40: Athletic Performance Enhancers

Interest in extraordinary athletic achievement fuels the modern day science of performance enhancement in sports. The desire to improve athletic performance in a scientific manner is a relatively recent development. At one time, the focus on maximizing human physical and mental potential centered on the importance of manual work and military service. The role of sport was inconsequential, except for its potential in improving ­military preparedness.⁹² Today, “sports doping” refers to the use of a prohibited xenobiotic to enhance athletic performance. The word doping comes from the Dutch word doop, a viscous opium juice used by the ancient Greeks.²⁸

Controversy surrounding the systematic use of performance enhancing xenobiotics by the participating athletes has marred many sporting events. Since the International Olympic Committee (IOC) began testing during the 1968 Olympic games, prominent athletes have been sanctioned and stripped of their Olympic medals because they tested positive for banned xenobiotics. However, from a public health perspective, the use of performance-enhancing drugs among athletes of all ages and abilities is a far more serious concern than the highly publicized cases involving world class athletes. The majority of studies on the epidemiology of performance enhancing xenobiotics have investigated androgenic anabolic steroid use. Androgenic means masculinizing, and anabolic means tissue building. An anabolic process stimulates protein synthesis, promotes nitrogen deposition in lean body mass, and decreases protein breakdown. Studies of high school students document that 6.6% of male seniors have used anabolic steroids, and 35% of these individuals were not involved in organized athletics.³¹ Others find rates of androgenic steroid use in adolescent athletes range from 3% to 19%.^{101,114,170,226,231,232} Of the 243,193 samples analyzed by accredited laboratories for the presence of banned performance enhancing xenobiotics in Olympic and non-Olympic sports, 2% had positive findings. The majority of these findings are specific for anabolics and stimulants.

Performance enhancers can be classified in several ways. Some categorize performance enhancers according to the expected effects. For example, some xenobiotics increase muscle mass, whereas others decrease recovery time, increase energy, or mask the presence of other xenobiotics. However, one xenobiotic may have several expected effects. For example, diuretics may be used to mask the presence of other xenobiotics by producing dilute urine, or they may be used to reduce weight. Clenbuterol is an anabolic xenobiotic, but it also is a stimulant because of its β₂-adrenergic agonist effects. Depending on the xenobiotic, it is used either during training to improve future performance or during competition to improve immediate results.²⁸

According to the 2013 World Anti-Doping Agency (WADA) World Anti-Doping Code, a xenobiotic or method constitutes doping and can be added to the Prohibited List if it is a masking xenobiotic or if it meets two of the following three criteria: it enhances performance, its use presents a risk to the athlete’s health, and it is contrary to the spirit of sport²³⁰ (Table 40–1). Some of the prohibited substances and methods on the WADA 2013 Prohibited List are used to treat legitimate medical conditions of athletes.²³⁰ Athletes with documented medical conditions requiring the use of a prohibited substance or method may request a therapeutic use exemption (TUE). For example, the use of a β-adrenergic agonist other than albuterol, salbutamol, or formoterol in an athlete with documented asthma requires a TUE.

Anabolic Androgenic Steroids

Anabolic-androgenic steroids (AASs) increase muscle mass and lean body weight, and cause nitrogen retention.¹⁴⁵ The androgenic effects of ­steroids are responsible for male appearance and secondary sexual characteristics such as increased growth of body hair and deepening of the voice. Testosterone is the prototypical androgen, and most AASs are synthetic testosterone derivatives. The WADA categorizes AASs into two groups: exogenous, referring to substances that are not ordinarily capable of being produced by the body naturally, and endogenous, referring to those capable of being produced by the body naturally. As such, there is sometimes discordance in categorization schemes with other governing entities. In this document, the term anabolic steroid means any xenobiotic, chemically and pharmacologically related to testosterone, other than estrogens, progestins, corticosteroids, and DHEA.

In the 1970s and 1980s, federal regulation of anabolic steroids was under the direction of the Food and Drug Administration (FDA). Because of increasing media reports on the use of AASs in sports, particularly by high school students and amateur athletes, Congress enacted the Anabolic Steroid Control Act of 1990, which amended the Controlled Substances Act and placed anabolic steroids in schedule III. Schedule III implies that a xenobiotic has a currently accepted medical use in the United States and has less potential for abuse than the drugs categorized as schedule I or II. The Anabolic Steroid Control Act of 2004 adds certain steroid precursors, such as androstenedione and dihydrotestosterone, to the list of controlled substances that are considered illegal without a prescription. However, DHEA is exempted. Possession of androstenedione or other metabolic precursors called prohormone drugs is considered a federal crime. Nevertheless, AASs are still available illicitly over the Internet from international marketers, veterinary pharmaceutical companies, and some legitimate US manufacturers (Table 40–2).

Antiestrogens and Antiandrogens

In male athletes using androgens, avoiding the unwanted side effects of feminization, such as gynecomastia, or in female athletes, avoiding masculinization and features such as facial hair and deepening voice, requires manipulation of the metabolic pathways of androgen metabolism. Creating a xenobiotic that completely dissociates the desired from the unwanted effects has not been possible. However, xenobiotics with properties capable of manipulating metabolic pathways associated with undesirable side effects are divided into four main groups, all on the Prohibited List.

1. Aromatase inhibitors such as anastrozole and aminoglutethimide prevent the conversion of androstenedione and testosterone into estrogen.

2. The antiestrogen clomiphene blocks estrogen receptors in the hypothalamus, opposing the negative feedback of estrogen, causing an increase in gonadotropin-releasing hormone, thereby increasing testosterone release.

3. Selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene bind to estrogen receptors and exhibit agonist or antagonist effects at the estrogen receptors. By indirectly increasing gonadotropin release, SERMs restore endogenous testosterone production upon discontinuation of AASs.¹⁷⁸

4. Selective androgen receptor modulators (SARMs) are nonsteroidal ­tissue ­selective anabolic xenobiotics. SARMS are neither not aromatized and are not substrates for 5α-reductase, nor do they undergo the same metabolic pathways as testosterone. Therefore, they have fewer unwanted androgenic side effects.^73,209

Administration

Approximately 50% of AASs are taken orally. The remainder are administered by intramuscular injection, with one-fourth of intramuscular users sharing needles.^53,152 One third of the needles and syringes exchanged in a needle-exchange program in Wales were used for AAS injections.¹⁵⁹ Unlike therapeutically indicated regimens, which consist of fixed doses at regular intervals, athletes typically use AASs in cycles of 6 to 8 weeks.¹² For example, the athlete may use steroids for 2 months and then abstain for 2 months. Cycling is based on the athlete’s individual preferences and not on any validated protocol. Stacking implies combining the use of several AASs at one time, often with both oral and intramuscular administration. To prevent plateauing, or developing tolerance, to any one xenobiotic, some athletes use an average of five different AASs simultaneously. The doses used are frequently hundreds of times in excess of scientifically based therapeutic recommendations.^3,227Pyramiding implies starting the AASs at a low dose, increasing the dose many times, and then tapering once again. Fat soluble steroids may require several months to be totally eliminated, whereas water soluble steroids may require only days to weeks to be cleared by the kidney. Water soluble testosterone esters are used for “bridging therapy.” Bridging refers to the practice of halting the administration of long lasting alkylated testosterone formulations so that urine analyses at a specific time offer no evidence of use, whereas injections of shorter acting testosterone esters are used to replace the orally administered alkylated formulations. This strategy, which was used extensively in the German Democratic Republic, is documented in a review of the subject based on extensive research of previously classified records.⁶⁵ Clearance profiles for testosterone congeners were determined for each athlete. In general, the daily injection of testosterone esters was used when termination of the more readily detectable synthetic alkylated testosterone derivatives was necessary to avoid a positive doping test. These daily injections of testosterone propionate were halted 4 to 5 days before competition. Corrupt officials involved in doping were sure that the values would decrease to acceptable concentrations in time for the event, based on the science of athletes’ clearance of testosterone esters.⁶⁵

Clinical Manifestations of Anabolic-Androgenic Steroid Use

Cancer.

An association between AAS use and the development of cancer is observed in experimental animals.¹⁸⁰ Testicular and prostatic carcinomas are reported in more frequent users of anabolic-androgenic steroids.^61,71,179 Hepatocellular carcinoma,^100,153 cholangiocarcinoma,^12,80 Wilms tumor, and renal cell carcinoma are also reported in young AAS users.^30,169 The relationship between the dose of AASs and cancer is unknown.

Cardiovascular.

Cardiac complications include acute myocar­dial infarction, venous thromboembolism, and sudden cardiac arrest.^{7,64,87,96,127,129,131,141} Autopsy examination of the heart may reveal biventricular hypertrophy, extensive myocardial fibrosis, and contraction-band necrosis. Myofibrillar disorganization as well as hypertrophy of the interventricular septum and left ventricle are present.¹²⁹ Intense training and use of AASs impair diastolic function by increasing left ventricular wall thickness. Animal models and in vitro myocardial cell studies show similar pathologic changes.^{45,113,142,208,216,217} Doppler echocardiography shows that several years after strength athletes discontinue using AASs, excessive concentric left ventricular hypertrophy remains. Growth hormone may potentiate the effects of AASs and further increase concentric remodeling of the left ventricle.¹⁰⁵ In addition to direct myocardial injury, vasospasm or thrombosis may occur.¹⁴² Alkylated androgens lower the concentration of high-density lipoprotein (HDL) cholesterol and may increase platelet aggregation.^3,64 Thromboembolic complications include pulmonaryembolus,^50,72 stroke,^109,110,196 carotid arterial occlusion,¹²⁰ cerebral sinus thrombosis,¹¹⁷ poststeroid balance disorder,²⁶ and popliteal artery entrapment.¹²⁵

Dermatologic/Gingiva.

Cutaneous side effects are common and include keloid formation, sebaceous cysts, comedones, seborrheic furunculosis, folliculitis, and striae.¹⁹² Acne is associated with steroid use and sometimes is referred to as “gymnasium acne.”^39,162 A common triad of acne, striae, and gynecomastia occurs. The production of sebum is an androgen-dependent process, and dihydrotestosterone is active in sebaceous glands.¹² Gingival hyperplasia is reported.¹⁵⁴

Endocrine.

Conversion of AASs to estradiol in peripheral tissues results in feminization of male athletes. Gynecomastia may be irreversible. AAS use causes negative feedback inhibition of gonadotropin-releasing hormone, luteinizing hormone, and follicle-stimulating hormone from the hypothalamus. This process results in testicular atrophy and decreased spermatogenesis, which may be reversible. In women, menstrual irregularities and breast atrophy may occur.²⁰⁶

Hepatic.

Hepatic subcapsular hematoma with hemorrhage is reported.¹⁹¹ Peliosis hepatis, a condition of blood-filled sinuses in the liver that may result in fatal hepatic rupture, occurs most commonly with alkylated androgens and may not improve when androgen use is stopped.^{13,91,198,225} This condition is not associated with the dose or duration of treatment.^12,99,201 Cyproterone acetate is a chlorinated progesterone derivative that inhibits 5α-reductase and reportedly causes hepatotoxicity.^12,70,75

Infectious.

Local complications from injection include infected joints,⁶⁰ cutaneous abscess,^137,173 and Candida albicans endophthalmitis.²²⁶ Injection of AASs using contaminated needles has led to transmission of infectious diseases such as human immunodeficiency virus and hepatitis B and C.^{152,155,172,175,193,197} Severe varicella is reported in long-term AAS users.¹⁰¹

Musculoskeletal.

Supraphysiologic doses of testosterone, when combined with strength training, increase muscle strength and size.²² The most common musculoskeletal complications of steroid use are tendon and ligament rupture.^{67,89,121,126}

Neuropsychiatric.

Distractibility, depression or mania, delirium, irritability, insomnia, hostility, anxiety, mood lability, and aggressiveness (“roid rage”) may occur.^{17,68,166,167,207} These neuropsychiatric effects do not appear to correlate with serum AAS concentrations.^196,207 Withdrawal symptoms from AAS include decreased libido, fatigue, and myalgias.^108,232

Specific Anabolic Xenobiotics

Dehydroepiandrosterone.

DHEA is a precursor to testosterone (Fig. 40–1). Because it is produced endogenously, DHEA most commonly is not categorized as an AAS. However, DHEA is weakly anabolic and weakly androgenic. Although banned by the FDA in 1996, this xenobiotic subsequently was marketed as a nutritional supplement and is available for purchase without a prescription.²⁰⁶ DHEA is converted to androstenedione and then to testosterone by the enzyme 17β-hydroxysteroid dehydrogenase.^94,128,132 Administration of androstenedione in dosages of 300 mg/d increases testosterone and estradiol concentrations in some men and women.¹²⁴ Women with adrenal insufficiency given DHEA replacement at a dose of 50 mg/d orally for 4 months demonstrated increased serum concentrations of DHEA, androstenedione, testosterone, and dihydrotestosterone. Serum total and HDL cholesterol concentrations simultaneously decreased. Some women experienced androgenic side effects, including greasy skin, acne, and hirsutism.¹⁰ Sense of well-being and sexuality increased in men and women after 4 months of treatment.^10,146,147 The neuropsychiatric effects of DHEA have been demonstrated in animals. Increased hypothalamic serotonin, anxiolytic effects, antagonism at the γ-aminobutyric acid type A (GABA_A) receptor, and agonism at the N-methyl-d-aspartate receptor (NMDA) are demonstrated.^10,133,143

Clenbuterol.

Clenbuterol is a β₂-adrenergic agonist that decreases fat deposition and prevents protein breakdown in animal models.^6,38,90 Clenbuterol is also a potent nutrient partitioning agent, a term implying it can increase the amount of muscle and decrease the amount of fat produced per pound of feed given to cattle and other animals.^68,177 Use of clenbuterol in cattle farming is illegal in many countries. Nevertheless, the consumption of veal liver contaminated with clenbuterol has resulted in sympathomimetic symptoms and positive urine tests in affected individuals.¹⁸³ Clenbuterol is composed of a racemic mixture of (+) and (–) stereoisomers, eliminated in urine in approximately equal amounts. As clenbuterol accumulates in animal meat, stereoisomer ratios change over time and (–) clenbuterol is depleted. By analyzing urinary ratios of clenbuterol stereoisomers, it is possible to differentiate administration of therapeutic clenbuterol preparations from inadvertent ingestion of clenbuterol in meat products.²¹¹ Clenbuterol increases the glycolytic capacity of muscle and causes hypertrophy, enhancing the growth of fast-twitch fibers^134,234 (Chap. 66).

Creatine

Creatine is an amino acid formed by combining the amino acids methionine, arginine, and glycine. It is synthesized naturally by the liver, kidneys, and pancreas. Creatine is found in protein-containing foods such as meat and fish.¹⁵⁴ In its phosphorylated form, it is involved in the rapid resynthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by acting as a substrate to donate phosphorus.²⁰⁵ Because ATP is the immediate source of energy for muscle contraction, creatine is used by athletes to increase energy during short, high-intensity exercise.²²⁰ More than 2.5 million kilograms of creatine are consumed annually in the United States.⁵⁶ Exceptional athletes have admitted to using creatine as part of their training nutritional regimen, leading to interest by athletes at all levels. Numerous studies demonstrate improved performance with creatine supplementation, particularly in sports requiring short, high-intensity effort.^{25,31,84,116,140,220} Creatine is found in skeletal muscle and in the heart, brain, and kidney. Two-thirds of creatine is stored primarily as phosphorylated creatine and the remainder as free creatine.¹⁴ Consuming carbohydrates with creatine supplements increases total creatine and phosphorylated creatine stores in skeletal muscle.⁸³ This process explains why creatine is marketed in combination with carbohydrate. Human endogenous creatine production is 1 g/d, and normal diets containing meat and fish offer another 1 to 2 g/d as dietary intake. One to two grams of creatine is eliminated daily by irreversible conversion to creatinine.²²⁴

Creatine supplementation is most commonly accomplished with creatine monohydrate. A dose of 20 to 25 g/d can increase the skeletal muscle total creatine concentration by 20%.^84,97 Creatine stores do not increase in some individuals despite creatine supplementation. Creatine uptake in skeletal muscle occurs via the creatine transporter proteins at the sarcolemma. Creatine transporter expression and activity, as well as exercise and training, influence the uptake of creatine and the effect of creatine loading on athletic performance.^199,200,202

One adverse effect of creatine supplementation is weight gain, which is thought to result primarily from water retention.^84,140 However, evidence indicates that net protein increase is partially responsible for the weight gain associated with long-term creatine use.¹⁰⁴ Diarrhea was the most commonly reported side effect of creatine use in one study of 52 male college athletes. Other complaints were muscle cramping and dehydration, although many subjects had no complaints.¹⁰³

Creatine supplementation increases urinary creatine and creatinine excretion and may increase serum creatinine concentrations by 20%.^84,104 Long- and short-term creatine supplementation does not appear to have an adverse effect on kidney function.^164,165 One patient who had been taking creatine 5 g/d for 4 weeks developed interstitial nephritis that improved with cessation of creatine use. Whether ingestion of creatine caused the nephritis is unknown.¹¹⁵ A young man with focal segmental glomerular sclerosis developed an elevated creatinine concentration and decreased glomerular filtration rate when creatine supplementation was started. The values returned to baseline upon cessation of creatine supplementation.¹⁷¹ The possibility of developing decreased kidney function is a theoretical concern. Ingestion of large amounts of creatine may result in formation of the carcinogenic substance N-nitrososarcosine, which induces esophageal cancer in rats.^8,9

Human Growth Hormone

Human growth hormone (hGH) is an anabolic peptide hormone secreted by the anterior pituitary gland. It stimulates protein synthesis and increases growth and muscle mass in children. Recombinant hGH (rhGH) has been available since 1984. It is commonly used therapeutically for children with growth hormone deficiency in daily doses of 5 to 26 µg/kg body weight.²²¹

Growth hormone secretion is stimulated by growth hormone–­releasing hormone and is inhibited by somatostatin. Growth hormone receptors are found in many tissues, including the liver. Binding of hGH to hepatic receptors causes secretion of insulinlike growth factor-1 (IGF-1), which has potent anabolic effects and is the mediator for many of the actions of hGH.

Release of hGH, which occurs mainly during sleep, occurs in a pulsatile manner. Exercise stimulates hGH release, and more intense exercise causes proportionately more hGH release.^27,43,206 Amino acids such as ornithine, l-arginine, tryptophan, and l-lysine, increase hGH release through an unknown mechanism and often are ingested for this purpose.^43,86

By causing nitrogen retention and increased movement of amino acids into tissue, hGH stimulates protein synthesis and tissue growth. The effects on increasing muscle mass and size are demonstrated in growth hormone–deficient individuals. Some studies do not support an increase in strength secondary to the increase in muscle size in athletes,^41,130 whereas others demonstrate lean body mass, strength, and power increases.⁸² Growth hormone improves muscle and cardiac function, increases red cell mass and oxygen-carrying capacity, stimulates lipolysis, normalizes serum lipid concentrations, and decreases subcutaneous fat. It also improves mood and sense of well-being.^{41,42,88,185,206,221}

Growth hormone is used by athletes for its anabolic potential. As a xenobiotic of abuse, it is particularly attractive because laboratory detection is difficult. In one survey, 12% of people in gyms used hGH for body building.⁵⁹ In another survey of adolescents, 5% of 10th-grade boys had used hGH.¹⁷⁶ It may be sold illicitly as recombinant hGH.

Administration of hGH may cause myalgias, arthralgias, carpal tunnel syndrome, and edema.⁹⁵ The effects of hGH on skeletal growth depend on the user’s age. In preadolescents, excessive hGH may cause increased bony growth and gigantism. In adults, excessive hGH may cause acromegaly.^217,219 Growth hormone may cause glucose intolerance and hyperglycemia. Skin changes, such as increased melanocytic nevi and altered skin texture, occur.¹⁶³ Lipid profiles may be adversely affected. HDL concentrations are decreased, a change associated with increased risk of coronary artery disease.²³⁵ Because hGH must be given parenterally, there is risk of transmission of infection.¹³⁰ The illicit sale of cadaveric human pituitary-derived growth hormone is associated with a risk of Creutzfeldt-Jakob disease.⁴⁹ Long-term users of hGH may be at increased risk for prostate cancer because of the complications associated with IGF-1⁷⁹ (see below).

Testing for hGH is plagued by the difficulties inherent in testing for exogenous peptide doping in general—the identical amino acid sequences of both rhGH and hGH; the fluctuating, pulsatile secretion; short half-life; and variation in normal concentration depending on sleep as well as stress and exercise status. Unlike the ability to use the differing pattern of N-linked glycosylation in hEPO produced in the human kidney to distinguish it from rhEPO produced in the hamster, hGH has no N-linked glycosylation sites to facilitate differentiation.

There are currently two approaches to detection of hGH currently: the marker approach and the isoform approach.⁹³ The marker approach uses an immunoassay to measure hGH-dependent factors through which hGH exerts its effect, such as IGF-1 and insulinlike growth factor binding proteins, as well as other markers of bone growth and turnover, such as the N-terminal extension peptide of procollagen type III.^23,168 The isoform approach refers to the measurement of the various forms of growth hormone. Whereas rhGH is primarily a 22-kDa monomeric form, pituitary hGH contains multiple isoforms. In athletes using rhGH, endogenous hGH with its multiple isoforms is suppressed through negative feedback on the pituitary. Therefore, the 22-kDa form characteristic of rhGH becomes predominant. The ratio of isoforms, as measured by immunoassay, changes.

Insulinlike Growth Factor-1

IGF-1 is a peptide chain structurally related to insulin. A recombinant form is available.¹⁷⁶ Parenteral administration of IGF-1 is approved for clinical treatment of dwarfism and insulin resistance. Children who develop antibodies to rhGH may respond to IGF-1.

The primary stimulus for release of IGF-1 is hGH, although insulin, DHEA, and nutrition play a role.^146,182 The actions of IGF-1 can be classified as either anabolic or insulinlike.¹⁸² The effects of growth hormone are primarily mediated by IGF-1. IGF-1 is produced in the liver and many other cell types. IGF-1 binds principally to the type I IGF receptor, which has 40% homology with the insulin receptor and a similar tyrosine kinase subunit.²¹³ IGF-1 also binds to insulin receptors, but it has only 1% of insulin affinity for the insulin receptor. IGF-1 increases glucose utilization by causing the movement of glucose into cells, increasing amino acid uptake and stimulating protein synthesis.

Side effects are similar to those associated with use of growth hormone and include acromegaly. Other effects include headache, jaw pain, edema, and alterations in lipid profiles. A potentially serious side effect of IGF-1 is hypoglycemia. High endogenous plasma IGF-1 concentrations are associated with an increased risk for prostate cancer.³⁶

Few studies on the efficacy of IGF-1 in improving the conditioning of athletes are available. IGF-1 can be considered favorably by female athletes because it does not cause virilization.²⁰⁶

Insulin

Insulin is used by body builders for its anabolic properties. Of 20 self-­identified AAS users in a single gym, 25% who had no medical reason to take insulin reported using it to increase muscle mass.¹⁷⁴ These individuals stated that they had injected insulin from 20 to 60 times over the 6 months prior to the study.¹⁷⁴ Their practice was to inject 10 units of regular insulin and then eat sugar-containing foods after injection. As expected, hypoglycemia is reported in body builders using insulin.^56,98,171

Insulin inhibits proteolysis and promotes growth by stimulating movement of glucose and amino acids into muscle and fat cells. It increases the synthesis of glycogen, fatty acids, and proteins⁴⁴ (Chap. 53).

Human Chorionic Gonadotropin (hCG)

In men, the glycoprotein hCG stimulates testicular steroidogenesis. In women, hCG is secreted by the placenta during pregnancy. It may be used by male athletes to prevent testicular atrophy during and after androgen administration.¹¹¹ Analysis of hCG in 740 urinary specimens of male athletes revealed abnormal concentrations in 21 individuals. This finding prompted the IOC ban on hCG use in 1987.^30,43 Presently, distinguishing exogenous hCG administration from hCG production in early pregnancy is not possible, so the urine samples of women are not tested.¹¹¹

Very small amounts of hCG are normally present in men and nonpregnant women.¹²¹ Currently, measurement is made by immunoassay. The decision limit, the concentration at which the test is considered positive, is set at 5 IU/mL urine. Trophoblastic tumors and nontrophoblastic tumors can increase hCG concentrations, and this possibility must be considered in the evaluation of elevated urinary hCG concentration.⁴⁸ Administration of hCG causes an increase in the total testosterone and epitestosterone produced.

Erythropoietin (EPO)

Human erythropoietin (hEPO) is a hormone that, through a receptor-mediated mechanism, induces erythropoiesis by stimulating stem cells. EPO has been available since 1988 as recombinant human erythropoietin (rhEPO), and its use in international competition has been prohibited since 1990. While hEPO is produced primarily by the kidneys, rhEPO is produced in hamsters⁵²; this results in differing glycosylation patterns, an important piece in the laboratory detection of EPO in sports doping.

Because EPO increases exercise capacity and hemoglobin production, it is used by athletes, often with additional iron supplementation. The clinical effects of increased hemoglobin occur several days after administration.^69,156 EPO increases maximal oxygen uptake by 6% to 7%, an effect that lasts approximately 2 weeks after rhEPO administration is completed.⁵⁵

Two EPO analogs exist. Darbepoetin, also known as new erythropoiesis-­stimulating protein, differs from EPO by five amino acids. It has a much longer half-life and can be injected weekly.¹⁵⁷ Another protein, known as synthetic erythropoiesis protein, has a similar protein structure to EPO. The protein polymers created in this molecule have less immunogenicity, fewer biologic contaminants, and more predictable pharmacokinetics.¹⁵⁷

Human EPO is secreted primarily by the kidney, although some is produced by the liver. The mean apparent half-lives of rhEPO are 4.5 hours following intravenous administration and 25 hours after sub­cutaneous administration.¹⁸⁴

EPO enhances endothelial activation and platelet reactivity and increases systolic blood pressure during submaximal exercise.^21,204 These effects, in addition to the increase in hemoglobin, increase the risk of thromboembolic events, hypertension, and hyperviscosity syndromes.^21,139,156 Nineteen Belgian and Dutch cyclists died of uncertain causes between 1987 and 1990.⁵⁴ Increases in hematocrit subsequent to EPO use are believed to have contributed to these deaths. The 1998 Tour de France was marred by the discovery of widespread EPO use by members of several different cycling teams.

An EPO overdose occurred in a patient who self-administered 10,000 units/d for an unknown period of time as a result of a dosing error. The patient presented to the hospital with confusion, a plethoric appearance, blackened toes, decreased pulses, and a hematocrit of 72%. Emergent erythropheresis was performed and resulted in rapid reduction of hematocrit and improvement in the patient’s condition.²³³ Another report of deliberate daily self-administration of an unknown dose of rhEPO resulted in a hematocrit of 70%. The patient was treated emergently with phlebotomy and intravenous hydration and improved.²⁹

Testing for Erythropoietin.

EPO is directly measured by a monoclonal anti-EPO antibody test, which does not distinguish between endogenously produced and exogenously administered recombinant EPO. Therefore, indirect methods of EPO detection are used, such as measurement of hemoglobin or hematocrit.

Previously, some sports-governing bodies, such as the International Cycling Federation and the International Skiing Federation, selected a hematocrit of 50% in men and 47% in women as the action level above which an athlete may be disqualified for presumed EPO use. However, normal hematocrit values vary greatly among athletes. Several studies have shown that hematocrits above the action values of 50% in men and 47% in women are common in athletes. From 3% to 6% of athletes who did not use EPO had hematocrits greater than 50%.²²² Of those athletes living and training at altitudes between 2000 and 3000 meters above sea level, 20.5% had hematocrit values higher than 50%.²²² Other studies confirm the increased hematocrits of athletes training at altitudes of 1000 to 6000 meters.^{19,188,189,222}

Although many endurance athletes may have increased blood volume, the hematocrit may be lowered because of the increased plasma volume, which exceeds the RBC volume. This dilutional pseudoanemia is sometimes called sports anemia.¹⁹⁵ Additionally, hematocrit measurements are affected by hydration status, posture (upright versus supine), and nutrition, and they demonstrate an approximately 3% diurnal variation.¹⁸⁷ Because of natural variations among individuals, postural effects, and the ease of manipulation through saline infusion, indirect detection of EPO use by hematocrit measurement is fraught with potential for error.¹⁵⁶

Several methods have been studied to detect the use of rEPO by athletes. The ratio between serum soluble transferrin receptors (sTfr) and ferritin was used as an indirect method for detection of EPO use. The sTfr is released from RBC progenitors. EPO stimulates erythropoiesis and causes an increase in sTfr and a decrease in ferritin.⁷⁴ Individuals with other causes of polycythemia or accelerated erythropoiesis also can exhibit increased ratios and be falsely accused of EPO use. An increased hematocrit with sTfr greater than 10 µg/mL and sTfr-to-serum protein ratio greater than 153 has been proposed as an indirect measurement of EPO use.¹²

At the 2000 Olympics in Sydney, Australia, a combination of multiple indirect markers was developed for detection of altered erythropoiesis and rhEPO use.¹⁵⁶ Current EPO use, known as the “ON-model,” and recent, but not current, use of EPO, known as the “OFF-model,” were identified by measured laboratory values. For example, five variables predict current rhEPO use: reticulocyte count, serum EPO concentration, sTfr, hematocrit, and percentage of macrocytes. The three variables in combination, including hematocrit, reticulocyte count, and serum EPO concentration, were the best mechanism for detecting recent rhEPO use.¹⁵⁶ A major drawback to this method is the instability of these variables in whole blood, so that confirmatory testing of the split blood sample is impossible.¹⁵⁷

State-of-the-art detection of EPO doping is accomplished by two techniques: isoelectric focusing and immunoblotting performed on urine samples. The two isoforms of EPO, recombinant and endogenous, have different glycosylation patterns and glycan sizes, resulting in differing molecular charges.¹⁵⁷ An immunoblotting procedure takes advantage of these different net charges, and the proteins can be separated by their charges when they are placed in an electric field.¹²³ Subsequently, by isoelectric focusing, this method obtains an image of EPO patterns in the urine.¹²² WADA considers a positive urine test result by this method definitive, even without the blood testing of indirect markers.¹⁵⁷ Because of the structural similarity of darbepoetin to EPO, these detection techniques also are effective for darbopoietin.¹⁵⁷

Athlete’s Biological Passport.

A significant development in the detection of blood transfusion, known as blood doping, is the development of a longitudinal record of an athlete’s red blood cell (RBC) parameters called the athlete’s biological passport. The detection of plasticizer metabolites from blood storage and alteration of gene expression resulting from infusion of autologous blood are proposed adjuncts to detection of autologous blood transfusion.¹⁴⁸ The observation of physiological parameters over time has made it easier to detect use of performance enhancing agents.¹⁶

Caffeine

Caffeine is a central nervous system stimulant that causes a feeling of decreased fatigue and increases endurance performance^58,158 (Chap. 66). These changes may occur through several different mechanisms, including increased calcium permeability in the sarcoplasmic reticulum and enhanced contractility of muscle, phosphodiesterase inhibition and ­subsequent increased cyclic nucleotides, adenosine blockade leading to blood vessel dilation, and inhibited lipolysis. Caffeine is no longer prohibited by World Anti-Doping Code 2005 Prohibited List. Caffeine and pseudoephedrine are included in a monitoring program that was implemented by the WADA to detect patterns of misuse for substances that are no longer on the Prohibited List.²¹⁵

Amphetamines

The beneficial effects of amphetamines in sports result from their ability to mask fatigue and pain.⁴⁶ Initial studies done in soldiers showed that they could march longer and ignore pain when taking amphetamines (Chap. 76).²¹⁴ In one study in college students, resting and maximal heart rate, strength, acceleration, and anaerobic capacity increased. However, although the perception of fatigue decreased, lactic acid continued to accumulate and maximal oxygen consumption was unchanged.³⁷ Other studies have shown no significant effects on exercise performance¹⁰⁷.

Sodium Bicarbonate

Sodium bicarbonate loading, known as “soda loading,” has a long history of use in horse racing.¹⁵ Sodium bicarbonate may buffer the metabolic acidosis associated with an elevated lactate caused by exercise, thereby delaying fatigue and enhancing performance.⁷⁶

During high-intensity exercise, metabolism becomes anaerobic and lactic acid is produced. Intracellular acidosis is said to contribute to muscle fatigue by reducing the sensitivity of the muscle contractile apparatus to calcium.¹⁶⁰ Several studies demonstrated improved performance in running when sodium bicarbonate was ingested 2 to 3 hours before competition.^40,181 The study dose was 0.2 to 0.3 g/kg body weight of sodium bicarbonate, approximately 160 mEq of sodium bicarbonate per day. The effects of sodium bicarbonate are greatest when periods of exercise last longer than 4 minutes because anaerobic metabolism contributes more to total energy production and energy from aerobic metabolism diminishes.^76,77 Adverse effects of bicarbonate loading include diarrhea, abdominal pain, and the possibility of hypernatremia.⁷⁶

An animal model demonstrated that intracellular acidosis associated with lactate production reversed muscle fatigue.^4,160 Previously, intracellular acidosis was thought to contribute to muscle fatigue by reducing the sensitivity of the muscle contractile apparatus to calcium, decreasing the force of muscle contraction. However, the mechanism of excitation-contraction is complex. Because it permeates membranes easily, chloride is an important ion in maintaining and stabilizing the muscle fiber resting membrane potential at normal pH. Because of this, a large sodium current is needed to overcome membrane stabilization and produce an action potential. In the case of intracellular acidosis, the permeability of the membrane to the chloride ion is reduced, the resting membrane potential is no longer stabilized, and less inward sodium influx is needed to produce an action potential. The excitability of the muscle T-tubule system is therefore increased by acidosis, protecting against muscle fatigue.¹⁶⁰

The WADA bans nonmedical diuretic use.²²⁹ Diuretics are used in sports in which the athlete must achieve a certain weight to compete in discrete weight classes. In addition to weight loss, body builders find that diuretic use gives greater definition to the physique as the skin draws tightly around the muscles.² In one report, a professional body builder attempted to lose weight using diuretics including bumetanide and spironolactone as well as potassium supplements. He presented with hyperkalemia and hypotension² (Chap. 19). Diuretics also result in increased urine production, thereby diluting the urine and making the detection of other banned xenobiotics more difficult.^32,47

Enormous amounts of energy and money are expended to determine the presence or absence of performance-enhancing xenobiotics. Analysis of samples on the international level is performed by a limited number of accredited laboratories. The majority of tests are performed on urine, with careful procedural requirements regarding handling of samples. Attention must be paid to proper storage of specimens, because bacterial metabolism may increase urinary steroid concentrations.^24,51 Upon arrival of a sample at the testing laboratory, the integrity of the sample is checked, including the code, seal, visual appearance, density, and pH. Registration of the sample is completed, and the sample is divided into two aliquots. All testing is done on the first aliquot, and any positive results are confirmed on the second aliquot. The aliquots are commonly referred to as sample A and sample B. Sample preparation is difficult and time consuming.

The complexity of the laboratory testing and continuous attempts to evade detection is illustrated in the discovery of an AAS previously undetectable by standard sport doping tests of urine. In the following situation, there was no preexisting reference data for the unknown xenobiotic. In the summer of 2003, a used syringe was provided anonymously to the US anti-doping authority. Through a painstaking process of analyses, a previously unrecognized chemical in the syringe was identified as a derivative of the AAS norbolethone, a known reference compound, leading to the discovery, synthesis, and detection of tetrahydrogestrinone (THG), a new chemical unknown as a pharmaceutical or veterinary compound.³⁴ Since the discovery of THG, the identity of another designer steroid, madol, or desoxymethyltestosterone, was similarly discovered and the structure synthesized.¹⁹⁴

Capillary Gas Chromatography–Mass Spectrometry

Capillary gas chromatography allows the determination of approximately 95% of all doping positive results. Gas chromatography typically is combined with mass spectrometry for detection of the majority of substances.¹⁵⁰ Analysis of the urine by gas chromatography–mass spectrophotometry (GCMS) is the current standard for detection of AASs.³² Such analysis relies on a large amount of previously derived reference data.³⁴

Testosterone-to-Epitestosterone Ratio

GCMS cannot distinguish endogenous testosterone from pharmaceutically derived exogenous testosterone. Therefore, other methods of detection are needed. One way of detecting the use of exogenous testosterone is to measure the testosterone-to-epitestosterone (T/E) ratio. Epitestosterone is not a metabolite of testosterone, but its a 17-α epimer, differing from testosterone only in the configuration of the hydroxyl group on C-17. Men produce 30 times more testosterone than epitestosterone; however, 1% of testosterone and 30% of epitestosterone is excreted unchanged in the urine. Therefore, the normal T/E ratio in the urine is about 1:1.²⁰³ A T/E ratio less than 4:1 is considered acceptable; a T/E ratio greater than 4:1 is considered evidence of doping using testosterone. In order to maintain a normal T/E ratio, an athlete may self-administer both testosterone and epitestosterone.³³

The overall pattern of the T/E ratio over time is important. Athletes subjected to testing will have previous measurements of their T/E ratios on record with antidoping authorities, or additional tests may be obtained to establish a pattern of T/E ratios. These results are plotted against time. The mean, standard deviation, and confidence values are calculated.³³ The confidence values of three or more samples taken over months will be less than 60% unless the athlete is using testosterone.³⁴ Sudden variations in an athlete’s pattern of T/E ratios over time is a cause for further testing.

In one high-profile doping case where the T/E ratio was elevated, the athlete suggested that ethanol consumption the previous night caused the elevated T/E ratio. In this regard, there is evidence that at very high doses, for example, 2 g/kg, ethanol increases the T/E ratio, although the T/E ratio did not exceed 6:1.⁶² This effect of ethanol is more pronounced in women and is limited to 8 hours postingestion of ethanol. The mechanism of this effect of ethanol may be that it increases the NADH-to-NAD⁺ ratio and many steroid oxidation-reduction reactions are dependent on the relative abundance of NADH to NAD⁺.⁶² Ketoconazole inhibits testosterone synthesis and may cause a decrease in the T/E ratio within 6 hours of administration.¹¹²

Isotope Ratio Mass Spectrometry

Carbon is made up of six protons and six neutrons, giving it an atomic weight of 12 (¹² C). Sometimes carbon has an extra neutron, giving it an atomic weight of 13 (¹³ C). Carbon is derived from carbon dioxide in the atmosphere. Warm climate plants, such as soy, process carbon dioxide differently than other plants, using different photosynthetic pathways for carbon dioxide fixation, causing the depletion of ¹³C.¹⁸⁶ Pharmaceutical testosterone is made from plant sterols, primarily soy plants, and therefore has less ¹³C isotope than endogenous testosterone, made in the body from a typical human diet based in corn and not soy. This difference in isotope ratios is measured by isotope ratio mass spectrometry. An athlete’s natural carbon makeup is determined by analysis of an endogenous reference compound such as the testosterone precursor cholesterol. Cholesterol may be called an “autostandard” because it represents the athlete’s ¹³C/¹² C ratio.²⁰ Finally, it is the difference between the ratio of the athlete’s ¹³C/¹² C ratio and an international standard ratio that is measured and reported.¹ Values are negative because both endogenous and pharmaceutical testosterone contain less ¹³C than the international standard.⁸⁵

Insulin

Laboratory detection methods for insulin are not yet standardized and accredited by WADA. Therefore, athletes are not currently tested for insulin use. The technology for testing for insulin uses immunoaffinity purification followed by liquid chromatography–tandem mass spectrometry to identify analytes including urinary metabolites of insulin.²¹² When insulin is modified to improve its receptor selectivity or give it other favorable properties, the change in molecular weight or amino acid profile from human insulin makes it detectable by GCMS.²¹²

Masking Xenobiotics

Any chemical or physical manipulation done with the purpose of altering the integrity of a urine or blood sample is prohibited by WADA.²²⁹ For example, use of intravenous fluids for dilution of the sample, or urine substitution, is prohibited. Some xenobiotics are added to the urine for the sole purpose of interfering with urine testing and are easily detected. Examples include “Klear,” which is 90% methanol, and Golden Seal tea, which produces colored urine.²⁸ Other commercially available products include “Xxtra Clean,” which contains pyridinium chlorochromate, and “Urine Luck,” which contains glutaraldehyde.

Niacin has been used to alter urine test results, although there is no evidence it is effective for this purpose. There are reports of niacin toxicity, including skin reactions such as itching, flushing, and burning when niacin is used for this purpose.^35,144 More serious symptoms, including nausea, elevated liver enzymes, hypoglycemia, and anion gap metabolic acidosis, are reported as a result of ingesting niacin in large amounts, in the 2.5 to 5.5 g range over 1 to 2 days.^35,144

A significant issue in the analysis of urine for the presence of prohibited peptides such as rEPO is the masking potential of proteases surreptitiously added to urine specimens slated for doping analysis. The proteases are packaged in “grains” known as protease granules or “rice grains” and placed in the urethra.²⁰⁹ Upon urination for the purpose of providing a specimen for doping analysis, the grain flows with urine into the specimen cup. The proteases, including trypsin, chymotrypsin, and papain, will quickly degrade renally excreted peptides, most notably EPO, making them undetectable. By the process of autolysis, proteases may themselves become undetectable over time. In one report, urine with elevated protease concentrations greater than 15 µg/mL underwent further analysis to identify urinary proteins such as albumin.²⁰⁹ Normally, the presence of urinary proteins creates the image of a visible band by gel electrophoresis. However, the urine with elevated protease activity may demonstrate something called trace of burning, a term indicating an absence of proteins.²¹⁰ In this report, suspicious specimens were subjected to further testing using liquid chromatography–­mass spectrometry. After further molecular sequencing of derived proteins, human proteases can be distinguished from nonhuman proteases, such as bovine chymotrypsin or papain. The addition of a protease inhibitor to urine samples immediately after collection may be a future strategy to control the effectiveness of protease addition as a masking method.²¹⁰

In approximately 15% of urine samples performed in anti-doping laboratories, there is no endogenous or rEPO detectable by immunoelectrophoresis.¹¹⁹ Undetectable EPO occurs more commonly in competition, the “competition effect.” This is due in part to circumstances that may be unrelated to doping, such as physiological variation in EPO production, gender, and very low or very high urine specific gravity. However, doping with exogenous rEPO and inhibition of endogenous hEPO production or addition of proteases are other possible causes. Strenuous effort causes a shift in the isoforms of EPO yielding a more basic isoelectric point, a result known as “effort” urine.¹¹⁸ In any case, a urine deemed as suspicious may ultimately not result in a positive doping test result.^119,209

The list of prohibited masking xenobiotics includes diuretics, epitestosterone, probenecid, plasma expanders such as albumin, dextran, and hydroxymethyl starch, and α-reductase inhibitors such as finasteride and dutasteride.²²⁹ Probenecid blocks urinary excretion of the glucuronide conjugates of AAS.

Gene Doping

The discovery of the genetic codes for some diseases has made gene therapy of medical conditions, such as muscular dystrophy, a reality. It is now conceivable that this technology can be used to enhance athletic performance. Gene doping is included on the WADA 2013 Prohibited List.²³⁰ Gene doping is defined as “the non-therapeutic use of cells, genes, genetic elements, or of the modulation of gene expression, having the capacity to enhance athlete performance.”²²⁹ For example, insertion of a gene sequence could produce a desired effect, such as large muscles or increased body production of potentially advantageous substances such as testosterone or growth hormone. In animal models, genes for EPO lead to erythropoiesis and genes for IGF-1 produce increased muscle size and strength.¹⁸ Myostatin, which belongs to a family of proteins that control growth and differentiation of tissues in the body, inhibits skeletal muscle growth.^102,190 Mutations of the myostatin gene may result in muscle hypertrophy. In dogs, the athletic performance of racing whippets is enhanced in those animals with a myostatin gene mutation.¹⁴⁹ In humans, a report of an extremely muscular baby born with a mutation in the myostatin gene illustrates the potential effect of gene alterations on athletic performance. The mother of this infant was a professional athlete, and other members of the family were known for their strength.¹⁹⁰ Transcription regulators such as AMP-activated protein kinase (AMPK) are exercise mimetics and increase muscle endurance when administered orally in animal models.^81,151 Influencing the expression of the transcription factor peroxisome proliferator-activated receptor-δ (PPAR-δ), a nuclear hormone receptor hormone protein, leads to increased formation of slow twitch skeletal muscle. Angiotensin II receptor blockers such as telmisartan influence both the AMPK pathway and up-regulate PPAR-δ expression improving muscle performance.⁶³

Many unexpected deaths in certain groups of young competitors have occurred in the absence of obvious medical or traumatic causes. In some of these cases, the use of performance-enhancing drugs was linked to the deaths. The use of EPO, introduced in Europe in 1987, may have contributed to the large number of deaths in young European endurance athletes over the next several years.^{57,78,138,161,223} In young healthy athletes experiencing cerebrovascular events or myocardial infarction, the temporal link between the use of cocaine, ephedrine, or performance enhancers such as AASs suggests a role for these xenobiotics as precipitants of these adverse events.¹³⁵ Nevertheless, the leading cause of nontraumatic sudden death in young athletes is most often associated with cardiac anomalies.¹³³ In autopsy studies of athletes in the United States with sudden death, hypertrophic cardiomyopathy is the most common structural abnormality, accounting for more than one-third of the cardiac arrests, followed by coronary artery anomalies.¹³⁵ In Italy, dysrhythmogenic right ventricular cardiomyopathy is implicated in one fourth of these deaths.^{66,106,136,218} Medical causes of sudden death other than cardiac causes include heat stroke (Chap. 30), sickle cell trait, and asthma.

• Although the press spotlights a few world-class athletes, the vast majority of individuals using performance-enhancing substances are not in the public view. Some individuals suffer adverse consequences. The knowledgeable clinician will identify these health effects when they occur and educate susceptible individuals on the health risks of using performance enhancing substances.

• Continuous development and refinement of anti-doping laboratory methods broadly benefits our understanding of the physiology of exercise.

• WADA is the international body responsible for coordinating anti-doping efforts nationally and internationally, and as such the WADA Prohibited List sets the standard for methods and substances barred in sport.

References