26 minutes reading time (5239 words)

    On Track to the Olympic Games: Chemistry Giving the Winning Edge

    Dr Mark Holmes
    Central Queensland University

    2012 is an exciting time for sports fans. From July 27 to August 12, all eyes will be on London, the host city of the Games of the XXX Olympiad. The 2012 Paralympic Games will follow from August 29 to September 9. Logistically, the staging of these Games is huge. The London Organising Committee of the Olympic and Paralympic Games (LOCOG) anticipates that there will be around 17000 athletes from 205 countries attending the XXX Olympiad to compete for over 300 gold medals in 26 different sports (1). Coverage of the games will involve 20000 media personnel as they attempt to reach a worldwide audience of around four billion people.

    Sports historians often point out that there is no sporting occasion quite like the Olympic Games (2). There is no other multi-sport event that has captured its international appeal, its intensity and excellence of competition amongst elite-level athletes in a myriad of sports, and its basic ideology. Little-known athletes become super-stars and household names all over the world as the result of competing and winning in an Olympic event.

    The first modern Olympic Games was staged in 1896 at Athens in Greece. They were revived about 2000 years after the original ancient Olympic Games had collapsed. It was a French Baron, Pierre de Coubertin (1863-1937), who took the initiative of reviving the Olympic Games for athletes from all countries of the world (2,3). He founded the International Olympic Committee (IOC) in 1894, which consisted of members chosen by de Coubertin for their devotion to the concept of the Olympic movement. The modern Olympic Games are now held every four years, in keeping with the ancient Greek Olympiad tradition.

    World records keep tumbling
    A fascinating aspect about the modern Olympic Games has been the trend in which world records keep falling in most of the sports. For instance, in 2009 in Berlin, Atlanta Olympic Games, Jamaican sprinter Usian Bolt set a new world record of 9.58 seconds for the 100 metre sprint. At the Paris Games in 1900 , however, the world record for this event was 11.0 seconds, which means that the modern 100 metre sprinter is over one second faster than their predecessors (4). Similar stories abound in many other Olympic sports, and no doubt this trend will continue at the London 2012 Olympiad. Australians are certainly anticipating great feats and possibly new world records from many of our own athletes, especially in some of the swimming and athletic events if the results from recent national and international championships are any guide.

    One of the major factors contributing to the trend in which world records keep falling has been the development of sports science over the past century. Indeed sports scientists are today helping elite-level athletes find the tiny margins required to win in world class competition, such as the Olympic Games (5). The development if sports science and sports technology has helped refine the basic principles of training and preparation of athletes for competition. Our elite Australian athletes and their coaches, for example, are now supported by a multitude of sports scientists from the Australian Institute of Sport (AIS) and other sporting academies across the nation. These sports scientists are seeking out the latest advances from scientific disciplines that could give our athletes the winning edge in London.

    Chemistry in sport science
    Chemistry has become an integral part of modern sports science. Chemists are now providing athletes with a new tool in the search for the winning edge that they require. Of course, there are many people who think that "chemistry giving the winning edge" means that chemists are designing performance-enhancing drugs for athletes. This is certainly not the case. Chemistry is helping athletes improve their performance in beneficialways such as in the design of Hi-tech clothing fabrics and structural materials for sporting equipment (eg carbon fibre and kevlar for the 'superbikes'); the development of performance tests for athletes; and in the drug testing of elite-level athletes.

    Performance testing is all about an athlete gaining a better understanding of their own body function and chemistry. Nowadays there are many elite-level athletes who use knowledge of their body chemistry to assist them with the fine-tuning of training programs for major competition. The outcomes of performance tests can tell an athlete whether they are overtraining, and help them to peak at competition, rather than before or after the event. This review will provide an overview of the chemistry behind a very important performance test designed to determine the blood lactate threshold. This performance test provides endurance athletes with information about how the energy systems in their skeletal muscles are developing as they prepare for major competition.

    Chemists are involved extensively in the testing of athletes for the use of banned performance-enhancing drugs. It is certainly more challenging to a chemist to catch a cheat, than to be involved with the design of drugs that may enhance an athlete's performance, but at the same time may have disastrous side-effects. Drug-testing of athletes is now a highly controlled and sophisticated process, and Australia is a world leader in this area. The role of Australian chemists in drug testing also will be outline in this review.

    Energy for muscle contractionIn exercise, the most important part of our anatomy that contributes to locomotion are the skeletal muscles (6). Of course, this does not mean that other parts of our anatomy such as the heart and lungs are not important. But it is our skeletal muscles that make us move and perform when we undertake physical activity. They also respond quickly to the correct training in preparation for competition (6).

    Our skeletal muscles contain hundreds to thousands of long cylindrical cells called muscle fibres (7). Although muscle fibres predominate, blood vessels, nerve fibres and substantial amounts of connective tissue are also present within each muscle. Blood flow through a muscle can increase up to 100 times during exercise. This ensures that the muscle fibres are receiving a continuous supply of oxygen and nutrients to meet the energy demands of muscle contraction, while at the same time removing carbon dioxide and other waste products.

    Muscle fibres contain protein molecules that run lengthwise called actin and myosin (7,8). These proteins are the major components of the thin and thick filaments in muscles. A single muscle fibre may contain 15 billion thin and thick filaments. Upon stimulation by the nervous system, it is thought that the actin and myosin molecules slide towards each other to shorten (of contract) the muscle. This process is called the Sliding Filament Theory of muscle contraction (7,8).

    The energy required for muscle contraction comes from a special high-energy molecule called adenosine triphosphate (ATP). ATP is a molecule that can store energy temporarily and is often referred to as the "energy currency of cells". The structure of ATP can be divided into three parts (7,8). A heterocyclic amine (or nitrogenous base) called adenine that is also found in other important molecules such as DNA and RNA. A sugar (furanose) called ribose, which is sometimes assumed to be the energy source in ATP because people equate sugars with energy. Finally, ATP contains three inorganic phosphate groups. Phosphate is often represented as Pi. It is the triphosphate part of the of ATP that stores the energy for biological work. 

     The first phosphate on ATP is attached to the ribose by an inorganic ester bond, whereas the other two phosphates are joined by phosphoanhydride bonds. A phosphoanhydride bond contains a considerable amount of energy and is usually referred to as a high energy phosphate bond.

     Our body stores energy in the phosphoanhydride bonds of ATP. Cleavage of the terminal phosphate bond of ATP yields a molecule with two inorganic phosphate groups called adenosine diphosphate (ADP), together with an inorganic phosphate (Pi). In the process, a large amount of free energy is released (about 30.5 kjoules per mole).

     ATP is the most abundant high energy molecule in the human body. The chemical energy stored in ATP provides just the right amount of energy for the body to carry out biological work such as digestion, nerve transmission, active transport across cells, biosynthesis and powering muscle contraction. ATP has been compared to a tightly coiled spring, ready to uncoil with tremendous energy when released (7,8).

    Considering the importance of ATP to muscle contraction, you may think that a well-trained athlete would have a huge store of ATP in their muscles before they compete. However, this is not the case. Our cells have a limited store of ATP, and even an elite-level sprinter only has about 2 to 3 seconds supply of ATP in their muscle fibres. Of course, without ATP, our muscles cannot contract. Why then can a sprinter complete 100 metres in 10 seconds, and yet other athletes continue exercising for several hours before fatiguing?

    The answer is that muscle fibres are able to regenerate ATP. This means that ATP is constantly being reformed and reused during exercise - its turnover rate is high. To replenish their ATP stores, cells must reattach a Pi to ADP. Some sports scientists have estimated that during strenuous exercise the human body produces and uses as much as 1kg of ATP every 2 minutes. You can buy ATP from chemical companies at about $10 per gram. This means that every time you run hard for about 2 minutes, you are turning over about $10 000 worth of ATP. And you thought that sporting equipment was expensive!

    Energy required to regenerate ATP during exercise is gained from the breakdown of food nutrients such as carbohydrate (glucose), lipid (fatty acids) and protein (amino acids). These "fuel molecules" contain a huge amount of stored chemical energy that is harnessed by the energy systems in muscles to replenish the stores of ATP. This can be likened to putting petrol (fuel) into a car to keep the engine going (7,8).

     The carbohydrate glucose is a particularly important fuel molecule for the contracting muscles of athletes. It is the major monosaccharide in the food we eat and contains about 2780 kjoules per mole of free energy. We only need about 30kjoules per mole of free energy to attach a Pi to ADP to reform ATP. A cell is capable of using up to 40% of the energy stored in a glucose molecule. In other words, there is plenty of energy in glucose for a cell to make lots of ATP (7,8).

     Glucose is delivered to the muscle fibres via the blood circulation. We always have plenty of glucose for energy in our blood. While resting, glucose is stored in our muscles in the form of a large polysaccharide called glycogen. This ensures a ready supply of glucose in the muscles for exercise. You may have heard about "carbohydrate loading". This is where athletes increase the intake of carbohydrates in their diet a few days prior to competition by eating foods such as rice, pasta, bread and potatoes (9). This ensures that the athlete has plenty of glucose stored in the "glycogen warehouse" in their muscles before they exercise.

    When we exercise, the turnover rate of ATP increases dramatically. In response, our muscles break down their stores of glycogen or use the glucose delivered by the blood to obtain the energy they need to replenish the ATP store. Two important energy systems in out muscles involved in the process of extracting energy out of glucose to make ATP are the aerobic energy system and the anaerobic energy system (6,7,8). Which of these energy systems predominates depends on the power requirements of our muscles and on the duration of our physical activity.

    The aerobic energy system has a strong reliance on oxygen. This energy system is important during long-term moderate intensity exercise (eg marathon running, triathlon, long-distance cycling, walking) and extracts energy from not only glucose, but also fatty acids and amino acids, in order to make lots of ATP. During these forms of exercise, our lungs and blood circulation are capable of delivering a continuous supply of oxygen to the skeletal muscles.

    In contrast, the anaerobic energy system in muscles (Figure 1) is particularly important when we undertake short-term high intensity exercise such as sprinting. The term anaerobic means "without oxygen", which suggests that some of the energy in glucose is used to make ATP without the requirement for oxygen. This is important during forms of exercise which are short-term and of high intensity because our lungs and blood circulation cannot deliver sufficient energy to the skeletal muscles.

    Lactic acid and the anaerobic energy system
    Lactic acid is an end product of the anaerobic energy system in our muscles (Figure 1). If we exercise hard and fast, we produce lots of lactic acid (6,7). However, if lactic acid builds up too quickly, it will eventually slow down or stop the muscles from contracting. This is one cause of muscle fatigue. You may have experienced the burning sensation in your muscles during exercise, the cause of which is a buildup of lactic acid in your muscles.

    A sports scientist can tell whether an athlete is producing lots of lactic acid in their muscles because the lactic acid spills over into the blood. In other words, there will be a high concentration of lactic acid in our blood when we exercise hard. Thanks to some remarkable developments in chemistry and electronics, sports scientists can now measure the concentration of lactic acid in a drop of blood from an athlete using a hand-held analyser and a test-strip. These hand-held analysers are similar to glucometers used by diabetics to monitor their blood glucose concentrations before and after a meal.

    In sports science, the terms lactic acid and lactate are often used interchangeably. However, it is important to keep in mind that lactic acid and lactate are not the same compound (7). Lactic acid produced during exercise quickly dissociates to the lactate ion and a hydrogen ion (H+). The H+ ions contribute to acidity in the muscles during exercise, which in turn contributes to muscle fatigue. Intense muscular activity usually results in the accumulation of lactate and H+ ions in the blood as well.

    We have an important acid-base balance in our body. Under normal resting conditions, our body fluids have more bases than acids. This results in a resting arterial blood pH of between 7.35 and 7.45, and a resting muscle pH of 7.1 to 7.2 (Figure 2).

    However, arterial blood pH and muscle pH change considerably with moderate to high intensity exercise due to the production of lactic acid. It is common for arterial blood pH values in elite-level athletes to drop to 7.0 or lower following a maximal sprint-type exercise (7). The pH in muscles during exhaustion can drop even more to about 6.6 (Figure 2). Such disturbances in acid-base balance impair the ability of muscles to contract and generate energy in the form of ATP. The drop in blood and muscle pH experienced by athletes during exhaustion can only be tolerated for a few minutes. An athlete's acid-base balance is usually returned to normal within about 30 to 40 minutes of recovery from high-intensity exercise.

    To minimise to acidic effect of the free H+ ions released from the production of lactic acid, our body fluids contain special substances called buffers. The four major buffers in our body are bicarbonate (HCO3-), phosphate (Pi), haemoglobin in red blood cells, and proteins (7). In simple terms, these buffers are able to "mop up" H+ ions and therefore resist a change in pH. The buffers in blood and tissues play an important role in maintaining the acid-base balance of an athlete during exercise and also returning it to normal during recovery from intense exercise.

     Some athletes consume sodium bicarbonate (baking soda) just prior to competition in an attempt to gain additional buffering capacity in their blood against the free H+ ions produced during exercise. By increasing the levels of blood carbonate, athletes are able to increase their blood pH, making their blood slightly more alkaline. This is called "bicarb loading" and is not a banned practice. However, while beneficial effects for bicarb-loading have been observed, a down-side is that some athletes have reported severe gastrointestinal discomfort (eg. diarrhoea, cramps, bloating and nausea) after consuming a high dose of sodium bicarbonate (7).

    Blood lactate (anaerobic) threshold

    Lactic acid is present in the blood circulation even at rest, usually a concentration of about 0.5 to 1.0 mM(6). During high intensity exercise, the anaerobic energy system in the muscles produces large amounts of lactic acid, some of which diffuses in to the blood. As exercise continues, the lactic acid begins to accumulate in the muscles and blood, and eventually the lactic acid reduces the ability of the muscles to contract. 

     Blood concentrations can rise to as high as 25 mM in an elite power athlete after a short bout of high intensity exercise such as the 400 metre run and 100 metre swim (6).

    The point in the intensity of exercise where lactic acid (or lactate) begins to accumulate in the blood is referred to by sports scientists as the "blood lactate threshold" or "anaerobic threshold" (6,7). Sports scientists can determine an athlete's blood lactate threshold in a sports laboratory or in the field by gradually increasing the level of intensity of exercise, and measuring the blood lactic acid (or lactate) concentration, work output, and heart rate at each level. The blood lactate threshold is the point (work output or heart rate) at which the concentration of lactic acid in the blood begins to accumulate (6).

    For endurance athletes such as long distance runners, cyclists and swimmers, the determination of blood lactate threshold is a very important performance test. These sportsmen and sportswomen rely on the aerobic energy system in their muscles to supply ATP. It is essential that they produce and accumulate very little lactic acid during exercise. Therefore, for endurance athletes, the blood lactate threshold represents a level of exercise intensity which can be maintained for long periods of time without fatigue setting in (6).

    A typical blood lactic acid-heart rate curve is presented in Figure 3. The curve shows that the blood lactic acid concentration at rest is less than 1 mM. As the level of exercise becomes more intense, so the lactic acid concentration begins to rise. The blood lactate threshold is the point on the curve where lactic acid begins to accumulate in the blood (6). Heart rate at the blood lactate threshold is determined and becomes useful information to the athlete. They will know that if they raise their heart rate above that determined at threshold, then lactic acid will reach high levels in the muscles and blood, and exercise performance will deteriorate quickly. Many athletes monitor their heart rate during competition and develop race strategies based on a knowledge of their blood lactate threshold.

    The blood lactate threshold also allows the endurance athlete to gauge the effectiveness of their training program. With correct training, endurance athletes can improve their blood lactate threshold. The heart rate at blood lactate threshold for an average untrained person is around 60-70% of maximum heart rate (6). However, an elite- level endurance athlete can achieve a heart rate at blood lactate threshold as high as 90% of their maximum heart rate (6).

    The future of performance testing
    Chemistry will most likely have a hand in future technological developments associated with the testing of athletic performance. For example, one day athletes may be using a "non-invasive lactate meter". This will probably take the form of a watch worn by the athlete that shines a beam of near-infrared light through their skin and accurately measures the concentration of lactate in their bloodstream. Gone will be the days of finger-pricking and test-strip technology. Moreover, an athlete will be able to continuously monitor their blood lactate concentrations during both training and competition.

    Sound like something out of Star Trek? Maybe not. There is already a considerable amount of research being undertaken around the world into the development of a non-invasive blood glucose meter for people with diabetes mellitus (10,11). the technology is based on near-infrared (NIR)spectroscopy, a technique widely used in chemistry. NIR spectroscopy involves wavelengths of light that are beyond those that can be seen and felt by people. Scientists are working on the principle that selected wavelengths of NIR light emitted by the meter will penetrate the skin and tissues of a person and be absorbed by the glucose in their bloodstream. Each substance in the bloodstream has a unique "spectral signature" and, after giving the NIR wavelengths a few seconds to be absorbed, the meter will then determine the number of glucose spectral signatures in the blood (10). A mini-computer in the meter will perform a series of complicated mathematical equations in order to calculate the concentration of blood glucose. Not a drop of blood will have been taken. As Tom Fortin, Vice-President of Rio Grande Medical, America, recently stated about the NIR technology - "basically it's right that you can't see, you can't feel, but you shine it into human tissue, and the wavelengths bounce around in there and see how much glucose there is" (10).

    The modern lactate analyser used by sports scientists that requires test-strips and a drop of blood is based on the technology used in the design of glucometers for diabetics. If the NIR technology being trialled for the non-invasive measurement of blood glucose is successful, it is most likely that a non-invasive analyser for blood lactate will appear on the market soon after.

    Drugs in sport
    The abuse of drugs by athletes is not only a form of cheating, but can be dangerous or even fatal (12). Some athletes have died from taking drugs intended to enhance their performance. Athletes caught taking banned drugs can have their sporting careers destroyed. When Ben Johnson was caught at the 1988 Seoul Olympic Games for taking the banned anabolic steroid, stanozolol, he was stripped of his gold medal for the 100 metre sprint and had almost $12 million of endorsements cancelled. More recently in the 2006 Tour de France, cyclist Floyd Landis was disqualified as winner of the prestigious race after he was found to have administered testosterone.

    It is interesting that one of the reasons for the demise of the ancient Olympic Games about 2000 years ago (in addition to political interference) was the abuse of drugs by the athletes (13). The "win at all costs" attitude in sport was prevalent even in ancient times. While many of the substances used in ancient times were harmless, some such as hallucinogens extracted from mushrooms and plant seeds, had terrible side effects.

    According to the World Anti-Doping Agency (WADA), "doping" is the use by athletes of banned substances or methods that may enhance performance. The substances and methods that are now banned by most sporting organisations around the world are based on those by the WADA (14). They include:
    1. Stimulants (e.g. amphetamines, cocaine) that act on the central nervous system to make athletes feel more awake and alert. They also hide the natural feelings of fatigue.
    2. Narcotic analgesics (e.g. pethidine, morphine) are painkillers used to treat injury and illness but could be used illegally by athletes in contact sports such as boxing.
    3. Anabolic steroids (e.g. stanozolol, testosterone) are used to build muscle bulk and are popular with athletes competing in power sports such as weightlifting. Endurance athletes may also illegally use anabolic steroids to assist them with recovery from injury and intensive training periods.
    4. Beta-2 agonists (e.g. clenbuterol, salbutamol, terbutaline) can increase muscle mass and are often referred to as non-steroidal anabolic agents. Salbutamol and terbutaline are used as inhalants by asthmatics so athletes must have a therapeutic use exemption from their national sporting organisation to use these drugs (14).
    5. Diuretics (e.g. frusemide) can be used to control body weight by regulating water and salt retention via increased urine production. They can be illegally used by athletes to dilute urine a urine sample before a drug test.
    6. Masking agents (e.g. probenecid) similar to diuretics can hide a performance enhancing drug such as reducing the clearance of anabolic steroids by the kidneys into urine.
    7. Peptide hormones (e.g. erythropoietin (EPO), human growth hormone (hGH)) are natural hormones that may stimulate many body processes such as red blood cell production (EPO) and muscle growth (hGH).
    8. Blood doping involves the taking of 1-2 litres of blood from an athlete's body several weeks before a competition, storing the blood by refrigeration, then re-infusing the blood about one week before competition. Blood doping has the desired effect of increasing an athlete's red blood cells so that their body can carry more oxygen.
    9. Catheterisation is the drawing of urine from the bladder with a tube. It is banned because it reduces the chance of being caught for the taking of drugs by the standard urinary drug testing procedures.

    The WADA has a list of "restricted substances" that may be banned in certain forms or may be tested for only in certain sports (14). These include alcohol, marijuana, local anaesthetics, corticosteroids, beta-blockers and sedatives. Special permission is required before some of these drugs can be used during training or competition. It is essential that athletes and their coaches are aware of the substances banned in their sport. For instance, sedatives are banned in the modern pentathlon and beta-blockers are banned in archery, diving and shooting.

    Drug testing in Australia
    Drug testing in Australia is administered by the Australian Sports and Anti-Doping Authority (ASADA). This agency was formed in 1989 and now tests over 4,000 athlete samples each year from about 50 sports. ASADA has the task of testing Australian athletes before they depart for the London Olympic Games. Athletes are randomly selected by ASADA for drug testing either during competition or out-of-competition. The athletes are normally given minimal notice and are expected to provide a 80 mL urine sample during a highly controlled sample collection procedure conducted by ASADA officials.

    The urine collected from an athlete is poured into two sample bottles (A and B), security sealed, then sent to the Australian Sports Drug Testing Laboratory (ASDTL) at the National Measurement Institute in Sydney. The ASDTL is one of only 34 WADA-accredited laboratories in the world for sports drug testing, and uses a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). Sample A of the urine collected from an athlete is screened by the scientists at ASDTL for any banned substances. If the scientists find that the urine sample tests positive, the B-sample is kept for future testing in the presence of the athlete or a representative of the athlete. A positive test for a banned drug can lead to the disqualification of an athlete from a sporting event and possibly a ban from further competition.

    The GC-MS equipment used in sports testing laboratories is unerringly accurate, and can detect drugs in urine to a level as low as one part per billion or nanogram per millilitre (ng/mL). An analogy is that if you were to dissolve teaspoon of sugar into an Olympic size swimming pool, the GC-MS will be able to detect traces of sugar in the water. Sports testing laboratories began using GC-MS around 1982.

    A drug is placed on the WADA prohibited list if it has been shown to have a performance-enhancing effect and/or harmful side-effects. Trace amounts of an anabolic steroid will lead to a severe penalty for an athlete. Anabolic steroids such as stanozolol are an artificial version of the male sex hormone, testosterone, responsible for the androgenic effects producing secondary male sex characteristics such as greater hair distribution, a deeper voice and stronger body (12,19,20). It has an anabolic effect because it builds up muscles by improving body nitrogen retention and increasing muscle protein synthesis. Women also have testosterone circulating in their body, but at a level that is about one-twentieth of the amount produced by men.

    Q. Name the three key chemistry functionalities of the testosterone molecule.
    A. Ketone (C=O), alkene (C=C) and alcohol or hydroxyl (OH).

    Chemical modifications of testosterone have been attempted to alter its anabolic to androgenic ratio ("muscle per whisker"), reduce its virilizing androgenic effects, and to make it available as an oral preparation (20). This has led to the production of stanozolol. Despite the chemical modifications, it has not been possible to eliminate the virilizing androgenic effects from these artificial steroids. Athletes are affected by anabolic steroids in several ways. Sportsmen can develop breasts and bad acne, have decreased testis size and sperm count, and become more aggressive. On the other hand, sportswomen may start to grow hair in places where they never had hair before, have problems with menstruation, notice a deepening of the voice, and also become aggressive. For women, these changes can be permanent, even though they may have stopped taking the anabolic steroid. 

     Q. Would stanozolol be considered more or less polar than testosterone?
    A. Stanozolol contains heterocyclic nitrogen atoms as part of a pyrazole ring that makes it more polar than testosterone.

    References
    1. http://www.london2012.com. Official Website of the 2012 London Olympic Games.
    2. Tyler, M.(1974). The History of the Olympic Games. From 1896 to 1976. Marshall Cavendish Publications Ltd, London, UK.
    3. Astrand, P-O. (1996). Man as an Athlete. In Oxford Textbook of Sports Medicine. Eds. Harries, M., Williams, C., Stanish, W.D. and Mitchell, L.J. pp. 1-11. Oxford University Press, New York, USA.
    4. Famighetti, R. (1997). The World Almanac and Book of Facts 1997. World Almanac Books, K-III Reference Corporation, New Jersey, USA.
    5. Kearney, J.T. (1996). Training the Olympic Athlete. Scientific American. June Issue, pp. 44-55.
    6. Davis, P. (1996). Sports Physiology. In Smart Sport. The Ultimate Reference Manual for Sports People. Eds. de Castella, R. and Clews, W. pp. 2.1-2.24. RWM Publishing Pty Ltd, Chapman, ACT, Australia.
    7. Wilmore, J.H. and Costill, D.L. (1994). Physiology of Sport and Exercise. Human Kinetics, Champaign, IL, USA.
    8. Maugham, R., Gleeson, M. and Greenhaff, P.L. (1997). Biochemistry of Exercise and Training. Oxford University Press, Oxford, UK.
    9. Cox, G. (1996). Sports Nutrition. In Smart Sport. The Ultimate Reference Manual for Sports People. Eds. de Castella, R. and Clews, W. pp. 6.1-6.21. RWM Publishing Pty Ltd, Chapman, ACT, Australia.
    10. http://www.diabetes.org/DiabetesForecast/95nov/default.htm. Roberts, S.L. (1995). The Evasive Noninvasive Meter. Diabetes Forecast.
    11. http://www.castleweb.com/Diabetes/d_06_e30.htm. The Glucowatch. A Painless, Continuous Glucose Meter. Children with Diabetes. Fourth Quarter, 1996.
    12. Australian Sports Drug Agency Infopac (1996). Second Edition. Canberra, ACT, Australia.
    13. Kempnich, J. (1993). Drugs In Sport Isn't Just About Steroids (Second Edition). Australian Sports Drug Agency. National Capital Printing, Fyshwick, ACT, Australia.
    14. Corrigan, B. (1989). Doping in Sport. Coaching Director. Vol. 4, No. 4, pp. 24-37.
    15. Readshaw, D. (1995). Drugs in Sport Handbook (Second Edition). Australian Sports Drug Agency. Imprint Limited, Canberra, Australia.
    16. Marston, K. and Haire, M. (1996). Measurement in Sport. Student Booklet. National Standards Commission. Maxwell Printing, Sydney, NSW, Australia.
    17. Catlin, D., Cowan, D., Donike, M., Fraisse, D., Oftebro, H. and Rendic, S. (1992). Testing Urine for Drugs. Journal of Automatic Chemistry. Vol. 14, pp. 85-92.
    18. Park, J. (1991). Doping Test Report of 10th Asian Games in Seoul. The Journal of Sports Medicine and Physical Fitness. Vol. 31, No. 2, pp. 303-317.
    19. Clarkson, P.M. and Thompson, H.S. (1997). Drugs and Sport. Research Findings and Limitations. Sports Medicine. Vol. 24, No. 6, pp. 366-384.
    20. Cowan, D.A. (1996). Drug Abuse. In Oxford Textbook of Sports Medicine. Eds. Harries, M., Williams, C., Stanish, W.D., and Micheli, L.J. pp. 314-329. Oxford University Press, New York, USA.
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