Free Radical Introduction

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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 atom (13).

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 (14).

PEROXIDATION

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 RADICALS

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 free radical.

CATALYSTS

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.

MEASUREMENT OF FREE RADICALS

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 (9).

PHYSIOLOGICAL EFFECTS

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.

REQUIREMENT

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 immune to disease and injury. Therefore, the need for antioxidant supplementation is discussed.

FATIGUE

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.

RECOVERY

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.

IMPORTANCE OF FREE RADICALS

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 DEFENSES

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.

REFERENCES

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  2. 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. [Abstract]
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  5. Del Mastero, R.F. An approach to free radicals in medicine and biology. Acta. Phyiol. Scand. 492: 153-168, 1980.
  6. 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]
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  8. Halliwell, B., and S. Chirico. Lipid peroxidation: Its mechanism, measurement, and significance. Am. J. Clin. Nutr. 57: 715S-725S, 1993. [Abstract]
  9. 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.
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  11. 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]
  12. Karlsson J. Exercise, muscle metabolism and the antioxidant defense. World Rev Nutr Diet. 82:81-100, 1997. [Abstract]
  13. Karlsson, J. Introduction to Nutraology and Radical Formation. In: Antioxidants and Exercise. Illinois: Human Kinetics Press, 1997, p. 1-143.
  14. 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]
  15. 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]


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