What Are Free Radicals

What are free radicals? Why are they damaging to the human body? And how does vitamin E and the other antioxidant nutrients help protect the body against free radical damage? We?ll attempt to answer these questions and help you understand why eating 5-8 servings per day of anti-oxidant rich fruits and vegetables can benefit your health. But first, a little background?

Background: A Brief Look at Chemical Bonding To understand the way that free radicals and antioxidants interact, you must first understand a bit about cells and molecules. So here's a (very) brief refresher course in Physiology/Chemistry 101: The human body is composed of many different types of cells. Cells are composed of many different types of molecules. Molecules consist of one or more atoms of one or more elements joined by chemical bonds. As you probably remember from your old high school days, atoms consist of a nucleus, neutrons, protons and electrons. The number of protons (positively charged particles) in the atom?s nucleus determines the number of electrons (negatively charged particles) surrounding the atom. Electrons are involved in chemical reactions and are the substance that bonds atoms together to form molecules. Electrons surround, or "orbit" an atom in one or more shells. The innermost shell is full when it has two electrons. When the first shell is full, electrons begin to fill the second shell. When the second shell has eight electrons, it is full, and so on. The most important structural feature of an atom for determining its chemical behavior is the number of electrons in its outer shell. A substance that has a full outer shell tends not to enter in chemical reactions (an inert substance). Because atoms seek to reach a state of maximum stability, an atom will try to fill it?s outer shell by: • •

Gaining or losing electrons to either fill or empty its outer shell Sharing its electrons by bonding together with other atoms in order to complete its outer shell

Atoms often complete their outer shells by sharing electrons with other atoms. By sharing electrons, the atoms are bound together and satisfy the conditions of maximum stability for the molecule.

How Free Radicals are Formed Normally, bonds don?t split in a way that leaves a molecule with an odd, unpaired electron. But when weak bonds split, free radicals are formed. Free radicals are very unstable and react quickly with other compounds, trying to capture the needed electron to gain stability. Generally, free radicals attack the nearest stable molecule, "stealing" its electron. When the "attacked" molecule loses its electron, it becomes a free radical itself, beginning a chain reaction. Once the process is started, it can cascade, finally resulting in the disruption of a living cell. Some free radicals arise normally during metabolism. Sometimes the body?s immune system?s cells purposefully create them to neutralize viruses and bacteria. However, environmental factors such as pollution, radiation, cigarette smoke and herbicides can also spawn free radicals. Normally, the body can handle free radicals, but if antioxidants are unavailable, or if the free-radical production becomes excessive, damage can occur. Of particular importance is that free radical damage accumulates with age.

How Antioxidants May Prevent Against Free Radical Damage The vitamins C and E, are thought to protect the body against the destructive effects of free radicals. Antioxidants neutralize free radicals by donating one of their own electrons, ending the electron-"stealing" reaction. The antioxidant nutrients themselves don?t become free radicals by donating an electron because they are stable in either form They act as scavengers, helping to prevent cell and tissue damage that could lead to cellular damage and disease. Vitamin E ? The most abundant fat-soluble antioxidant in the body. One of the most efficient chain-breaking antioxidants available. Primary defender against oxidation. Primary defender against lipid peroxidation (creation of unstable molecules containing more oxygen than is usual). Vitamin C ? The most abundant water-soluble antioxidant in the body. Acts primarily in cellular fluid. Of particular note in combating free-radical formation caused by pollution and cigarette smoke. Also helps return vitamin E to its active form.

The Antioxidants and Disease Prevention • •

Heart Disease ? Vitamin E may protect against cardiovascular disease by defending against LDL oxidation and artery-clogging plaque formation. Cancer ? Many studies have correlated high vitamin C intakes with low rates of cancer, particularly cancers of the mouth, larynx and esophagus.

The Lesson: Eat Your Fruits and Vegetables! The antioxidants are believed to help protect the body from free-radical damage. But before you go out and stock your pantry with mega-doses of these vitamins, be warned: more is not always better. The long-term effect of large doses of these nutrients has not been proven. Other chemicals and substances found in natural sources of antioxidants may also be responsible for the beneficial effects. So for now, the best way to ensure adequate intake of the antioxidant nutrients is through a balanced diet consisting of 5-8 servings of fruits and vegetables per day. For Phone Orders, call toll-free 888-337-4684 (Monday - Friday 9 am - 6 pm E.S.T) All other inquiries, please phone 718-339-6212 FAX 718-336-5570

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 peroxy radical. The peroxy 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 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 shortest­lived 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 1. 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. 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] 3. Clarkson P. M. Antioxidants and physical performance. Crit.Rev. Food Sci. Nutr. 35: 131-141, 1995. [Abstract] 4. 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] 5. Del Mastero, R.F. An approach to free radicals in medicine an 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] 7. Goldfarb, A. H. Nutritional antioxidants as therapeutic and preventive modalities in exercise-induced muscle damage. Can. J. Appl. Physiol. 24: 249-266, 1999. [Abstract] 8. Halliwell, B., and S. Chirico. Lipid peroxidation: Its mechanism, measurement, and signficance. 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.

10. 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]

Antioxidants and Free radicals Antioxidants are intimately involved in the prevention of cellular damage -- the common pathway for cancer, aging, and a variety of diseases. The scientific community has begun to unveil some of the mysteries surrounding this topic, and the media has begun whetting our thirst for knowledge. Athletes have a keen interest because of health concerns and the prospect of enhanced performance and/or recovery from exercise. The purpose of this article is to serve as a beginners guide to what antioxidants are and to briefly review their role in exercise and general health. What follows is only the tip of the iceberg in this dynamic and interesting subject.

It's the radicals, man Free radicals are atoms or groups of atoms with an odd (unpaired) number of electrons and can be formed when oxygen interacts with certain molecules. Once formed these highly reactive radicals can start a chain reaction, like dominoes. Their chief danger comes from the damage they can do when they react with important cellular components such as DNA, or the cell membrane. Cells may function poorly or die if this occurs. To prevent free radical damage the body has a defense system of antioxidants. Antioxidants are molecules which can safely interact with free radicals and terminate the chain reaction before vital molecules are damaged. Although there are several enzyme systems within the body that scavenge free radicals, the principle micronutrient (vitamin) antioxidants are vitamin E, beta-carotene, and vitamin C. Additionally, selenium, a trace metal that is required for proper function of one of the body's antioxidant enzyme systems, is sometimes included in this category. The body cannot manufacture these micronutrients so they must be supplied in the diet. Vitamin E : d-alpha tocopherol. A fat soluble vitamin present in nuts, seeds, vegetable and fish oils, whole grains (esp. wheat germ), fortified cereals, and apricots. Current recommended daily allowance (RDA) is 15 IU per day for men and 12 IU per day for women. Vitamin C : Ascorbic acid is a water soluble vitamin present in citrus fruits and juices, green peppers, cabbage, spinach, broccoli, kale, cantaloupe, kiwi, and strawberries. The

RDA is 60 mg per day. Intake above 2000 mg may be associated with adverse side effects in some individuals. Beta-carotene is a precursor to vitamin A (retinol) and is present in liver, egg yolk, milk, butter, spinach, carrots, squash, broccoli, yams, tomato, cantaloupe, peaches, and grains. Because beta-carotene is converted to vitamin A by the body there is no set requirement. Instead the RDA is expressed as retinol equivalents (RE), to clarify the relationship. (NOTE: Vitamin A has no antioxidant properties and can be quite toxic when taken in excess.)

Preventing cancer and heart disease -- do antioxidants help? Epidemiologic observations show lower cancer rates in people whose diets are rich in fruits and vegetables. This has lead to the theory that these diets contain substances, possibly antioxidants, which protect against the development of cancer. There is currently intense scientific investigation into this topic. Thus far, none of the large, well designed studies have shown that dietary supplementation with extra antioxidants reduces the risk of developing cancer. In fact one study demonstrated an increased risk of lung cancer in male smokers who took antioxidants vs. male smoker who did not supplement. Whether this effect was from the antioxidants is unknown but it does raise the issue that antioxidants may be harmful under certain conditions. Antioxidants are also thought to have a role in slowing the aging process and preventing heart disease and strokes, but the data is still inconclusive. Therefore from a public health perspective it is premature to make recommendations regarding antioxidant supplements and disease prevention. New data from ongoing studies will be available in the next few years and will shed more light on this constantly evolving area. Perhaps the best advice, which comes from several authorities in cancer prevention, is to eat 5 servings of fruit or vegetables per day.

Exercise and oxidative damage Endurance exercise can increase oxygen utilization from 10 to 20 times over the resting state. This greatly increases the generation of free radicals, prompting concern about enhanced damage to muscles and other tissues. The question that arises is, how effectively can athletes defend against the increased free radicals resulting from exercise? Do athletes need to take extra antioxidants? Because it is not possible to directly measure free radicals in the body, scientists have approached this question by measuring the by-products that result from free radical reactions. If the generation of free radicals exceeds the antioxidant defenses then one

would expect to see more of these by-products. These measurements have been performed in athletes under a variety of conditions. Several interesting concepts have emerged from these types of experimental studies. Regular physical exercise enhances the antioxidant defense system and protects against exercise induced free radical damage. This is an important finding because it shows how smart the body is about adapting to the demands of exercise. These changes occur slowly over time and appear to parallel other adaptations to exercise. On the other hand, intense exercise in untrained individuals overwhelms defenses resulting in increased free radical damage. Thus, the "weekend warrior" who is predominantly sedentary during the week but engages in vigorous bouts of exercise during the weekend may be doing more harm than good. To this end there are many factors which may determine whether exercise induced free radical damage occurs, including degree of conditioning of the athlete, intensity of exercise, and diet.

Can antioxidant supplements prevent exercise induced damage or enhance recovery from exercise? Although it is well known that vitamin deficiencies can create difficulties in training and recovery, the role of antioxidant supplementation in a well nourished athlete is controversial. The experimental studies are often conflicting and conclusions are difficult to reach. Nevertheless, most of the data suggest that increased intake of vitamin E is protective against exercise induced oxidative damage. It is hypothesized that vitamin E is also involved in the recovery process following exercise. Currently, the amount of vitamin E needed to produce these effects is unknown. The diet may supply enough vitamin E in most athletes, but some may require supplementation. There is no firm data to support the use of increased amounts of the other antioxidants.

Performance In general, antioxidant supplements have not been shown to be useful as performance enhancers. The one exception to this is vitamin E which has been shown to be useful in athletes exercising at high altitudes. A placebo controlled study done on mountaineers demonstrated less free radical damage and decline in anaerobic threshold in those athletes supplemented with vitamin E. Although difficult to generalize, this finding suggests that supplementation with vitamin E might be beneficial in those triathletes who are adapting to higher elevations.

How much is enough?

Although there is little doubt that antioxidants are a necessary component for good health, no one knows if supplements should be taken and, if so, how much. Antioxidants supplements were once thought to be harmless but increasingly we are becoming aware of interactions and potential toxicity. It is interesting to note that, in the normal concentrations found in the body, vitamin C and beta-carotene are antioxidants; but at higher concentrations they are pro-oxidants and, thus, harmful. Also, very little is known about the long term consequences of megadoses of antioxidants. The body's finely tuned mechanisms are carefully balanced to withstand a variety of insults. Taking chemicals without a complete understanding of all of their effects may disrupt this balance.

Recommendations •

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Follow a balanced training program that emphasizes regular exercise and eat 5 servings of fruit or vegetables per day. This will ensure that you are developing your inherent antioxidant systems and that your diet is providing the necessary components. Weekend warriors should strongly consider a more balanced approach to exercise. Failing that, consider supplementation. For extremely demanding races (such as an ultradistance event), or when adapting to high altitude, consider taking a vitamin E supplement (100 to 200 IU, approximately 10 times the RDA) per day for several weeks up to and following the race. Look for upcoming FDA recommendations, but be wary of advertising and media hype. Do not oversupplement.

Selected References 1. The Effect of Vitamin E and Beta Carotene on the Incidence of Lung Cancer and Other Cancers in Male Smokers New England Journal of Medicine (NEJM). vol 330 (15) Apr. 14, 1994. pp 1029-1035. 2. A Clinical Trial of Antioxidant Vitamins to Prevent Colorectal Adenoma NEJM, vol 331 (3). July 21, 1994. pp 141-147 3. Antioxidant Vitamins -- Benefits Not Yet Proved (editorial) NEJM vol 330 (15) Apr. 14, 1994. p 1080 - 1081 4. Antioxidants and Physical Performance (review) Critical Reviews in Food Science and Nutrition, 35(1&2):131-141 (1995). 5. Increased blood antioxidant systems of runners in response to training load. Clinical Science (1991). 80, 611-618. 6. Exercise, Oxidative Damage and Effects of Antioxidant Manipulation (review). Journal of Nutrition 122(3 suppl): 766-73, 1992 Mar. 7. Antioxidants: role of supplementation to prevent exercise-induced oxidative stress (review). Medicine and Science in Sports and Exercise. 25(2):232-6, 1993 Feb.

8. Prospects for the use of antioxidant therapies.(Review). Drugs 49(3):345-61, 1995 Mar.