals. However, until further more reliable
techniques are established, it is generally accepted that two
or more assays be utilized whenever possible to enhance validity
Under normal conditions (at rest), the antioxidant
defense system within the body can easily handle free radicals
that are produced. During times of increased oxygen flux (i.e.
exercise) free radical production may exceed that of removal
ultimately resulting in lipid peroxidation. Free radicals have
been implicated as playing a role in the etiology of cardiovascular
disease, cancer, Alzheimer's disease, and Parkinson's disease.
While worthy of a discussion, these conditions are not the focus
of the current literature review. This literature review will
only examine the current literature addressing the relationship
between free radicals and exercise, which is introduced below.
The driving force behind these topics is lipid peroxidation.
By preventing or controlling lipid peroxidation, the concomitant
effects discussed below would be better controlled.
Oxygen consumption greatly increases during
exercise, which leads to increased free radical production. The
body counters the increase in free radical production through
the antioxidant defense system. When free radical production
exceeds clearance, oxidative damage occurs. Free radicals formed
during chronic exercise may exceed the protective capacity of
the antioxidant defense system, thereby making the body more
susceptible to disease and injury.
Therefore, the need for antioxidant supplementation is discussed.
A free radical attack on a membrane, usually
damages a cell to the point that it must be removed by the immune
system. If free radical formation and attack are not controlled
within the muscle during exercise, a large quantity of muscle
could easily be damaged. Damaged muscle could in turn inhibit
performance by the induction of fatigue. The role individual
antioxidants have in inhibiting this damage has been addressed
within the review of the four antioxidants that follows.
One of the first steps in recovery from
exercise induced muscle damage is an acute inflammatory response
at the site of muscle damage. Free radicals are commonly associated
with the inflammatory response and are hypothesized to be greatest
twenty-four hours after completion of a strenuous exercise session.
If this theory were valid, then antioxidants would play a major
role in helping prevent this damage. However, if antioxidant
defense systems are inadequate or not elevated during the post-exercise
infiltration period, free radicals could further damage muscle
beyond that acquired during exercise. This in turn would increase
the time needed to recover from an exercise bout.
This section has focused only on the negatives
associated with free radical production. However, free radicals
are naturally produced by some systems within the body and have
beneficial effects that cannot be overlooked. The immune system
is the main body system that utilizes free radicals. Foreign
invaders or damaged tissue is marked with free radicals by the
immune system. This allows for determination of which tissue
need to be removed from the body. Because of this, some question
the need for antioxidant supplementation, as they believe supplementation
can actually decrease the effectiveness of the immune system.
Antioxidant means "against oxidation."
Antioxidants work to protect lipids from peroxidation by radicals.
Antioxidants are effective because they are willing to give up
their own electrons to free radicals. When a free radical gains
the electron from an antioxidant, it no longer needs to attack
the cell and the chain reaction of oxidation is broken (4). After
donating an electron, an antioxidant becomes a free radical by
definition. Antioxidants in this state are not harmful because
they have the ability to accommodate the change in electrons
without becoming reactive. The human body has an elaborate antioxidant
defense system. Antioxidants are manufactured within the body
and can also be extracted from the food humans eat such as fruits,
vegetables, seeds, nuts, meats, and oil. There are two lines
of antioxidant defense within the cell. The first line, found
in the fat-soluble cellular membrane consists of vitamin E, beta-carotene,
and coenzyme Q (10). Of these, vitamin E is considered the most
potent chain breaking antioxidant within the membrane of the
cell. Inside the cell, water soluble antioxidant scavengers are
present. These include vitamin C, glutathione peroxidase, superoxide
dismutase (SD), and catalase (4). Only those antioxidants that
are commonly supplemented (vitamins A, C, E and the mineral selenium)
are addressed in the literature review that follows.
- Acworth, I.N., and B. Bailey. Reactive
Oxygen Species. In: The handbook of oxidative metabolism. Massachusetts:
ESA Inc., 1997, p. 1-1 to 4-4.
- Alessio, H.M., and E.R. Blasi. Physical
activity as a natural antioxidant booster and its effect on a
healthy lifestyle. Res. Q. Exerc. Sport. 68 (4): 292-302, 1997.
- Clarkson P. M. Antioxidants and physical
performance. Crit.Rev. Food Sci. Nutr. 35: 131-141, 1995. [Abstract]
- Dekkers, J. C., L. J. P. van Doornen, and
Han C. G. Kemper. The Role of Antioxidant Vitamins and Enzymes
in the Prevention of Exercise-Induced Muscle Damage. Sports Med
21: 213-238, 1996. [Abstract]
- Del Mastero, R.F. An approach to free radicals
in medicine and biology. Acta. Phyiol. Scand. 492: 153-168, 1980.
- Dillard, C.J., R.E. Litov, W.M. Savin,
E.E. Dumelin, and A.L. Tappel. Effects of exercise, vitamin E,
and ozone on pulmonary function and lipid peroxidation. J. Appl.
Physiol. 45: 927, 1978. [Abstract]
- Goldfarb, A. H. Nutritional antioxidants
as therapeutic and preventive modalities in exercise-induced
muscle damage. Can. J. Appl. Physiol. 24: 249-266, 1999. [Abstract]
- Halliwell, B., and S. Chirico. Lipid peroxidation:
Its mechanism, measurement, and significance. Am. J. Clin. Nutr.
57: 715S-725S, 1993. [Abstract]
- Halliwell, B., and J.M.C. Gutteridge. The
chemistry of oxygen radicals and other oxygen-derived species.
In: Free Radicals in Biology and Medicine. New York: Oxford University
Press, 1985, p. 20-64.
- Kaczmarski, M., J. Wojicicki, L. Samochowiee,
T. Dutkiewicz, and Z. Sych. The influence of exogenous antioxidants
and physical exercise on some parameters associated with production
and removal of free radicals. Pharmazie 54: 303-306, 1999. [Abstract]
- Kanter, M.M., G.R. Lesmes, L.A. Kaminsky,
J. LaHam-Saeger, and N.D. Nequin. Serum creatine kinase and lactate
dehydrogenase changes following an eighty-kilometer race. Eur.
J. Appl. Phsyiol. 57: 60-65, 1988. [Abstract]
- Karlsson J. Exercise, muscle metabolism
and the antioxidant defense. World Rev Nutr Diet. 82:81-100,
- Karlsson, J. Introduction to Nutraology
and Radical Formation. In: Antioxidants and Exercise. Illinois:
Human Kinetics Press, 1997, p. 1-143.
- Sjodin, T., Y.H. Westing, and F.S. Apple.
Biochemical mechanisms for oxygen free radical formation during
exercise. Sports Med. 10: 236-254, 1990. [Abstract]
- Wong, S.H.Y., J.A. Knight, S.M. Hopfer,
O. Zaharia, C.N. Leach, and F.W. Sunderman. Lipoperoxides in
plasma as measured by liquid-chromatographic separation of malondialdehyde-thiobarbituric
acid adduct. Clin. Chem. 33(2): 214-220, 1987. [Abstract]