Accepted Manuscript Detection of anabolic androgenic steroid use by elite athletes and by members of the general public Bradley D. Anawalt PII:
S0303-7207(17)30512-9
DOI:
10.1016/j.mce.2017.09.027
Reference:
MCE 10087
To appear in:
Molecular and Cellular Endocrinology
Received Date: 20 September 2017 Accepted Date: 20 September 2017
Please cite this article as: Anawalt, B.D., Detection of anabolic androgenic steroid use by elite athletes and by members of the general public, Molecular and Cellular Endocrinology (2017), doi: 10.1016/ j.mce.2017.09.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Detection of Anabolic Androgenic Steroid Use by Elite Athletes and by Members of the
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Bradley D. Anawalt, MD Professor of Medicine Department of Medicine University of Washington Box 356420 1959 NE Pacific Street Seattle, WA 98195
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General Public
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Key words: Anabolic androgenic steroids, testosterone, sex steroids, anti-doping, biological passport
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Abstract
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Because national and international sports competitions are sources of community pride and financial revenue, there have been great efforts to prevent and detect the use of performanceenhancing drugs such as anabolic androgenic steroids by elite athletes. The World Anti-Doping
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Agency and its national affiliate anti-doping agencies have created sophisticated monitoring systems and advanced testing techniques to detect the use of banned substances including
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anabolic androgenic steroids by participants in international and national athletic competitions. The creation of a longitudinal monitoring program known as the biological passport is a recent, important development in the efforts to prevent and detect the use of banned performanceenhancing drugs and methods. The biological passport program consists of the measurement
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of urinary and blood markers of anabolic androgenic steroid use (and other banned drugs or methods) at baseline and at random times. A panel of experts reviews the longitudinal data and interprets the likelihood of the use of banned drugs and methods. These advances in anti-
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detection process.
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doping appear to be highly effective, but some athletes persist in their efforts to cheat the
In addition, some members of the general public use anabolic androgenic steroids for a variety of reasons including to improve physical appearance or to enhance performance in athletics. Clinicians must depend on clinical acumen and the measurement of serum testosterone and gonadotropins to guide them in making a tentative diagnosis of anabolic androgenic steroid use. Definitive diagnosis requires that the patient disclose the use of the drugs.
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Because anabolic androgenic steroids are effective for improving certain aspects of physical performance, some elite athletes (and members of the general public) will continue to use
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these drugs. Effective efforts to curtail the use of AAS will require decreasing the ease of access to them, continued advancements in laboratory techniques, further research into their effects, and effective education of the general public about the known and potential adverse health
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athletic performance and muscular appearance.
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effects. Most importantly, there will need to be a shift from the current societal adulation for
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1.1 Introduction Detection of unapproved use of anabolic androgenic steroids (AAS) is important in competitive
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sports where the governing agencies have opted to ban the use of these agents or to restrict the use of such drugs to specific, approved clinical scenarios through a process known as
“therapeutic use exemption”. The rationale for restriction of the use of AAS is to prevent male
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and female athletes from achieving a competitive advantage via the performance-enhancing
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effects of AAS. In clinical practice, detection of AAS use is also important primarily when clinicians are evaluating men for management of side effects of AAS such as infertility. Because AAS remain readily available through non-prescription sources, clinicians also need to recognize and manage the use of AAS in men who are not elite athletes, but who are using AAS to
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increase musculature, improve performance in community athletic events or for non-evidencebased indications including improved sexual function or longevity.
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In this review, I focus on the World Anti-Doping Association (WADA) process of screening and detection of the use of AAS or pharmacological agents that increase endogenous androgens.
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This WADA process is the basis for regulation of AAS use in many international athletic competitions including the Olympic games. Governing bodies of international and national sports athletics that wish to regulate the use of AAS in their sport generally agree to follow the WADA guidelines although they may apply internal rules about the frequency and type of monitoring. I will also discuss a pragmatic approach to the detection of AAS use in clinical practice.
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The barriers to detection of AAS use are formidable for several reasons. First, there is significant variation in normal production and metabolism of sex steroid hormones in humans
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as well as a range of sensitivity to circulating sex steroid hormones. These natural variations lead to a broad range of circulating sex hormone concentrations in genotypic men and women with normal, congruent phenotypes and the establishment of normal ranges of serum hormone
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concentrations that are based on the statistics of normal distributions in populations and not
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on the physiology of an individual. Distinguishing between an athlete with naturally high testosterone concentrations and an athlete using testosterone or testosterone precursor supplements is difficult. Second, measurement of AAS in blood and urine samples must be accurate and precise (i.e., measure AAS analytes correctly and reproducibly). There have been
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technological advancements that have dramatically improved the measurement of AAS, but there remain challenges, particularly as the development of novel, designer AAS advances rapidly. Third, collection of the biological sample for analysis must be done in a manner that
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reflects the individual’s physiology. Collection of such biological samples is often inconvenient (e.g., early morning blood sample), invasive to privacy (e.g., witnessed urine collections to
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prevent substitution of a different urine sample) and costly (due to costs of locating, traveling to and collecting specimen from the athlete for random testing). Fourth, to avoid the possibility of tampering, the biological samples must be kept secure at every step of the process after collection: transportation, processing, testing and storage.
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Finally, there are sociological barriers to creating effective screening and identification of AAS use in elite athletes. These barriers include extraordinary personal incentives for elite athletes to use AAS; a centimeter or a few milliseconds mean the difference between gold and
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adulation or ignominy. Many elite athletes who have spent years training will do almost
anything to win. A survey of 212 participants in a Canadian national track meet demonstrated that greater than 5% of participants would take a legal performance-enhancing drug that would
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result in death within 5 years if the drug would guarantee an Olympic gold medal.1 In addition,
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greater than 10% of these elite athletes would take an illegal, but undetectable, performanceenhancing drug if the drug would guarantee an Olympic gold medal. With the personal stakes so high, athletes will attempt to cheat detection of AAS use with a variety of methods such as the following: 1) use of AAS with drugs that interfere with metabolism of AAS (known or novel aromatase inhibitors or 5-α reductase inhibitors)2; 2) use of AAS with diuretics or probenecid to
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lower urinary concentrations (by dilution or decreased excretion of acidic AAS conjugates respectively); 3) use of designer AAS that are not currently detectable; 4) discontinuous use of
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AAS with very short half-lives (to increase total exposure to AAS but have undetectable concentration at testing times). The WADA screening and identification process (Figure 1) is
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intended to thwart these pharmacological efforts to cheat.
Additional sociological and cultural barriers to detection of AAS use include the increasingly fluid definitions of sex and gender and the recent appropriate focus on the rights and privileges of individuals with disorders of sex determination or of those individuals with endocrinopathies associated with alterations in circulating sex steroid concentrations. Designation of different threshold values of circulating sex steroids as normal for women and men creates significant
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debate among experts and members of the general public about fairness and justice for all athletes: genotypically and phenotypically concordant athletes, athletes with disorders of sexual development and transgender athletes. As a result of a challenge by an Indian track athlete,
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Dutee Chand, the Court of Arbitration for Sports has temporarily suspended the International Association of Athletic Federations’ regulations (that included meeting a criterion for a normal
2.1 Detection of AAS use by elite athletes
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female range for serum testosterone) for participation as a female in international competitions.3
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For the WADA guidelines, the key principle for detection of AAS use is the establishment of an athlete’s “biological passport”. The concept of a biological passport is simple. An athlete’s urinary and serum testosterone, dihydrotestosterone, epitestosterone (another endogenous testicular steroid) and their precursors are measured at baseline (prior to the first competition
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that has anti-doping regulations). Repeat measurements of the athlete’s steroid and steroid metabolite concentrations are followed longitudinally for any significant deviation from
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baseline. The biological passport is intended to detect attempts to use exogenous AAS or attempts to increase endogenous AAS by drugs that stimulate endogenous production of AAS.
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All exogenous AAS suppress endogenous testosterone, dihydrotestosterone and epitestosterone production, and administration of any agent to stimulate supranormal endogenous androgen production alters the urinary concentrations of testicular steroids and/or testicular steroid precursors.
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WADA has developed a sophisticated monitoring and detection process for banned performance enhancing drugs (“doping”) by elite athletes competing in national and international events (Figure 1).4 For AAS doping, this process includes the following:
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1) establishment of a secure database for the biological passports of individual athletes 2) a network that includes personnel in charge of monitoring individual athletes,
experts to interpret the testing results
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personnel to collect biological samples from athletes (e.g., blood and urine) and
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3) A system for locating the whereabouts of athletes and notifying them of a required sample collection at a specified time on the day of notification (i.e., without advanced notice)
4) Collection of the sample by an approved, trained person (“collection officer”) within
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a defined window of time on the day of notification
5) Secure transportation of the sample to an approved laboratory for testing and storage
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6) Analysis of the sample for markers of AAS 7) Entry of results into the secure database for longitudinal interpretation of individual
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data
8) Interpretation of results by experts
Each step must be documented. The collection officer must document that the athlete did not train within 2 hours of the blood sample collection and the circumstances of training in the 2week interval prior to blood or urine collection (e.g., training at altitude or using hypoxic tent). There are strict criteria regarding timing and processing of sample collection (e.g., how long the
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athlete must sit before venipuncture, custody of the specimen (e.g., prevention of tampering), transportation of the sample (e.g., temperature of sample container) and laboratory specimen
“B”), but typically only one blood sample is collected.
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handling and testing procedures. Two urine samples are always collected (samples “A” and
2.2. Specific tests for detection of use of exogenous AAS drugs or precursors of endogenous AAS by elite athletes
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For a given sport, the governing body of the sport may determine the screening process for
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detection of AAS use, but the 2017 WADA guidelines stipulate that the minimal screening process for AAS consists of measurement of the following core panel of urinary steroid markers: testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstane-3α,17β-diol (5αAdiol) and 5β-androstane-3α,17β-diol (5βAdiol). WADA requires that these markers be
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measured by gas chromatography combined with mass spectroscopy.
The normal range of these urinary steroids has been well defined in men and women with
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normal gonadal and adrenal function, and their absolute concentrations and ratios can be used to detect AAS (including testosterone) use. The best known use of these urinary markers of
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AAS use is the testosterone to epitestosterone ratio. A high testosterone to epitestosterone ratio suggests exogenous testosterone administration. Low or undetectable testosterone and epitestosterone concentrations suggest use of an AAS other than testosterone. Exogenous AAS use is also associated with abnormal concentrations and/or ratios of the remainder of the WADA core panel of urinary steroid markers.
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Any of the following urinary test results are considered suspicious: testosterone/epitestosterone > 4, androsterone/testosterone < 20 or 5αAdiol/5βAdiol > 2.4.
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High concentrations of testosterone, epitestosterone, androsterone, etiocholanolone, or
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5αAdiol are also considered suspicious.4
2.3. Screening and detection of the use of drugs that increase production of endogenous
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AAS.
There are a number of substances that increase circulating endogenous androgens by stimulating production. These agents include luteinizing hormone (LH), human chorionic gonadotropin (hCG), androgen precursors, aromatase inhibitors and selective estrogen receptor
of androgens to estrogens.
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modulators. Aromatase inhibitors also increase serum androgens by inhibiting the metabolism
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Urinary immunoassays that have met strict performance criteria are used to detect elevated concentrations of LH and the α−β hCG.5 Assays using liquid or gas chromatography and mass
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spectroscopy are used to detect use of aromatase inhibitors and selective estrogen receptor modulators.6,7
Use of high dosages of precursors of endogenous AAS raise endogenous AAS production by mass action. The use of high dosages of endogenous AAS precursors is also detected by the WADA core panel of urinary markers because such use of endogenous AAS precursors alters the
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absolute concentrations and ratios of these markers. For example, administration of dehydroepiandrosterone (also known as DHEA or prasterone), a WADA-banned precursor of testosterone, significantly increases urinary androsterone, etiocholanolone, 5αAdiol and
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5βAdiol conjugate concentrations and results in a high androsterone/testosterone ratio.8
2.4. Confirmatory testing for AAS use by elite athletes
Confirmatory testing consists of re-measurement of any suspicious markers in an additional
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aliquot of the athlete’s original “A” sample; the “B” sample is used as necessary for additional confirmatory testing if the “A” sample tests positively for a suspicious substance.4. Concentrations are adjusted for the specific gravity of the urine specimen. The laboratory will
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also assess for the presence of compounds (or their metabolites) known to affect the metabolism or measurement of AAS including ethanol, 5-α reductase inhibitors and
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ketoconazole.4,9 Although there have been some in vitro studies suggesting that green and white teas and nonsteroidal anti-inflammatory drugs might affect the urinary steroid profile, these observations have not been confirmed by in vivo experiments.10 In addition, the laboratory will assess for signs of severe degradation of the sample by microbes. Signs of severe degradation include increased concentrations of 5α-androstanedione and/or 5β-
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androstanedione. Signs of severe degradation due to microbes may invalidate the sample for further studies.
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If re-measurement confirms the abnormal marker concentrations or ratios, then the laboratory performs gas chromatography-combustion-isotope ratio mass spectrometry to quantify the prohibited substance(s) and performs DNA analysis to confirm the source of the sample.4,11-13
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The final steps of the process of determination of whether an athlete has used an AAS involves
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expert interpretation of the laboratory results with comparison of the results to previous results in the athlete’s biological passport.4,14,15 The athlete’s identity is withheld from the experts during the interpretation process, but the experts may be provided information (“intelligence”) about the athlete. Such information includes information from any criminal
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investigation by law enforcement agencies in pursuit of suspected trafficking of prohibited substances. Determination of an AAS use violation requires a unanimous conclusion of “doping
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likely” by three independent experts.
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2.4.1. Specific additional tests for AAS use by elite athletes Although the WADA guidelines state that gas chromatography plus mass spectroscopy is satisfactory for initial testing, gas chromatography plus tandem mass spectroscopy generally is required to meet WADA stated lower limit of detection for AAS, and this assay technique is likely to become the new standard.16
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Historically, carbon isotope testing has been used to distinguish between endogenous and exogenous sources of testosterone, epitestosterone, dihydrotestosterone, nandrolone and dehydroepiandrostenedione.18-20 Endogenous androgens have a higher percentage of 13C than
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synthetic forms of androgens. Human androgens are produced from endogenous cholesterol or exogenous cholesterol using carbon that is ultimately derived from a mixture of plant
sources including plants such as maize, sugar cane millet and pineapple that contain high
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amounts of 13C .20 The carbon source for synthetic testosterone is derived from yams and soy
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that are low in 13C and high in 12C.20 Thus, the ratio of 13C/12C is higher in endogenous androgens than pharmaceutical androgens. This ratio can be determined by isotope ratio mass spectrometry. The test can be individualized by measuring the athlete’s cholesterol 13C/12C ratio to use as the comparator. This test is too expensive for routine use, and it is no longer a
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selected cases.
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formal component of the WADA testing algorithm. This technique remains potentially useful in
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2.5. Novel tests for AAS use by elite athletes In addition, AAS testing has typically depended on the detection of the glucuronidated forms of AAS, but sulfonated forms of AAS have longer half-lives and might prove to be useful or even superior for detection of AAS use.21,22 Novel assay techniques, such as high resolution liquid chromatography plus high resolution mass spectrometry, show promise for rapid screening for AAS.23,24
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2.6. Potential pitfalls with the WADA passport system of detecting AAS use in elite athletes There are many sources of potential error or uncertainty with the WADA passport system.
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Sources of error include mishandling of collection, processing, transportation or storage of blood or urine specimens. An important potential source of error is ensuring that the baseline sample for the biological passport reflects the athlete’s unadulterated, normal state. The
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passport paradigm also does not fully account for athletes with biological variants or female
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athletes with disorders of sexual development. For example, there are known genetic polymorphisms of the principal enzymes (uridine diphosphate glucuronosyltransferases) that glucuronidate endogenous androgens. One specific polymorphism in the gene coding for uridine diphosphate glucuronosyltransferase 2B significantly lowers the urinary testosterone to
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epitestosterone glucuronide ratio, and ethnic variations in this polymorphism are associated with significant differences in the urinary testosterone to epitestosterone ratio in normal, nonAAS using individuals.25-27 Determining the correct baseline concentrations and ratios of
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testosterone to epitestosterone can be difficult due to these polymorphisms. Likewise, for athletes with disorders of sexual development, what is the correct baseline hormone
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concentrations for the biological passport? Must such athletes have hormone concentrations within the normal range of the general population?
The passport also does not account for the effects of aging in male athletes. Several studies have demonstrated that serum testosterone concentrations decline in many (but not all men)
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beginning as early as the fourth and fifth decade of life.28-31 How does one interpret unchanging serum androgen (and metabolite) concentrations in a man between ages 18-45?
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Finally, despite the significant progress in the development of testing protocols and assays for the detection of AAS, the athletes continue to have high incentive to use AAS and attempt to evade detection. Novel AAS and masking agents are likely to be under continual development,
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and even the passport process coupled with advanced assay techniques such as gas
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chromatography with tandem mass spectroscopy might not be fool-proof.
2.6. Detection of AAS use in men and women who are not elite athletes Men (and a very small number of women) may use AAS to enhance their appearance or to
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improve performance in non-elite athletic competition. The public has access to AAS through the internet and illicit sites, and there is no mechanism or monitoring program to detect AAS use by non-elite athletes or other members of the general public. Thus, if an individual who is
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not an athlete monitored by the WADA passport system wishes to use AAS, it is not possible to
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prevent or reliably detect such use.
There is no practical diagnostic test to detect AAS use in this clinical setting for two reasons. First, the tests used by WADA to detect AAS use are not clinically available. Second, if a patient wishes to avoid detection, he or she can easily do so by any of the mechanisms that elite athletes might use (e.g., avoiding use of the AAS at the time of testing); patients are not subject
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to random testing. The diagnosis generally can only be confirmed by the patient disclosing a history of AAS use.
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Although there is no definitive test for AAS use in a non-athlete, the diagnosis should be
suspected when a well-virilized, muscular man presents with infertility or gynecomastia or, less commonly, in a hirsute, muscular woman with amenorrhea. The most useful of the commonly
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available tests in this clinical setting are measurement of serum testosterone, FSH and LH
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concentrations. Because exogenous testosterone, non-testosterone AAS or hCG suppress circulating FSH and LH concentrations, measurement of serum testosterone, FSH and LH concentrations are useful for determining the likelihood of AAS use.29,30 Depending on clinical assessment of patient and the results of these tests, measurement of hCG also might be useful.
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Table 2 provides an overview of the interpretation and further management of the results of these tests. In general, the most useful “diagnostic test” is a gentle and non-accusatory query
Conclusions
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about the use of AAS.
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Since Ben Johnson shattered the world record for the 100 meters in 1988 and then tested positive for anabolic androgen steroid use, athletes who are willing to use performance enhancing AAS and anti-doping agencies have been in a race. The athletes who are willing to use banned AAS have worked with chemists and scientists willing to create novel AAS that are not detectable with conventional testing methods. Anti-doping agencies have funded the development of sophisticated laboratory methods for detecting known and novel AAS. Equally importantly, the 2009 advent of the WADA athlete biological passport appears to be a highly
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effective mechanism for detection of AAS use. According to the 2015 WADA Anti-Doping Violations report, only 1,649 of 229,412 (.007%) athletes tested had anti-doping rule violations based on an “adverse analytical finding” plus a judgement of “doping likely” from the expert
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panel review of passport results. Another 280 athletes of the 229,412 (.001%) were judged to have an anti-doping rule violation related to failure to follow the rules of mandatory sample collection or for evidence of an intent, attempt or event of administration of a banned
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substance or prohibited method (e.g., blood transfusion) to any athlete. About 50% of the
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“adverse analytical findings” (i.e., positive tests for banned substances methods) were related to AAS.
One can regard these findings as evidence of success for the WADA anti-doping program;
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99.99% of athletes appear to be adhering to the WADA regulations. On the other hand, some athletes continue to use AAS and other banned substances. These data suggest that athletes believe that they can avoid detection of AAS use despite the biological passport and random
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collection system. A cynic would note that the WADA data cannot include the false negatives— athletes who have used AAS without detection. Nonetheless, the combination of random
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testing, advancements in laboratory testing techniques and the biological passport appears to be an effective method of preventing and detecting AAS use among athletes.
References
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1. Connor J, Woolf J, Mazanov J. Br J Sports Med. Would they dope? Revisiting the Goldman dilemma. 2013;47:697-700. 2. Ventura R, Segura J. Masking and manipulation.Handb Exp Pharmacol. 2010;(195):327-
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54.
3. CAS Interim Arbitral award: D. Chand vs AFI and IAAF. http://www. tas- cas. org/fileadmin/ user_ upload/ award_ internet. pdf.
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biological-passport-abp-operating-guidelines.
SC
4. https://www.wada-ama.org/en/resources/athlete-biological-passport/athlete-
5. https://www.wada-ama.org/en/resources/laboratories/guidelines-reporting-andmanagement-of-hcg-and-lh-findings.
6. Mareck U, Sigmund G, Opfermann G, Geyer H, Thevis M, Schanzer W. Identification of
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the aromatase inhibitor letrozole in urine by gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom. 2005;19:3689–3693 7. Mareck U, Geyer H, Guddat S, Haenelt N, Koch A, Kohler M, Opfermann G, Thevis M,
EP
Schanzer W. Identification of the aromatase inhibitors anastrozole and exemestane in human urine using liquid chromatography/tandem mass spectrometry. Rapid Commun
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Mass Spectrom 2006;20:1954–1962.
8. Dehennin L, Ferry M, Lafarge P, Pérès G, Lafarge JP. Oral administration of dehydroepiandrosterone to healthy men: alteration of the urinary androgen profile and consequences for the detection of abuse in sport by gas chromatography-mass spectrometry. Steroids. 1998;63:80-7.
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9. Albeiroti S, Ahrens BD, Sobolevskii T, Butch AW. The influence of small doses of ethanol on the urinary testosterone to epitestosterone ratio in men and women. Drug Test Anal. 2017 Jul 3. [Epub ahead of print].
RI PT
10. Kuuranne T, Saugy M, Baume N. Confounding factors and genetic polymorphism in the evaluation of individual steroid profiling. Br J Sports Med. 2014;48:848-55.
11. Ouellet A, LeBerre N, Ayotte C. A simplified and accurate method for the analysis of
SC
urinary metabolites of testosterone-related steroids using gas
Spectrom. 2013;27:1739-50.
M AN U
chromatography/combustion/isotope ratio mass spectrometry. Rapid Commun Mass
12. WADA Technical Document TD2016IRMS 2016 Detection of Synthetic Forms of Endogenous Anabolic Androgenic Steroids by GC-C-IRMS. https://www.wada-
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ama.org/sites/default/files/resources/files/wada-td2016irmsdetection_synthetic_forms_eaas_by_irms-en.pdf 13. WADA Technical Document TD2015IDCR: 2015 Minimum Criteria for Chromatographic-
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Mass Spectrometric Confirmation of the Identity of Analytes for Doping Control Purposes. https://www.wada-ama.org/sites/default/files/resources/files/td2015idcr_-
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_eng.pdf
14. Ponzetto F, Mehl F, Boccard J, Baume N, Rudaz S, Saugy M, Nicoli R. Longitudinal monitoring of endogenous steroids in human serum by UHPLC-MS/MS as a tool to
detect testosterone abuse in sports. Anal Bioanal Chem. 2016;408:705-19. 15. Ayotte C. Detecting the administration of endogenous anabolic androgenic steroids. Handb Exp Pharmacol. 195:77-98, 2010.
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16. WADA Technical Document TD2016EAAS: 2016 Endogenous Anabolic Androgenic Steroids Measurement and Reporting. https://www.wadaama.org/sites/default/files/resources/files/wada-td2016eaas-eaas-measurement-and-
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reporting-en.pdf.
17. Balcells G, Pozo OJ, Esquivel A. Screening for anabolic steroids in sports: analytical
strategy based on the detection of phase I and phase II intact urinary metabolites by
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J Chromatogr A. 2015;1389:65-75.
SC
liquid chromatography tandem mass spectrometry.
18. E Strahm, C Emery, M Saugy, J Dvorak, C Saudan. Detection of testosterone administration based on the carbon isotope ratio profiling of endogenous steroids: international reference populations of professional soccer players Br J Sports
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Med. 2009;43:1041–1044.
19. Aguilera R, Hatton CK, Catlin DH 2002 Detection of epitestosterone doping by isotope ratio mass spectrometry. Clin Chem 48:629–636
EP
20. Cawley AT, Trout GJ, Kazlauskas R, Howe CJ, George AV. Carbon isotope ratio (delta13C) values of urinary steroids for doping control in sport. Steroids. 2009;74:379-92.
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21. Strahm E, Baume N, Mangin P, Saugy M, Ayotte C, Saudan C. Profiling of 19norandrosterone sulfate and glucuronide in human urine: implications in athlete's drug testing. Steroids. 2009;74:359-64.
22. Balcells G, Gómez C, Garrostas L, Pozo ÓJ, Ventura R. Sulfate metabolites as alternative markers for the detection of 4-chlorometandienone misuse in doping control. Drug Test Anal. 2017;9:983-993.
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23. Görgens C, Guddat S, Thomas A, et al. Simplifying and expanding analytical capabilities for various classes of doping agents by means of direct urine injection high performance liquid chromatography high resolution/high accuracy mass spectrometry. J Pharm
RI PT
Biomed Anal. 2016;131:482-496.
24. Ponzetto F, Boccard J, Baume N, et al. High-resolution mass spectrometry as an alternative detection method to tandem mass spectrometry for the analysis of
SC
endogenous steroids in serum. J Chromatogr B Analyt Technol Biomed Life
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Sci. 2017;1052:34-42.
25. Jakobsson J, Ekstrom L, Inotsume N, et al. Large differences in testosterone excretion in Korean and Swedish men are strongly associated with a UDP-glucuronosyl transferase 2B17 polymorphism. J Clin Endocrinol Metab 2006;91:687–93.
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26. Schulze JJ, Lundmark J, Garle M, et al. Doping test results dependent on genotype of uridine diphospho-glucuronosyl transferase 2B17, the major enzyme for testosterone glucuronidation. J Clin Endocrinol Metab 2008;93:2500–6.
EP
27. Okano M, Ueda T, Nishitani Y, et al. UDP-glucuronosyltransferase 2B17 genotyping in Japanese athletes and evaluation of the current sports drug testing for detecting
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testosterone misuse. Drug Test Anal 2013;5:166–81.
28. WADA 2015 Anti-doping Testing Figures. ama.org/sites/default/files/resources/files/2015_wada_antidoping_testing_figures_report_0.pdf
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29. Coviello AD, Matsumoto AM, Bremner WJ, et al. Low-dose human chorionic gonadotropin maintains intratesticular testosterone in normal men with testosteroneinduced gonadotropin suppression. J Clin Endocrinol Metab. 2005;90:2595–2602.
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30. McLachlan RI, de Kretser DM. Hypogonadotropism with elevated serum testosterone:
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TE D
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reversible causes of secondary infertility. Nat Clin Pract Urol. 2006;3:560–565.
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Table 1. Commonly used exogenous anabolic androgenic steroids and drugs that increase endogenous anabolic androgenic steroids Anabolic Androgenic Steroids (AAS) Drugs that increase endogenous AAS Designer AAS Gonadotropins Bolandiol Human chorionic gonadotropin Clostebol Recombinant human luteinizing hormone Danazol Drostanolol Aromatase inhibitors Dehydrochloromethyltestosterone Aminoglutethimide Gestrinone Anastrozole Metandienone Exemestane Metenolone Formestane Oxandrolone Letrozole Stanozol Testolactone Tetrahydrogestrinone Trenbolone Selective estrogen receptor modulators Endogenous AAS used as drugs Clomiphene Dihydrotestosterone Raloxifene Boldenone Tamoxifene Nandrolone Toremifene Testosterone Endogenous AAS precursors Androstenedione Androsterone Boldione Dehydroepiandrostenedione
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Table 2. Serum hormone concentrations in non-elite athletes and members of the general public using commonly used AAS or other substances to increase serum AAS AAS used Testosterone FSH LH Comments Evaluation for testicular or adrenal Testosterone ↑ ↓ ↓ tumor should be considered Evaluation for testicular or adrenal Testosterone ↑ ↓ ↓ precursors* tumor should be considered Evaluation for hCG-producing tumor hCG ↑ ↓ ↑ should be considered Nandrolone or other Evaluation for causes of non↓ ↓ ↓ hypogonadotropic hypogonadism testosterone should be considered AAS *High dosages of testosterone precursors such as androstenedione are required to raise serum testosterone concentrations above normal. **Further evaluation is based on clinical suspicion. It is often possible to elicit a history of AAS use by explaining that you are considering additional testing.
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Figure 1. The biological passport. The upper circles show the steps for creating the baseline measurements for an individual athlete’s biological passport. The low circles show the monitoring cycle after establishment of the baseline.
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Acknowledgements: I am receiving research funding from the National Institute of Health: HHSN2752013000251.
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