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Performance Enhancing Hormone Doping in Sport

, PhD, FRACP, FAHMS.

Author Information

Last Update: May 19, 2015.

ABSTRACT

Sport is the organized playing of competitive games according to rules. Hence doping represent drug cheating, a fraud on competitors, the sport and the public. The charter of the World Anti-doping Agency (WADA) forms a harmonized Code that authorizes an annually updated list of prohibited doping substances and methods as well as accrediting national anti-doping labs around the world. Sports performance has 4 major components: skill, strength, endurance and recovery, with each sport employing a distinct combination of these elements. These performance characteristics also correspond to the most potent and effective forms of doping. Sports requiring explosive power are most susceptible to androgen doping through their effect on increasing muscle mass and strength whereas sports that require endurance are most enhanced by hemoglobin (blood) doping which increases oxygen delivering capacity to exercising tissues. Performance in contact sports and those involving intense physical activity or training may also be enhanced by growth hormone and its secretagogues through speeding of tissue recovery from injury. Hormones remain the most potent and widely detected doping agents being responsible for about 2/3 of anti-doping rule violations detected by increasingly sophisticated detection methods. At present, the vast majority of positives are still due to a wide variety of androgens, including marketed and illicit (nutraceutical, designer) synthetic androgens as well as exogenous natural androgens, while the peptide hormones (erythropoiesis stimulating agents, growth hormone and its secretagogues) and autologous blood transfusion remain difficult to detect. For extensive up-to-the-minute review of all aspects of male reproductive endocrinology, visit www.endotext.org.

INTRODUCTION

Across the world, sport is a ubiquitous human social activity that forms an unique intersection of health, recreation, entertainment and industry1. It is both a major economic activity as well as a profound influence on social behaviour of individuals at home, work and play. One practical and concise definition of sport is the organised playing of competitive games according to rules. In that context, rule breaking is cheating to achieve an unfair competitive advantage whether it involves using illegal equipment, match fixing, banned drugs or other means.

The illicit use of banned drugs (doping) to influence the outcome of a sporting contest, constitutes a fraud against competitors, spectators, sport, sponsors and the public no different from other personal, professional or commercial frauds. While performance enhancement is almost invariably the intent of cheating, impairing performance is also well known in horse racing and even, rarely, in human elite sports (e.g. drink-spiking of banned drugs, physical assaults). Rules of sporting contest may change by agreement, but once set, represents the boundaries of fair competition. Nevertheless, fairness is an elastic, socially constructed concept which may change gradually over time. For example, a century ago deliberate training itself was considered an ungentlemanly breach of fairness as competition was then envisaged as a contest based solely on natural endowments and, similarly, some sports once maintained a distinction between amateurs and professionals. The philosophical foundations of the concept of fairness is a deep and complex issue 2,3 where the focus has been mainly on distributive justice. Less attention has been given to the philosophical basis of fair competition in sport where the prior distribution of talent and training and the outcome of contest are intended to provide equality of opportunity, but not of outcome, between contestants.

Naïve arguments have been made that deny doping is cheating, or unsafe or violates the spirit of sport and asserting that drugs should be freely available or under medical supervision4-6. However removing prohibition on doping would immediately render drug taking as pervasive as training in elite sport extending to promising underage and subelite athletes. Ensuing demands for excessive, often massive, drug doses without medical indications could not be ethically or safely prescribed by doctors. This could convert sporting participation into a potentially dangerous rather than a healthful activity. In practice, creating enforceable boundaries for drugs in sport is unavoidable whether it is prohibition or, even under the most idealistic libertarian scenarios, by age or dosage. These philosophical issues are not considered further here and, recognising that sport requires concrete, practical decisions, the establishment and enforcement of agreed rules is the basis of fair competition.

It is well understood that individual human genetic endowments are unequal and, among these, sporting prowess is at least partly genetically determined7. However, little is still known of the genotype-phenotype correlations that underlie beneficial genetic endowments for sports performance. Natural genetic advantages are recognized in height (tallness for basketball, shortness for jockeys and motor-cycle riders) and hereditary erythrocytosis where a high circulating hemoglobin (due to a high affinity EPO receptor8) for endurance sports, or conversely genetic disadvantage such as the common α-actinin-3 deletion genetic polymorphism which limits anaerobic, explosive power9. More examples of genetic (dis)advantages for sports performance are likely to be identified as genomics continues to expend our understanding of the biological basis of health, including natural human sporting prowess. In the context of sports doping, however, a person’s natural genetic endowment is a given creating a natural boundary whereby the use of exogenous drugs or chemicals (including DNA) may constitute drug cheating or doping.

WORLD ANTI-DOPING AGENCY (WADA) AND THE GLOBAL ANTI-DOPING REGULATORY ENVIRONMENT

Cheating is as old as sport itself, yet the present endemic of doping using pharmaceutical drugs to boost sports performance is largely a Cold War legacy. Eastern European national doping programs were established by governments aiming to achieve a short-cut propaganda victory over their Western rivals, a challenge quickly reciprocated and then taken up by individual coaches and athletes, starting with power sports10, from which the epidemic became entrenched as an endemic in sufficiently affluent circles. In 1967, following the introduction of anti-doping rules by some other federations, the International Olympic Committee (IOC) established its Medical Commission, which published their first list of prohibited substances. During the 1970’s the IOC Medical Commission took an increasingly active role by banning androgens which required developing standardized, valid methods to detect and deter androgen doping. After discarding alternatives such as immunoassays and blood sampling, mass spectrometry (MS)-based tests became the standard for detecting synthetic androgens in urine.

In 1999, the IOC established the WADA based in Montreal to be equally supported by governments and sporting organisations with its charter, the WADA Code, representing a harmonised set of global anti-doping rules introduced in 2004, revised in 2009 and 2015 11. WADA also publishes an annually updated Prohibited List of Substances and Methods, accredits anti-doping labs together with their operational anti-doping testing framework and established the Court for Arbitration in Sport (CAS) to settle anti-doping legal disputes as sport’s “Supreme Court”. By 2013, the Code was adopted by 204 Olympic Committees, 89 Olympic and 239 non-Olympic national federations and is implemented by 129 National Anti-Doping Organisations. The WADA Code prohibits substances or methods which meet 2 of 3 criteria comprising:

(i) enhance performance (cheating),

(ii) harmful to health (safety) or

(iii) violate the spirit of sport (unsporting).

Although the primacy of penalising cheating is widely understood, these criteria recognize ethical and practical difficulties in proving ergogenic effects of increasing numbers of illicit and/or non-approved substances for which safety is unknown so that human testing is not feasible and athlete safety is an additional important consideration. Crucially, the Code imposes strict liability on individual athletes so that a positive anti-doping test (including refusal or avoidance of testing or possession, attempts, trading and tampering with banned drugs) constitutes an anti-doping rule violation (ADRV), regardless of intent or negligence. Sanctions involve suspension from any elite competitive sport and extend to support personnel and teams. Suspensions were typically 2 years but were increased to 4 years from 2015, which is believed to be longer than the ergogenic benefits of doping, although recent evidence suggests that episodic androgen effects on muscle may have durable or even permanent effects12.

The Prohibited List bans, at any time either in or out of competition, the use of performance enhancing hormones, including androgens, EPO and growth hormone and related substances or drugs which stimulate endogenous production of these hormones (Table 1). Among the 15 categories of prohibited substances (12) and methods (3), hormones feature prominently in S1 (anabolic agents, mainly androgens), S2 (peptide hormones, growth factors and related substances), S4 (hormone and metabolic modulators) and S9 (glucocorticoids) with S1 and S2 having important “catch-all” provision for unnamed but related substances “with similar chemical structure or biological effects”. In addition, the S0 category bans non-approved substances, those without current regulatory approval for human therapeutic use. The prominence of hormones is reinforced by the WADA laboratory statistics for anti-doping tests where hormones remain the most frequently detected banned drugs (Table 2). In 2011, of ~250,000 anti-doping tests ~2% were positive with about 2/3 due to hormones, the vast majority (~99%) due to androgens. These findings confirm that the detection of androgen doping is effective whereas the low rate of detection of erythropoiesis-stimulating agents (ESA) and growth hormone may reflect the limitations of available tests for peptide hormones which require blood rather than conventional urine sampling and feature relatively low sensitivity and brief windows of detection, rather than their lack of abuse. Further use of out-of-competition testing and blood samples together with more sensitive detection tests having longer windows of detection are required particularly for peptide hormones.

Table 1 – 2015 WADA Prohibited List of Substances and Methods

Substances:

  • non-approved substances (S0)
  • anabolic agents (S1)
    • exogenous & endogenous androgens
    • “others with similar chemical structure or biological effects”
  • peptide hormones, growth factors and related substances (S2)
    • erythropoiesis-stimulating agents
    • hCG, LH (in men only)
    • GH, IGF-I, FGF, HGF, MGF, PDGF, VEGF
    • “others with similar chemical structure or biological effects”
  • beta2-agonists (S3), beta-blockers(P2)
  • hormone and metabolic modulators (S4)
    • aromatase inhibitors & anti-estrogens
    • myostatin inhibitors
    • metabolic modulators (insulin, PPARδ, AMPK agonists)
  • diuretics and other masking agents (S5)
  • stimulants (S6)
  • narcotics(S7), cannabinoids(S8), alcohol(P1)
  • glucocorticoids (S9)

Methods:

  • manipulation of blood & blood components (M1)
  • chemical and physical manipulation (M2)
  • gene doping (M3)

Substances and Methods as described in the WADA Prohibited List with the category label in brackets.

Table 2– Performance Enhancing Hormone Tests in WADA Labs

200320052007200920112013
Accredited Labs313334353333
Total tests151,210183,337223,898277,928243,193269,878
Positives
(% of total)
2,447(1.6%)3,909(2.1%)4,402(2.0%)5,610(2.0%)4,856(2.0%)5962(2.2%)
Hormones
(% of +ve)
46%55%55%73%68%57%
Androgens238938934375554148003352
Blood/Epo581627685063
GH/peptides000160

Source: WADA website report on laboratory testing figures. See http://www.wada-ama.org/en/Science-Medicine/Anti-Doping-Laboratories/Laboratory-Testing-Figures/

Therapeutic Use Exemption (TUE).

In rare cases, an elite athlete with a genuine medical need for therapeutic use of a prohibited drug may be granted a TUE 13. This exempts the athlete from the Code’s strict liability provision and permits them to compete during ongoing treatment. WADA provides medical guidelines that standardize the evaluation and management of TUE applications for a range of medical illnesses. A TUE is granted by a national anti-doping organisation based on an independent, expert review of valid, documented diagnosis, appropriate clinical indications and dose for hormonal treatment with a view to facilitating essential medical treatment but avoiding unjustified use or over-dosage. After stringent review TUE’s may be granted for treatment with testosterone, glucocorticoids and insulin but there are unlikely to be valid medical indications for EPO or, in adults, for growth hormone or IGF-1 in elite athletes. For example, TUE’s are usually justified for young male athletes with genuine androgen deficiency, occurring in ~1:200 men14, due to organic pituitary-testicular disorders with an established pathological basis (eg bilateral orchidectomy, severe mumps orchitis, Klinefelter’s syndrome) who require life-long testosterone replacement therapy 15. The TUE will approve, subject to regular review, a standard testosterone replacement regimen, including dosage and monitoring, with changes to regimen requiring approval. TUEs are not granted for men with functional decreases in blood T due to non-reproductive disorders including stress (“over-training”) or ageing (“andropause”, “LowT”,“late-onset hypogonadism”) or for women.

In principle, detection of prohibited substances is ideally aimed at identifying a xenobiotic substance or its distinctive chemical signature(s) which do not occur naturally in the body, thereby distinguishing it categorically from normal body constituents. Such identification of a non-natural substance that can’t be of endogenous origin is congruent with the strict liability onus in proving an ADRV. Proving an ADRV is more difficult to achieve with administration of natural hormones or their analogs which must be distinguished from their endogenous counterparts. In this situation, the alternative requires developing valid biomarkers to prove the use of banned substances through their distinctive effects on the body and tissues. It is a formidable challenge to validate an indirect biomarker as proof of an ADRV capable of withstanding vigorous medico-legal challenge when a proven ADRV would prevent an athlete from pursuing their profession. That requires rigorous standardization and harmonization of every stage of the anti-doping tests from sample collection, chain-of-custody, storage and analysis including accounting for any fixed (genetic, gender, age, ethnicity) or variable (exercise, hydration, masking vulnerabilities) factors which may impact on proposed test metrics.

Components of Sports Performance and Doping

Sports performance has 4 major dimensions – skill, strength, endurance and recovery (Figure 1). High performance in any sport requires a characteristic blend of these dimensions although individual sports differ widely in that balance. Similarly, the major ergogenic drug classes have distinctive effects aligned predominantly along one of these dimensions so that the most effective ergogenic drug classes used in doping are dictated by these dimensions of sports performance (Figure 2). While every sport requires an acquired skill, some are largely or solely based on skill and concentration (e.g. board games, target shooting, car driving, and motor-cycle riding) and may benefit from drugs that reduce anxiety, tremor, inattention or fatigue. Sports that are highly dependent on explosive, short-term anaerobic power (sprinting, throwing, boxing, wrestling), typically ones which favour a stocky, muscular build, are most susceptible to androgen-induced increases in muscle mass and strength. Other sports with an emphasis on aerobic effort and endurance (e.g. long distance or duration events), characteristically favoured by a lean build, may be boosted by hemoglobin doping (blood transfusion, erythropoietin (EPO) and its analogs or mimetics). Finally, sports that depend on recovery from major injury or recurrent minor injury during intensive training, notably contact sports, may benefit from tissue proliferative and remodelling effects of growth hormone and various growth factors.

Figure 1.

Figure 1.

Figure 2.

Figure 2.

ANDROGENS

Although the ergogenic effects of androgens were discovered empirically soon after the identification of testosterone in 193516, their applications to elite sport performance were mainly developed during the Cold War by trial and error experiments undertaken on unknowing elite athletes10, 17, 18; however, the scientific basis of androgen doping was only objectively proven in the 1990’s. Until that time, the settled consensus was that exogenous androgens had no effect in eugonadal men whose androgen receptors were already saturated by endogenous testosterone (T)10, 19, 20 and that the alleged benefits of androgen doping were misattributed placebo responses together with training and nutritional effects. Using a placebo-controlled, randomized clinical trial design with supra-physiological testosterone doses, Bhasin et al showed that T increased muscle mass and strength in eugonadal young men to a similar extent as exercise alone and with additive effects when combined with exercise21 (figure 3). Subsequent dose-response studies showed that administration of T increased muscle mass and strength by 10% without and 20-37% with exercise (where exercise alone increased them by 10-20%) together with additive effects from 3% increase in circulating hemoglobin. These benefits extended from below to well above physiological T doses or blood levels without evidence of plateau22, 23 and regardless of age24.

Figure 3.

Figure 3.

Androgen doping may be either direct or indirect (Table 3, figure 4). Direct androgen doping involves administration of synthetic androgens whereas indirect androgen doping includes a variety of non-androgenic drugs which increase endogenous T. Direct androgen doping originally involved all pharmaceutically marketed synthetic androgens but has extended to non-marketed designer and nutraceutical androgens as well as exogenous administration of natural androgens (T, DHT) and pro-androgens (androstenedione, DHEA). Indirect androgen doping involves use of hCG, LH, anti-estrogens (estrogen receptor blockers, aromatase inhibitors), opiate antagonists and neurotransmitters involved in neuroendocrine regulation of endogenous LH and T secretion25-27.

Table 3– Direct and Indirect Androgen Doping and Detection Methods

SubstanceDetection method
Direct
Synthetic androgensL/GC-MS
Natural androgensL/GC-MS, T/E, CIRMS
Designer & nutraceutical androgensL/GC-MS (bioassay)
Indirect
hCG (urinary or recombinant)hCG immunoassay (LC-MS)
hLH (recombinant)hLH immunoassay (LC-MS)
Anti-estrogensL/GC-MS
GnRH analogsL/GC-MS
Opioid antagonists & neurotransmittersL/GC-MS
Figure 4.

Figure 4.

Detection of direct androgen doping using steroids of known chemical structure is highly effective using gas or liquid chromatography MS28, 29. Traces of synthetic androgens or their metabolites may remain detectable for periods up to months after last administration. Recent developments including the identification of long-term metabolites has further widened the detection windows for synthetic androgens30-32. Challenges to detection of synthetic androgens have included the development of non-marketed designer and nutraceutical androgens, the use of natural androgens and pro-androgens, masking methods, restricting use to out-of-competition training or micro-dosing. Designer and nutraceutical androgens are typically non-marketed synthetic androgens based on structures and synthesis methods recovered from largely forgotten patent literature of the 1960-70’s. These are now synthesized by unregulated non-GMP chemical manufacturers to be sold over the internet or over-the-counter as nutritional supplements, which may contain undeclared steroids33. However, once the chemical structures of any synthetic androgens are known, they are easily detectable although the sheer profusion of such chemicals represents an ongoing challenge. Nevertheless, despite their novelty, there is little evidence designer androgens have been much used after they are discovered from when there is a high likelihood of detection so that virtually all ongoing androgen ADRVs are still due to conventional marketed synthetic androgens.

Distinguishing between the exogenous and endogenous steroids:

Administration of natural androgens (T or DHT) or pro-androgens (androstenedione, DHEA), raises the problem of distinguishing between the exogenous and endogenous steroids. Exogenous T administration can be detected by the urine T/E ratio, the ratio in urine of T to its 17α-epimer epitestosterone (E). Both T and E are co-secreted by Leydig cells and excreted in urine consistently so that the urine T/E is usually stable for any individual over time, being typically around 1. Administration of exogenous T, which is not converted to E, increases the urine T/E ratio and, when it exceeds a specified threshold, is evidence for administration of exogenous T. The urine T/E ratio thresholds were originally population-based, set initially at 6 and then subsequently lowered to 4. However, the possibility of false negatives and false positives of population-based thresholds are limitations which may require further analysis to confirm or refute T doping in individual cases. These considerations have led to establishment of the steroid module of the Athletes Biological Passport (ABP), a compendium of serial observation of any individual’s tests which creates adaptive individual-specific T/E ratio threshold. This substitution of an individual’s own person-specific, in place of the population-based, thresholds allows for more sensitive and accurate detection of individual deviations in urine T/E ratio as evidence of T doping.

One limitation of the urine T/E ratio is a genetic polymorphism of the uridine 5'-diphospho-glucuronosyltransferase (UGT) 2B17 gene which encodes a phase II hepatic enzyme that glucuronidates T rendering it more hydrophilic to facilitate urinary excretion. This polymorphism comprises a genetic deletion which, in homozygotes, produces a non-functional enzyme that reduces urinary T (but not E) excretion to near zero producing an extremely low T/E ratio (<0.1). While this genetic polymorphism has no apparent biological effect on T action, it is unevenly distributed geographically being much more frequent in South East Asian populations34. This biological false negative means that administration of exogenous T will not exceed the usual population-based T/E ratio thresholds35. On the other hand, it will exceed any individual’s own specific urine T/E ratio ABP threshold so that genotyping and/or Bayesian profiling of serial T/E ratio provide complementary evidence36, 37.

Administration of exogenous T may also be identified by carbon isotope ratio MS (CIRMS) which can distinguish endogenous from exogenous T according to the C13/C12 ratio of urinary T38, 39. Commercially, steroids are manufactured from starting material of plant sterols produced by photosynthesis which exhibit distinctly lower C13/C12 ratio (typically, -26‰ to -36‰ relative to the global standard) compared with mammalian T biosynthesis (between -16‰ to -26‰)40, 41. Hence, a significantly depleted C13/C12 ratio of urinary T, exceeding 3‰ relative to endogenous reference steroids, indicates that urinary T originates at least partly from exogenous chemical manufacture from plant sterols. CIRMS can also be applied to detect administration of other natural androgens or pro-androgens including DHT and DHEA42, androstenedione, or even attempted masking by administering E (to lower urine T/E ratio)40. A few T products have recently emerged with a less depleted, more mammalian-like C13/C12 ratio for urine T43 creating a challenge for CIRMS detection; nevertheless, extended isotope profiling of other steroid precursors and metabolites provides additional reference biomarkers44. Furthermore, development of hydrogen ion ratio mass spectrometry has further enhanced the ability to distinguish between endogenous and exogenous steroids even when the carbon isotope ratio is non-informative45-47. Suppression of urine LH excretion may also provide corroborative evidence for the use of exogenous T or other synthetic androgens48, 49.

While MS is highly effective for detecting specific androgens, it requires knowledge of the chemical structure to be detected and otherwise cannot be applied. This applies to never-marketed designer or nutraceutical androgens sold over the internet or in unregulated over-the-counter nutritional supplements with unlabelled steroid content. A potential solution is the modern in vitro androgen bioassay that incorporates the human androgen receptor together with a convenient transactivation chemical read-out signal into a host yeast or mammalian cell. This has the generic capacity to detect all bioactive androgens regardless of structure with a sensitive dose-response signal proportional to the potency of the bioactive androgen50-53. Yeast host cells have high specificity for detecting androgens but are less sensitive than mammalian cells, which express native steroid mechanisms including steroidogenic enzymes and/or other steroid receptors. Mammalian in vitro androgen bioassays can also detect pro-androgens, steroids lacking intrinsic androgenic bioactivity but which are converted into androgens by the mammalian cell. Hence, while mammalian host cells sacrifice specificity for higher sensitivity, they can also detect pro-androgens54. Hence yeast and mammalian in vitro androgen bioassays are complementary in detecting both androgens and pro-androgens.

The limitations of in vitro androgen bioassays are their susceptibility to matrix effects and difficulties in standardizing bioassay-based test so they may be best applied to characterize products and substances for androgens or pro-androgen content rather than to biological samples. Hence the yeast androgen bioassay was decisive in the first conviction for use of a designer androgen by proving that tetrahydrogestrinone (THG) was a potent androgen55 and has also been used to screen synthetic progestins to show that, unlike the original androgen-derived progestins, the modern generation of progestins are not androgenic56.

An attractive option to detect androgen doping is the use of hair samples. These have the advantages of easy, observable and minimally invasive sampling and simple, convenient storage featuring a potentially very long window of detection, according to hair growth rates57. MS-based methods have been reported to detect exogenous58-72 and endogenous61, 73-76 androgens in human hair following long-term, but not single dose77, exposure. However, hair analysis tests have yet to undergo sufficient standardization and validation to become acceptable anti-doping tests. Problems that remain to be fully overcome include matrix effects, low recovery and limited sensitivity as well as the impact of age, hair colour, alopecia, and shaving or passive chemical (cosmetic) contamination of hair. Additionally nails and skin could also provide analogous information on recent past androgen exposure but suitable tests are yet to be convincingly developed78. In theory, androgen-induced gene expression in circulating leukocytes might provide an additional biomarker of androgen action if specific and reproducible signatures can be defined79; however, as direct detection of androgens is feasible and preferable for proving an ADRV, a role for gene expression biomarkers of androgen action remains to be established for anti-doping.

Indirect androgen doping

This strategy aims to increase endogenous T production and thereby evades detection by routine screening tests for exogenous T such as urine T/E ratio or CIRMS. Urine hCG is detected by commercial hCG immunoassays using immunoassays specific for intact heterodimeric hCG (including its nicked variant) which, if positive by exceeding a detection threshold (>5 IU/L), requires confirmation by a second immunoassay for intact heterodimeric hCG which is required to prove hCG use. A key issue is to distinguish a positive hCG urine test, presumptively indicating hCG doping, from early trophoblastic tumor or immunoassay artefacts. As hCG doping is not effective in women and urine hCG screening can detect early pregnancy, an unwarranted privacy intrusion, hCG testing is restricted to male athletes27. Similarly, urine LH can be measured by some commercial LH immunoassays, none marketed for urine samples, validated by individual anti-doping labs. Although direct LH doping is an implausible doping threat80, suppressed48, 49, 80 or elevated urine LH may be useful for confirming any form of direct or indirect androgen doping26, 27, 48, 81. Anti-estrogens (estrogen receptor antagonists) or aromatase inhibitors, which can cause reflex increases in serum and urine LH and testosterone26, are detected by MS-based chemical detection methods.

Overall, detection of direct androgen doping is now so effective that in WADA-compliant elite competitions it is restricted to the ill-informed, often using counterfeit or unlabelled products82. Yet the potency of androgen doping in power sports continues to prompt development of novel androgen doping strategies. These will include use of undocumented synthetic androgens or novel indirect androgen doping methods, especially micro-dosing during out of competition training. There remains a need to maintain deterrence by effective detection methods for evolving new androgen doping threats.

HEMOGLOBIN (BLOOD) DOPING

Hemoglobin doping involves either direct blood transfusion or indirect methods of increasing hemoglobin via stimulating erythropoiesis by administration of erythropoietin, its analogs or mimetics (see excellent reviews83, 84) (Table 4). Boosting hemoglobin is advantageous in aerobic, endurance sports such as road cycling, distance running and cross-country skiing. In addition to cardiac output, maximal oxygen consumption (Vo2) is principally determined by blood oxygen transfer with a lesser contribution from tissue oxygen transfer85. Experiments on exercise tolerance and blood transfusion were first reported in 194586, 87 but the scientific basis of hemoglobin doping via enhanced tissue oxygen transfer was firmly established in 1972 by the work of Ekblom et al reporting experiments in healthy volunteers who underwent venesection and/or re-transfusion of 1, 2 or 3 units (400 mL) of blood with repeated testing of maximal exercise-induced oxygen consumption before and after each procedure88. This proved unequivocally that the maximal oxygen consumption was highly correlated with acute changes in hemoglobin (figure 5). Subsequently, during the 1970-80’s before its banning in 1988, blood transfusion became a prevalent surreptitious practice in road cycling and cross-country skiing and the apparently low prevalence among distance runners may be an underestimate89. Modelling of historical performance in European road cycling from 1993 onwards shows a unique progression averaging an improvement of 6.4% corresponding closely with the performance enhancement (6-7%) due to rhEPO administration, which is sustained for at least 4 weeks afterwards90-92.

Table 4– Direct and Indirect Hemoglobin Doping and Detection Tests

Doping MechanismDetection
Direct (Blood transfusion)
HeterologousFlow cytometry: bimodal population of blood group antigens
AutologousNo direct detection testBiomarkers:
Urine phthalate excretion
Total hemoglobin mass
Athletes Biological Passport
Indirect (Erythropoeisis stimulation)
Direct
rhEpo & biosimilars (>100)Epo analogsUrine double immunoblot, (LC-MS)
Indirect
Hypoxiaaltitude training, hypoxic sleep areaNot banned
Hypoxia-mimeticshypoxia-inducible factor & stabilizers, iron chelation, cobalt, 2,3 DPG analogsLC-MS/MS
Artificial O2 carriersHbOC, perfluorocarbonsLC-MS/MS
Figure 5.

Figure 5.

Blood transfusion

Transfusion may involve either another person’s (homologous) or the athlete’s own (autologous) blood administered prior to a contest to acutely increase circulating hemoglobin. Homologous blood can be transfused at any convenient time to enhance performance in competition but when performed by untrained personal in non-clinical environments risks transfusion reaction, blood-borne infectious disease and iron overload. By contrast, autologous transfusion reduces health risks but requires complex coordination as venesection itself is detrimental to performance, and it requires balancing recovery from blood withdrawal and loss of erythrocyte viability during long-term cryostorage with training and competition schedules. Although blood transfusion was first banned by the IOC in 1986, the first practical approach to banning blood doping was the introduction of hematocrit testing in 1997 by the international skiing and cycling federations which excluded athletes on health grounds from entering competition on the day if their hematocrit exceed a safety threshold (0.50). This encouraged hematocrit titration to just below threshold and only prevented competing until hematocrit returned under that threshold, which could be a very short period particularly if venesection was employed. The first ADRV’s for blood manipulation involving hematocrit threshold and titration were in 2001.

Homologous blood transfusion creates a bimodal population of blood group antigens which is detectable by flow cytometry using a panel of 12 minor blood group antigens93, from the wider array of blood group antigens94, which can detect a <5% contamination of exogenous erythrocytes. Subsequent refinements simplified and improved test sensitivity so that a panel of 8 antigens can detect contamination comprising a minor admixture population of 0.3-2.0% with no false positives but high sensitivity (~80%), the latter depending on the magnitude of the minor contaminating mixture95, 96. Alternatives based on genotyping for the admixture population of leukocytes have also been proposed97. As a test proving unequivocally the presence of non-endogenous erythrocytes in the circulation, this method is definitive if performed to the required standard. A remotely hypothetical defence against a positive test, based on stable marrow chimerism from a vanished twin, was raised by a cyclist who subsequently admitted transfusion97. Based on risk of detection as well as to health risks, homologous transfusion has now largely disappeared in favour of autologous transfusion98.

Figure 6.

Figure 6.

Autologous transfusion

The biggest gap in current anti-doping tests is the lack of a specific test to detect autologous transfusion99. Research to identify robust physico-chemical or biological markers for direct identification of a subpopulation of ex-vivo aged erythrocytes is underway but the dilution and rapid clearance of effete erythrocytes make for challenging detection problems100. In the interim, other indirect methods have been developed. These include measuring urinary excretion of phthalates, plasticizers that leach out from the polyvinylchloride blood packs used to store venesected blood101. This test has brief window of detection (2 day) so will detect auto-transfusion during or immediately before events (characteristic in road cycling, according to convicted dopers) but may miss earlier auto-transfusion. Furthermore, the ubiquity of low level environmental phthalate exposure requires establishing detection thresholds and non-plastic blood containers can be used. An alternative is the measurement of total hemoglobin mass102, a measure with good stability and reproducibility even during exercise and circumvents influence of variations in plasma volume such as due to dehydration or dilutional masking102, 103. However, as this requires inhalation of carbon monoxide, which has transient detrimental effects on performance, it is not ideal for routine anti-doping use and its sensitivity may be insufficient to detect all EPO micro-dosing104, 105. Nevertheless, alternative methods for serial measurement of total hemoglobin mass remain attractive. Other hypothetical methods include the detection of microRNA106 or immune reactions to transfusion107 but the sensitivity and specificity of these proposed tests remains to be fully evaluated.

The best detection test for autologous hemoglobin doping at present is the hematological module of the ABP introduced in 2009108. Conceptually, it is a biomarker test which adopts a Bayesian approach of creating serially-adaptive, person-specific reference limits, based on using all prior testing, to supplant population-based thresholds. Combining all of an individual’s previously collected hematological data creates a probabilistic test of whether any new result deviates significantly from that individual’s personal reference limits109. These person-specific thresholds allow for ongoing refinement and reinforcement by further testing. The thresholds are calculated by a variety of algorithms incorporating routine hematological parameters, notably hematocrit and reticulocyte counts. Those were developed over the last two decades to create the ABP hematological model which is sensitive to both direct and indirect hemoglobin doping110. The first attempts to regulate hemoglobin doping in the late 1990’s sought to prevent road cyclists or cross-country skiing athletes competing on health risk grounds when their hematocrit exceeded pre-determined, population-based safety criteria (e.g. hematocrit 0.50 or hemoglobin 170 g/L for cycling). However, while this excluded extreme hemoglobin doping only until the short period when the safety threshold was no longer exceeded, it allowed an increase in an athlete’s natural haematocrit, typically averaging ~0.45, up to the permitted ceiling threshold which fostered titrated hemoglobin doping and manipulations like hemodilution by saline or plasma volume expander infusions to avoid detection111. More sophisticated hematological algorithms were then developed to detect hemoglobin doping initially for the Sydney 2000 Olympics112, 113, the first generation of algorithms developing validated tests for ongoing and for recent cessation of hemoglobin doping, using a combination of biochemical variables related to erythropoiesis physiology. This approach was simplified by a second generation algorithm using only routine hematological parameters (hemoglobin, reticulocytes)114, and was subsequently combined with the concept of a sequential development of individual-specific reference ranges115 into a third generation algorithms116, 117 which were refined for the ABP108, 109. The hematological module of the ABP currently employs an algorithm involving 8 parameters derived from routine hematological profile (hemoglobin, hematocrit, erythrocyte count, reticulocyte count and percentage, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration)118. This is capable of detecting any form of hemoglobin doping, whether direct or indirect, with good but imperfect sensitivity103-105 and using only routine hematological tests. The reported increasing use of very low EPO doses (“micro-dosing”) would markedly reduce the magnitude of any dose-dependent ergogenic benefits105 while still carrying risks of detection, disqualification and disgrace.

Stimulation of Erythropoiesis

Indirect methods to increase hemoglobin include administration of recombinant human EPO or its analogs as well as hypoxia-mimetic drugs (hypoxia-inducible factor stabilisers, iron chelation, cobalt, 2,3 diphosphoglycerate analogs) or artificial oxygen carriers (perfluorocarbons, hemoglobin-based oxygen carriers). Related but non-banned methods include altitude training or its simulation by sleeping in hypoxic rooms which are less effective than hemoglobin doping91.

The identification of the human EPO gene in 1985 led to the marketing of recombinant human EPO (rhEPO) between 1987-9. Despite the IOC’s prohibition of EPO’s use in sports in 1990, the commercial availability of rhEPO created powerful new opportunities for indirect hemoglobin doping which were soon proven experimentally119. A drug, which circulates for hours to days, but with potent and long-lasting ergogenic effects after its disappearance due to the 4 month lifespan of erythrocytes, is both attractive for doping and a challenge to anti-doping testing. Expiry of the rhEPO patent in 2004 allowed marketing of a profusion of generic EPO (“biosimilar”) products, estimated globally at over 80120, as well as modified EPO analogs (darbepoeitin, pegylated EPO, peginesatide, EPO fusion proteins). A fatal cluster involving deaths of 18 Dutch and Belgian road cyclists, presumably due to inadvertent over-dosage during empirical attempts to maximise ergogenic effects of illicit rhEPO, was reported121, although difficult to verify122. A similar excess of unexpected deaths of road cyclists was also reported again in 2003-5 when novel EPO analogs and EPO biosimilars were marketed.

Detection of EPO in urine is difficult because of the prevailing low concentrations and need to distinguish exogenous recombinant from endogenous EPO. The first effective method for rhEPO in urine was a double immunoblot123, 124 which was capable of detecting urinary excretion of a variety of exogenous EPO products and analogs according to their differences in glycosylation side-chains, and differences in primary amino acid sequence where they exist, while distinguishing them from endogenous EPO. Although further refined125 and extended to other EPO analogs126, the immune-electrophoresis test is sensitive but relatively laborious and provides only a short window of detection of up to a week post-administration127. More sensitive methods based on proteomics (for EPO analogs with differences in primary structure) together with glycomics (for biosimilars and analogs which have host-cell specific variations in side-chain glycosylation but unchanged natural EPO primary structure128) are possible but not yet approved.

Other EPO mimetics such as hypoxia mimetic drugs including hypoxia-inducible factor (HIF) stabilisers and related small molecules represent growing threats as potential indirect hemoglobin doping agents129. These non-peptide chemicals interfere with various steps of the molecular oxygen sensing mechanism so as to mimic renal hypoxia and thereby induce EPO secretion to increase circulating hemoglobin. As a convenient orally active alternative to the lucrative pharmaceutical market for injectable erythropoiesis-stimulating peptides (~$7-8 billion130) to counteract anemias of chronic renal failure or marrow failure due to myeloproliferative disease or cytotoxic cancer therapy, they constitute a very active area of pre-clinical patent-based clinical drug development129. Experience suggests that such innovator products can enter the doping black market before marketing approval110, 129. Despite the profusion of pre-clinical leads, they represent families of related chemical structures disclosed in patents for which LC and/or GC-MS detection tests should, in principle, be effective. Understanding the metabolism of these drugs when they come to market may identify long-lasting metabolites that can extend the windows of detection. Coupled with evidence from the ABP, manipulation of the EPO pathway may be detected in conjunction with corroborative measurement of inappropriately suppressed or elevated endogenous EPO for the prevailing hemoglobin level.

HIF is a key generic biological mechanism for tissue sensing of hypoxia and triggering local (neovascularisation, angiogenesis) and systemic (EPO) defensive reactions. The promoter of the EPO gene contains enhancer and inhibitor regions with the hypoxia-responsive element which binds HIF and a GATA binding site which enhance and inhibit, respectively, EPO gene transcription. HIF is a heterodimer formed by constitutively expressed subunits with the β subunit in excess and availability of α subunit limiting formation of bioactive HIF. The 3 HIFα subunit isoforms are subject to hydroxylation of specific proline residues by prolyl hydroxylase enzymes which inactivate HIFα by ubiquitination, a tag which targets it to proteosomal degradation. HIFα subunit inactivation is strongly dependent on tissue oxygenation being active during normoxia but reduced during hypoxia when persistence of HIFα stabilises the HIF heterodimer. Notably, during hypoxia the expression of HIFα in renal cortical cells stimulates EPO gene expression so that HIF stabilisation by prolyl hydroxylase inhibitors leads to increased EPO secretion and circulating hemoglobin. Hence inhibiting prolyl hydroxylase activity via blocking its required cofactors (ascorbate, ketoglutarate, iron) using cobalt, nickel, iron chelation, ketoglutarate analogs or mechanism-based chemical inhibitors can result in increased hemoglobin via stimulation of EPO secretion129. Similarly, small molecule GATA inhibitors potently stimulate circulating EPO, hemoglobin and performance in mice131 although none have yet been marketed so their human efficacy and safety remain to be determined.

Another approach to increase oxygen delivery to muscle has been to exploit the ability of 2,3 diphophoglycerate (2,3 DPG), whose binding to hemoglobin reduces its affinity for oxygen with the left-shift of its oxygen dissociation curve as an oxygen unloading mechanism in tissues. 2,3 DPG analogs, developed as radiation sensitisers for hypoxic radio-resistant tumors, enhance tissue oxygen delivery in vivo132, 133 but would feature only short-term, acute effects readily detectable by mass spectrometry134, 135.

Adverse effects from use of rhEPO or its analogs are well known in medicine but poorly recognised in doping. They include immunogenicity (with risk of EPO autoantibody mediated pure red cell aplasia)136, 137, cardiovascular complications (including venous thromboembolism, stroke, hypertension and myocardial infarction) and premature death138-141. In routine clinical use of EPO to correct renal anemia, the goal is a gradual increase to subnormal hemoglobin targets so that the excessive and/or rapid rises in hematocrit and blood viscosity142 may explain the excess unexplained deaths among young European road cyclists in the late 1980s. In addition, use of rhEPO may deplete iron stores which limits hemoglobin synthesis so that athletes may also use oral or injectable iron supplements, which carry their own risks such as iv iron supplementation’s potentially adverse effects in enhanced tissue oxidative damage and excess mortality in chronic kidney disease143. Although clinical safety experience with ESAs is restricted to patients with serious medical disorders, there is evidence from the general community that higher natural hematocrit is associated with worse long-term cardiovascular health outcomes144-146.

GROWTH HORMONE

Growth hormone (GH) is a tissue growth promoter in children but after puberty it is predominantly a metabolic hormone although latent tissue growth promoting effects may be unleashed under non-physiological circumstances, such as during recovery from tissue injury. There is consistent anecdotal evidence that GH has been used in elite sports for decades147. Nevertheless, ergogenic effects of GH remain unproven and largely speculative as discussed in excellent recent reviews148-150. Claims of GH benefits in sport have included increases in muscle mass and strength, especially in conjunction with androgens, and/or improved tissue healing with more rapid recovery from either major injuries or minor repetitive injuries, such as from intense physical training allowing for more effective training. The biological basis of ergogenic effects of GH have been tested in these two different scenarios with largely inconclusive findings.

Evidence for direct enhancement of athletic performance by GH has been investigated in two well controlled RCTs with a primary focus on athletic performance. In one study, 96 recreational sub-elite athletes (63 male, 33 female, mean age 28 yr) were administered 8 weeks of daily sc injections of GH or placebo with the men also having weekly im injections of T enanthate or saline placebo for the last 5 weeks151. GH increased lean (muscle) mass (by +2.7 kg) and reduced fat mass (by -1.4 kg) while T increased lean mass (alone by +2.4 kg, by +5.8 kg with GH). The effects of GH were marginally significant for anaerobic sprint capacity (by +3.9%, p=0.05) when pooling male and female participants but this was due to significant effects in men only (by +5.5% alone and +8.3% with GH). However, there were no significant effects on maximal Vo2 consumption, dead lift or jump height151. A second study involved 30 healthy non-athletes (15 male, 15 female, mean age 25 yr) who were administered daily sc injections of GH at high (4.6 mg/day) or low (2.3 mg/day) doses or placebo152. There was no significant effect on muscle mass or maximal Vo2 consumption. Additional controlled studies of GH effects but with less focus on athletic performance have also shown that (a) a single dose of GH (~0.8 mg) in 9 recreational athletes did not affect maximal Vo2 or power output in repeated 30 min bursts of bicycle ergometry153, (b) short term (6 days), low dose GH (~1.7 mg/day) treatment of 48 male androgen abusers withdrawn from androgens for 12 weeks significantly increased maximal Vo2 more than placebo154, (c) daily sc injections of a GH receptor antagonist (pegvisomant) or placebo for 16 days to 20 sedentary men did not change maximal Vo2 although time to exhaustion at 90% maximal Vo2 was reduced155 and (d) 4 weeks of daily sc injections of GH (~5 mg/day) increased whole body protein synthesis156, lipolysis and glucose uptake157 with uncertain significance for athletic performance. Overall, these studies suggest that GH has, at most, a modest ergogenic effect in men only and there through enhancing T effects.

It is also claimed that GH may enhance injury healing, thereby facilitating more intensive training and/or recovery from muscle, connective tissue or bone injury, notably in contact sports. This claim is difficult to evaluate and no well controlled studies of recovery from sports injuries or tolerance of training intensity in elite athletes are reported. The most germane surrogate evidence available arises from investigations on the use of GH in recovery from injuries due to burns, fracture or for wound healing. A recent Cochrane meta-analysis review of GH treatment effects on recovery from burns injury and healing of donor skin graft sites suggests that GH has a small benefit in skin healing with large burns and reduced hospital stay but there was no benefit in reducing mortality or scarring and adverse effects, notably hyperglycemia, were increased158. In practice, the increased mortality due to administration of high dose GH in critical illness159 has led to GH treatment not being widely adopted in clinical practice of treatment of burns. Similarly, the only well controlled study of GH effects on bone healing from fracture reported that, among over 400 patients with tibial fractures treated for up to 16 weeks with GH (1, 2 or 4 mg/day) or placebo, there was no benefit of GH for overall healing160. Finally, while there are numerous experimental studies of GH or growth factors on wound healing in animal models a wide variety of findings are reported with detrimental, neutral or beneficial effects but no well-controlled human studies are available. In summary, the available evidence for improved tissue repair or regeneration is minimal.

Important caveats on interpreting these few well designed studies is that the effects of higher GH and T doses, as used in doping, have not been studied so that more potent higher dose and/or interactive effects cannot be excluded in the absence of well controlled studies. Anti-doping science history suggests that caution is required before rejecting evidence for claimed ergogenic effects without investigations replicating the pharmacological doses used.

Furthermore, safety analysis is not feasible based on the few, small, short-term studies of GH’s potential ergogenic effects; however, there are significant safety concerns about the long-term risk of cancer following GH administration. Even standard therapeutic GH doses administered to GH deficient children are associated with increased risk of second cancers in some 161-163 but not all 164 follow-up studies although these risks appear largely confined to survivors of childhood cancers and its treatment which render them GH deficient165-168. Although the significant cancer risk based on uncontrolled observational cohort data using standard GH doses remains contentious169, 170, the long-term risks of much higher GH doses used illicitly by athletes must be viewed with significant concern.

Detection of GH doping remains difficult171. A major challenge is the non-glycosylated primary structure of recombinant and endogenous 22 kDa GH, that lack the distinctive side-chain carbohydrate differences of exogenous glycoproteins EPO or hCG which provide a convenient basis for sensitive molecular detection tests. Nevertheless, minor infidelities in commercial manufacturing of GH may incorporate distinctive non-natural chemical features proving an exogenous origin172-174 although these findings have not been developed into detection tests. Challenges to the detection of GH doping arise from the physiological pattern of endogenous GH secretion with its intermittent, pulsatile pattern subject to prominent influence of exercise, stress, and nutritional effects together with GH’s brief circulating half-life and low urine concentrations175, 176. Like other major doping classes, there are both direct and indirect forms of GH doping, involving either direct administration of GH or IGF-I or their analogs and indirect GH doping involving drugs that aim to increase endogenous GH and IGF-I secretion (Table 5).

Table 5– Growth Factors, Growth Hormone Related and Other Peptides

Growth FactorsGrowth Hormone related peptidesOther Peptides
GHRH analogsGhrelin analogsOther
FGFGHRHGHRP-6IGF-1 & analogs (MGF, long R3 IGF-1)Thymosinß4
HGFCJC-1295GHRP-2 (pralmorelin)IGF-2
MGFGHRP-4Insulin & analogs
PDGFGHRP-5AOD-9604
VEGFGHRP-1
Hexarelin
Ipamorelin
Alexamorelin

The first test to detect administration of exogenous GH, the 22kD recombinant form of human GH, was based on blood sampling to measure the ratio of circulating isoforms of GH recognizing the fact that the pituitary secretes not only the major 22 kD isoform (65-80%) but also a variety of minor isoforms including a wide variety of minor isoforms and their multimeric variants 177. Administration of exogenous GH suppresses endogenous pituitary GH secretion leading to a predominance of circulating 22 kD GH. This is the basis for the GH isoform ratio test whereby a serum sample is measured by two different GH immunoassays, one with predominant 22 kD GH specificity (“rec” assay) and the other recognizing the broad spectrum of pituitary GH isoforms (“pit” assay) and the ratio of results (“rec”/”pit” ratio) is an index to detect administration of exogenous recombinant GH175, 178. This ratio test then serves to detect administration of exogenous recombinant human 22kD GH analogous to detection of exogenous T by the urine T/E ratio and exogenous insulin by analysis of serum C peptide 179. The differential GH isoform ratio test has undergone extensive validation involving standardization of the two GH immunoassays with distinctive immunoreactivities to quantify 20kD and 22kD epitopes as well as its application to various populations of elite athletes and evaluating physiological factors which might impact on the validity of test read-out. A strength of this test is that it is aimed at the exogenous doping agent itself, although it cannot definitively distinguish it from its endogenous counterpart. The major limitations of this differential isotope ratio test is its narrow window of detection (24-36 hr post administration) and its inability to detect indirect GH doping. While pituitary-derived human GH might not be detected, human pituitary GH, once obtained from national scale pituitary collection and purification programs, has not been available since 1985 when its risks of Creutzfeldt-Jakob disease were identified180, 181 with recombinant human GH replacing pituitary-extracted GH worldwide. This differential isoform test was first introduced for the 2004 Olympics182 and led in 2010 to the first successful detection of out of competition GH doping183.

A complementary detection test with a wider window of detection has been developed based on biomarkers of GH action. This uses two serum biomarkers of tissue GH effects, circulating IGF-1 as a short-term marker of hepatic GH action, and N-terminal peptide of procollagen type III (PIII-NP) as a long-term marker of GH-dependent collagen synthesis. In a study of 102 recreational athletes (53 male, 49 female, mean age 25 years, from 4 different European cities) randomly assigned to self-inject 2.7 mg or 5.4 mg GH or placebo once daily, measurement of serum IGF-1 and PIII-NP by specific immunoassays were able to correctly classify 86% of samples from males and 60% of samples from female using an empirical linear discriminant analysis of log-transformed serum IGF-1 and PIII-NP at the specificity of 1:10,000 required for a WADA biomarker threshold184. Subsequent studies have shown that additional collagen biomarkers, N-terminal propeptide and C-terminal telopeptide of type I collagen, further widen the window of detection for GH administration185, 186. This multiplex biomarker test, based on using standardized immunoassay antibodies, requires establishment of reliable reference range with specificity (false positive detection rate) of no more than 1:10,000 incorporating the impact of gender and age, although exercise, injury, ethnicity and sports type appear not to be confounding influences but is not yet in routine use by WADA anti-doping labs. The two GH doping test, the differential isoform and biomarker approaches, are considered ultimately complementary187.

IGF doping

IGF-1 is a circulating marker of hepatic GH effects and mediator of GH action so the marketing in 2005 of recombinant human IGF-I alone, and later with its major binding protein recombinant human IGF binding protein 3 (IGF-BP3)188, for treatment of diabetes, insulin or GH insensitivity or motor neuron disease, together with the availability of IGF-1 analogs for laboratory use, creates the possibility of IGF doping189. Time-series analysis of elite sports performance190 is consistent with the occurrence of IGF-1 doping but its prevalence is unknown33. As the biological basis for ergogenic effects of IGFs is due to its GH-like effects, this remains largely speculative and accompanied by the same safety concerns. IGF-1, IGF-2 and their analogs191 as well as insulin and its analogs192 are all readily detectable by LC-tandem MS and preliminary evidence suggests that biomarkers for IGF-1 administration (IGF-2, IGFBP2) may widen the window of detection193. However, a specific test to detect IGF doping remains to be established194.

MGF is a splice variant of IGF-I which, although not known to appear in the circulation, have any pharmacological effects or be approved for human use195, is advertised on the black-market and internet196 for alleged anabolic or tissue repair/regeneration benefits. Like other short peptide with known structure, it is readily detectable using LC- tandem MS196.

Growth factors, GH releasing and other peptides

For the unscrupulous in pursuit of the unlawful, the increasingly stringent detection of the most potent ergogenic drugs used in androgen and hemoglobin doping has created a new, more speculative form of doping involving peptide growth factors and GH releasing peptides. These are within the size range of automated bulk custom peptide synthesis and are marketed cheaply by chemical manufacturers. While notionally sold solely for laboratory research, these unregulated products are available for purchase over the internet. Promoted by speculative fantasies on their mode of action coupled with testimonials to their efficacy but without objective testing or assurance of safety in humans, they are believed to be widely used by gullible and/or desperate athletes and their trainers. As unregistered drugs, this growing range of peptides appears to constitute a greater threat to athlete’s health than a risk of effective cheating.

The S2.4 category of Prohibited Substances lists, in addition to GH and IGF-1, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF). The listing also contains a generic catch-all provision for unnamed growth factors and peptides which may affect connective, vascular, muscular or regenerative tissues or energy utilization and other substances with similar chemical structure or biological effects.

The major category of oligopeptides used for doping is the class of GH releasing peptides analogs of the endogenous GH releasing peptides, GHRH and ghrelin, whereby their analogs aim to increase endogenous GH secretion and are therefore banned (table 5). Most of these peptide were developed in the pharmaceutical industry from the 1990s aiming to provide cheaper, orally active, non-peptide agonists with capacity for sustained stimulation of endogenous GH secretion to “rejuvenate the GH/IGF-1 axis”197, an unusually explicit acknowledgment of the regular nexus between hormonal rejuvenation and doping198. However, none of these hormonal peptides have been registered for human therapeutic use with only one (pralmorelin) registered for single-dose, diagnostic use (for GH deficiency) in Japan. Although they may stimulate GH release initially, many failed to achieve sustained GH release due to desensitization and none achieved meaningful clinical improvements in any target diseases. If their unproven ergogenic benefits are due to sustained GH release this renders them unlikely to be beneficial; nevertheless, the caveat on not accepting negative conclusions without direct testing are also relevant to this class of peptides. Like other short peptides, once chemical structures are known, detection is readily feasible using LC-MS199, 200. The illicit nature of this market raises the risks of counterfeit and unsafe products with attendant risks of infection and residual toxic contaminants unlike the purity pharmaceutical product manufacturers are required to demonstrate by batch release testing.

PROGRESS, GAPS AND FUTURE PROSPECTS

Anti-doping science continues to make major progress over recent decades especially since the advent of WADA with its harmonization and focus on deterrence through standardized testing. Like any efforts to combat human malfeasance, the quest for drug-free and safe sport requires ongoing vigilance and continual renewal of intelligence-based detection testing. While great progress has been made in the two canonical forms of doping, androgen and haemoglobin doping, human ingenuity continually finds way to challenge the testing just as traditional frauds are supplanted by cyber-crime and ingenious computer hacking.

The major gaps remaining in anti-doping science are (a) the lack of a definitive test for autologous blood transfusion, (b) need for more sensitive detection tests for peptide doping with wider windows of detection and (c) more economical, affordable and robust sample handling and storage procedures. These challenges must be met by adapting novel technologies such as quantitative proteomics, genomics and metabolomics as well as implementing more out of competition and blood testing. Such progress depends on innovative applied research which is supported by WADA, Partnership for Clean Competition and certain national national anti-doping organisations together with regular peer-review research granting agencies. Finally, the development of effective forensic intelligence investigations, a slow, complex and costly process but which can have salutary effects (eg for road cycling in the Lance Armstrong case), is proving a valuable complementary approach as an adjunct to effective laboratory testing.

REFERENCES

  1. Handelsman, D.J. & Gooren, L.J. Hormones and sport: physiology, pharmacology and forensic science. Asian J Androl 10, 348-50 (2008).
  2. Rawls, J. Justice as Fairness: A Restatement (Belknap, Harvard University Press, Cambridge, MA, 2001).
  3. Ryan, A. Fairness and philosopy. Social Research 73, 597-606 (2006).
  4. Wiesing, U. Should performance-enhancing drugs in sport be legalized under medical supervision? Sports Med 41, 167-76 (2011).
  5. Shuster, S. & Devine, J.W. The banning of sportsmen and women who fail drug tests is unjustifiable. J R Coll Physicians Edinb 43, 39-43 (2013).
  6. Savulescu, J., Creaney, L. & Vondy, A. Should athletes be allowed to use performance enhancing drugs? BMJ 347, f6150 (2013).
  7. Eynon, N. et al. Genes and elite athletes: a roadmap for future research. J Physiol 589, 3063-70 (2011).
  8. de la Chapelle, A., Traskelin, A.L. & Juvonen, E. Truncated erythropoietin receptor causes dominantly inherited benign human erythrocytosis. Proc Natl Acad Sci U S A 90, 4495-9 (1993).
  9. Berman, Y. & North, K.N. A gene for speed: the emerging role of alpha-actinin-3 in muscle metabolism. Physiology (Bethesda) 25, 250-9 (2010).
  10. Wade, N. Anabolic Steroids: Doctors Denounce Them, but Athletes Aren't Listening. Science 176, 1399-403 (1972).
  11. Egner, I.M., Bruusgaard, J.C., Eftestol, E. & Gundersen, K. A cellular memory mechanism aids overload hypertrophy in muscle long after an episodic exposure to anabolic steroids. J Physiol 591, 6221-30 (2013).
  12. WADA. (2014).
  13. Allan, C.M. et al. Estradiol induction of spermatogenesis is mediated via an estrogen receptor-{alpha} mechanism involving neuroendocrine activation of follicle-stimulating hormone secretion. Endocrinology 151, 2800-10 (2010).
  14. WADA Medical Information to Support the Decisions of TUECs - Androgen Deficiency-Male Hypogonadism 2015 https://www.wada-ama.org/en/resources/science-medicine/medical-information-to-support-the-decisions-of-tuecs-adrenal#.VDhr4Bb92-Q.
  15. David, K., Dingemanse, E., Freud, J. & Laqueur, E. Uber krystallinisches mannliches Hormon aus Hoden (Testosteron), wirksamer als aus Harn oder aus Cholestrin bereitetes Androsteron. Hoppe Seylers Zeischrift Physiologische Chemie 233, 281-2 (1935).
  16. Yesalis, C.E., Courson, S.P. & Wright, J.E. in Anabolic Steroids in Sports and Exercise (ed. Yesalis, C.E.) 51-71 (Human Kinetics, Champaign, IL, 2000).
  17. Franke, W.W. & Berendonk, B. Hormonal doping and androgenization of athletes: a secret program of the German Democratic Republic government. Clinical Chemistry 43, 1262-79 (1997).
  18. Ryan, A.J. Anabolic steroids are fool's gold. Fed Proc 40, 2682-8 (1981).
  19. Elashoff, J.D., Jacknow, A.D., Shain, S.G. & Braunstein, G.D. Effects of anabolic-androgenic steroids on muscular strength. Annals of Internal Medicine 115, 387-393 (1991).
  20. Bhasin, S. et al. The effects of supraphysiologic doses of testosterone on muscle size and strength in normal men. New England Journal of Medicine 335, 1-7 (1996).
  21. Woodhouse, L.J. et al. Development of models to predict anabolic response to testosterone administration in healthy young men. Am J Physiol Endocrinol Metab 284, E1009-17 (2003).
  22. Storer, T.W. et al. Testosterone dose-dependently increases maximal voluntary strength and leg power, but does not affect fatigability or specific tension. J Clin Endocrinol Metab 88, 1478-85 (2003).
  23. Bhasin, S. et al. Older men are as responsive as young men to the anabolic effects of graded doses of testosterone on the skeletal muscle. J Clin Endocrinol Metab 90, 678-88 (2005).
  24. Stenman, U.H., Hotakainen, K. & Alfthan, H. Gonadotropins in doping: pharmacological basis and detection of illicit use. Br J Pharmacol 154, 569-83 (2008).
  25. Handelsman, D.J. Indirect androgen doping by oestrogen blockade in sports. Br J Pharmacol 154, 598-605 (2008).
  26. Handelsman, D.J. Clinical review: The rationale for banning human chorionic gonadotropin and estrogen blockers in sport. J Clin Endocrinol Metab 91, 1646-53 (2006).
  27. Kicman, A.T. Pharmacology of anabolic steroids. Br J Pharmacol 154, 502-21 (2008).
  28. Thevis, M. & Schanzer, W. Synthetic anabolic agents: steroids and nonsteroidal selective androgen receptor modulators. Handb Exp Pharmacol, 99-126 (2010).
  29. Sobolevsky, T. & Rodchenkov, G. Detection and mass spectrometric characterization of novel long-term dehydrochloromethyltestosterone metabolites in human urine. J Steroid Biochem Mol Biol 128, 121-7 (2012).
  30. Sobolevsky, T. & Rodchenkov, G. Mass spectrometric description of novel oxymetholone and desoxymethyltestosterone metabolites identified in human urine and their importance for doping control. Drug Test Anal 4, 682-91 (2012).
  31. Thevis, M., Piper, T., Beuck, S., Geyer, H. & Schanzer, W. Expanding sports drug testing assays: mass spectrometric characterization of the selective androgen receptor modulator drug candidates RAD140 and ACP-105. Rapid Commun Mass Spectrom 27, 1173-82 (2013).
  32. Kohler, M. et al. Confiscated black market products and nutritional supplements with non-approved ingredients analyzed in the Cologne Doping Control Laboratory 2009. Drug Test Anal 2, 533-7 (2010).
  33. Xue, Y. et al. Adaptive evolution of UGT2B17 copy-number variation. Am J Hum Genet 83, 337-46 (2008).
  34. Schulze, J.J. et al. Doping test results dependent on genotype of uridine diphospho-glucuronosyl transferase 2B17, the major enzyme for testosterone glucuronidation. J Clin Endocrinol Metab 93, 2500-6 (2008).
  35. Schulze, J.J. et al. Substantial advantage of a combined Bayesian and genotyping approach in testosterone doping tests. Steroids 74, 365-8 (2009).
  36. Sottas, P.E., Saugy, M. & Saudan, C. Endogenous steroid profiling in the athlete biological passport. Endocrinol Metab Clin North Am 39, 59-73, viii-ix (2010).
  37. Shackleton, C.H., Roitman, E., Phillips, A. & Chang, T. Androstanediol and 5-androstenediol profiling for detecting exogenously administered dihydrotestosterone, epitestosterone, and dehydroepiandrosterone: potential use in gas chromatography isotope ratio mass spectrometry. Steroids 62, 665-73 (1997).
  38. Cawley, A.T. & George, A.V. Complementary stable carbon isotope ratio and amount of substance measurements in sports anti-doping. Drug Test Anal 4, 897-911 (2012).
  39. Piper, T. et al. Determination of 13C/12C ratios of endogenous urinary steroids: method validation, reference population and application to doping control purposes. Rapid Commun Mass Spectrom 22, 2161-75 (2008).
  40. Cawley, A.T., Trout, G.J., Kazlauskas, R., Howe, C.J. & George, A.V. Carbon isotope ratio (delta13C) values of urinary steroids for doping control in sport. Steroids 74, 379-92 (2009).
  41. Van Renterghem, P., Van Eenoo, P., Sottas, P.E., Saugy, M. & Delbeke, F. Subject-based steroid profiling and the determination of novel biomarkers for DHT and DHEA misuse in sports. Drug Test Anal 2, 582-8 (2010).
  42. Cawley, A. et al. Stable isotope ratio profiling of testosterone preparations. Drug Test Anal 2, 557-67 (2010).
  43. Van Renterghem, P., Van Eenoo, P., Sottas, P.E., Saugy, M. & Delbeke, F. A pilot study on subject-based comprehensive steroid profiling: novel biomarkers to detect testosterone misuse in sports. Clin Endocrinol (Oxf) (2011).
  44. Piper, T., Emery, C. & Saugy, M. Recent developments in the use of isotope ratio mass spectrometry in sports drug testing. Anal Bioanal Chem 401, 433-47 (2011).
  45. Piper, T., Emery, C., Thomas, A., Saugy, M. & Thevis, M. Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing. Anal Bioanal Chem 405, 5455-66 (2013).
  46. Thevis, M., Piper, T., Horning, S., Juchelka, D. & Schanzer, W. Hydrogen isotope ratio mass spectrometry and high-resolution/high-accuracy mass spectrometry in metabolite identification studies: detecting target compounds for sports drug testing. Rapid Commun Mass Spectrom 27, 1904-12 (2013).
  47. Kicman, A.T. et al. Criteria to indicate testosterone administration. Br J Sports Med 24, 253-64 (1990).
  48. Goebel, C. et al. Screening for testosterone abuse in male athletes using the measurement of urinary LH, a revision of the paradigm. Drug Test Anal 1, 511-7 (2009).
  49. Handelsman, D.J. & Heather, A. Androgen abuse in sports. Asian J Androl 10, 403-15 (2008).
  50. Zierau, O., Lehmann, S., Vollmer, G., Schanzer, W. & Diel, P. Detection of anabolic steroid abuse using a yeast transactivation system. Steroids 73, 1143-7 (2008).
  51. Houtman, C.J. et al. Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassays. Anal Chim Acta 637, 247-58 (2009).
  52. Cadwallader, A.B., Lim, C.S., Rollins, D.E. & Botre, F. The androgen receptor and its use in biological assays: looking toward effect-based testing and its applications. J Anal Toxicol 35, 594-607 (2011).
  53. Akram, O.N. et al. Evaluation of androgenic activity of nutraceutical-derived steroids using mammalian and yeast in vitro androgen bioassays. Anal Chem 83, 2065-74 (2011).
  54. Death, A.K., McGrath, K.C., Kazlauskas, R. & Handelsman, D.J. Tetrahydrogestrinone is a potent androgen and progestin. J Clin Endocrinol Metab 89, 2498-500 (2004).
  55. McRobb, L. et al. Structure-activity relationships of synthetic progestins in a yeast-based in vitro androgen bioassay. Journal of Steroid Biochemistry and Molecular Biology 110, 39-47 (2008).
  56. Kintz, P. Testing for anabolic steroids in hair: a review. Leg Med (Tokyo) 5 Suppl 1, S29-33 (2003).
  57. Deng, X.S., Kurosu, A. & Pounder, D.J. Detection of anabolic steroids in head hair. J Forensic Sci 44, 343-6 (1999).
  58. Kintz, P., Cirimele, V., Sachs, H., Jeanneau, T. & Ludes, B. Testing for anabolic steroids in hair from two bodybuilders. Forensic Sci Int 101, 209-16 (1999).
  59. Kintz, P., Cirimele, V., Jeanneau, T. & Ludes, B. Identification of testosterone and testosterone esters in human hair. J Anal Toxicol 23, 352-6 (1999).
  60. Hold, K.M., Borges, C.R., Wilkins, D.G., Rollins, D.E. & Joseph, R.E., Jr. Detection of nandrolone, testosterone, and their esters in rat and human hair samples. J Anal Toxicol 23, 416-23 (1999).
  61. Gaillard, Y., Vayssette, F., Balland, A. & Pepin, G. Gas chromatographic-tandem mass spectrometric determination of anabolic steroids and their esters in hair. Application in doping control and meat quality control. J Chromatogr B Biomed Sci Appl 735, 189-205 (1999).
  62. Gaillard, Y., Vayssette, F. & Pepin, G. Compared interest between hair analysis and urinalysis in doping controls. Results for amphetamines, corticosteroids and anabolic steroids in racing cyclists. Forensic Sci Int 107, 361-79 (2000).
  63. Cirimele, V., Kintz, P. & Ludes, B. Testing of the anabolic stanozolol in human hair by gas chromatography-negative ion chemical ionization mass spectrometry. J Chromatogr B Biomed Sci Appl 740, 265-71 (2000).
  64. Kintz, P., Cirimele, V. & Ludes, B. Discrimination of the nature of doping with 19-norsteroids through hair analysis. Clin Chem 46, 2020-2 (2000).
  65. Kintz, P., Cirimele, V., Dumestre-Toulet, V. & Ludes, B. Doping control for nandrolone using hair analysis. J Pharm Biomed Anal 24, 1125-30 (2001).
  66. Dumestre-Toulet, V., Cirimele, V., Ludes, B., Gromb, S. & Kintz, P. Hair analysis of seven bodybuilders for anabolic steroids, ephedrine, and clenbuterol. J Forensic Sci 47, 211-4 (2002).
  67. Kintz, P., Cirimele, V., Dumestre-Toulet, V., Villain, M. & Ludes, B. Doping control for methenolone using hair analysis by gas chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 766, 161-7 (2002).
  68. Bresson, M., Cirimele, V., Villain, M. & Kintz, P. Doping control for metandienone using hair analyzed by gas chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 836, 124-8 (2006).
  69. Gambelunghe, C. et al. Analysis of anabolic steroids in hair by GC/MS/MS. Biomed Chromatogr 21, 369-75 (2007).
  70. Deshmukh, N., Hussain, I., Barker, J., Petroczi, A. & Naughton, D.P. Analysis of anabolic steroids in human hair using LC-MS/MS. Steroids 75, 710-4 (2010).
  71. Strano-Rossi, S. et al. Screening for exogenous androgen anabolic steroids in human hair by liquid chromatography/orbitrap-high resolution mass spectrometry. Anal Chim Acta 793, 61-71 (2013).
  72. Kintz, P., Cirimele, V. & Ludes, B. Physiological concentrations of DHEA in human hair. J Anal Toxicol 23, 424-8 (1999).
  73. Kintz, P., Cirimel, V., Devaux, M. & Ludes, B. Dehydroepiandrosterone (DHEA) and testosterone concentrations in human hair after chronic DHEA supplementation. Clin Chem 46, 414-5 (2000).
  74. Rambaud, L. et al. Development and validation of a multi-residue method for the detection of a wide range of hormonal anabolic compounds in hair using gas chromatography-tandem mass spectrometry. Anal Chim Acta 586, 93-104 (2007).
  75. Deshmukh, N.I., Barker, J., Petroczi, A. & Naughton, D.P. Detection of testosterone and epitestosterone in human hair using liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal 67-68, 154-8 (2012).
  76. Segura, J., Pichini, S., Peng, S.H. & de la Torre, X. Hair analysis and detectability of single dose administration of androgenic steroid esters. Forensic Sci Int 107, 347-59 (2000).
  77. Brown, H.G. & Perrett, D. Detection of doping in sport: detecting anabolic-androgenic steroids in human fingernail clippings. Med Leg J 79, 67-9 (2011).
  78. Schonfelder, M. et al. Gene expression profiling in human whole blood samples after controlled testosterone application and exercise. Drug Test Anal 3, 652-60 (2011).
  79. Handelsman, D.J. et al. Effects of recombinant human LH and hCG on serum and urine LH and androgens in men. Clinical Endocrinology 71, 417-28 (2009).
  80. Handelsman, D.J. et al. Detection and effects on serum and urine steroid and LH of repeated GnRH analog (leuprolide) stimulation. J Steroid Biochem Mol Biol 141, 113-20 (2014).
  81. Graham, M.R. et al. Counterfeiting in performance- and image-enhancing drugs. Drug Test Anal 1, 135-42 (2009).
  82. Elliott, S. Erythropoiesis-stimulating agents and other methods to enhance oxygen transport. Br J Pharmacol 154, 529-41 (2008).
  83. Reichel, C. & Gmeiner, G. Erythropoietin and analogs. Handb Exp Pharmacol, 251-94 (2010).
  84. di Prampero, P.E. & Ferretti, G. Factors limiting maximal oxygen consumption in humans. Respir Physiol 80, 113-27 (1990).
  85. Pace, N., Consolazio, W.V. & Lozner, E.L. The effect of transfusions of red blood cells on the hypoxia tolerance of normal men. Science 102, 589-91 (1945).
  86. Pace, N., Lozner, E.L. & et al. The increase in hypoxia tolerance of normal men accompanying the polycythemia induced by transfusion of erythrocytes. Am J Physiol 148, 152-63 (1947).
  87. Ekblom, B., Goldbarg, A.N. & Gullbring, B. Response to exercise after blood loss and reinfusion. J Appl Physiol 33, 175-80 (1972).
  88. Hoberman, J. History and prevalence of doping in the marathon. Sports Med 37, 386-8 (2007).
  89. Birkeland, K.I. et al. Effect of rhEPO administration on serum levels of sTfR and cycling performance. Med Sci Sports Exerc 32, 1238-43 (2000).
  90. Ashenden, M.J. et al. A comparison of the physiological response to simulated altitude exposure and r-HuEpo administration. J Sports Sci 19, 831-7 (2001).
  91. Russell, G., Gore, C.J., Ashenden, M.J., Parisotto, R. & Hahn, A.G. Effects of prolonged low doses of recombinant human erythropoietin during submaximal and maximal exercise. Eur J Appl Physiol 86, 442-9 (2002).
  92. Nelson, M., Popp, H., Sharpe, K. & Ashenden, M. Proof of homologous blood transfusion through quantification of blood group antigens. Haematologica 88, 1284-95 (2003).
  93. Cartron, J.P. & Colin, Y. Structural and functional diversity of blood group antigens. Transfus Clin Biol 8, 163-99 (2001).
  94. Giraud, S., Robinson, N., Mangin, P. & Saugy, M. Scientific and forensic standards for homologous blood transfusion anti-doping analyses. Forensic Sci Int 179, 23-33 (2008).
  95. Voss, S.C., Thevis, M., Schinkothe, T. & Schanzer, W. Detection of homologous blood transfusion. Int J Sports Med 28, 633-7 (2007).
  96. Manokhina, I. & Rupert, J.L. A DNA-based method for detecting homologous blood doping. Anal Bioanal Chem (2013).
  97. Giraud, S., Sottas, P.E., Robinson, N. & Saugy, M. Blood transfusion in sports. Handb Exp Pharmacol, 295-304 (2010).
  98. Segura, J., Monfort, N. & Ventura, R. Detection methods for autologous blood doping. Drug Test Anal 4, 876-81 (2012).
  99. Doctor, A. & Spinella, P. Effect of processing and storage on red blood cell function in vivo. Semin Perinatol 36, 248-59 (2012).
  100. Monfort, N., Ventura, R., Balcells, G. & Segura, J. Determination of five di-(2-ethylhexyl)phthalate metabolites in urine by UPLC-MS/MS, markers of blood transfusion misuse in sports. J Chromatogr B Analyt Technol Biomed Life Sci 908, 113-21 (2012).
  101. Pottgiesser, T. et al. Hb mass measurement suitable to screen for illicit autologous blood transfusions. Med Sci Sports Exerc 39, 1748-56 (2007).
  102. Morkeberg, J. et al. Detecting autologous blood transfusions: a comparison of three passport approaches and four blood markers. Scand J Med Sci Sports 21, 235-43 (2011).
  103. Pottgiesser, T., Echteler, T., Sottas, P.E., Umhau, M. & Schumacher, Y.O. Hemoglobin mass and biological passport for the detection of autologous blood doping. Med Sci Sports Exerc 44, 835-43 (2012).
  104. Ashenden, M., Gough, C.E., Garnham, A., Gore, C.J. & Sharpe, K. Current markers of the Athlete Blood Passport do not flag microdose EPO doping. Eur J Appl Physiol 111, 2307-14 (2011).
  105. Kannan, M. & Atreya, C. Differential profiling of human red blood cells during storage for 52 selected microRNAs. Transfusion 50, 1581-8 (2010).
  106. Pottgiesser, T. et al. Gene expression in the detection of autologous blood transfusion in sports--a pilot study. Vox Sang 96, 333-6 (2009).
  107. Sottas, P.E., Robinson, N. & Saugy, M. The athlete's biological passport and indirect markers of blood doping. Handb Exp Pharmacol, 305-26 (2010).
  108. Sottas, P.E., Robinson, N., Rabin, O. & Saugy, M. The athlete biological passport. Clin Chem 57, 969-76 (2011).
  109. Pottgiesser, T. & Schumacher, Y.O. Current strategies of blood doping detection. Anal Bioanal Chem (2013).
  110. Cazzola, M. A global strategy for prevention and detection of blood doping with erythropoietin and related drugs. Haematologica 85, 561-3 (2000).
  111. Parisotto, R. et al. A novel method utilising markers of altered erythropoiesis for the detection of recombinant human erythropoietin abuse in athletes. Haematologica 85, 564-72 (2000).
  112. Parisotto, R. et al. Detection of recombinant human erythropoietin abuse in athletes utilizing markers of altered erythropoiesis. Haematologica 86, 128-37 (2001).
  113. Gore, C.J. et al. Second-generation blood tests to detect erythropoietin abuse by athletes. Haematologica 88, 333-44 (2003).
  114. Malcovati, L., Pascutto, C. & Cazzola, M. Hematologic passport for athletes competing in endurance sports: a feasibility study. Haematologica 88, 570-81 (2003).
  115. Sharpe, K., Ashenden, M.J. & Schumacher, Y.O. A third generation approach to detect erythropoietin abuse in athletes. Haematologica 91, 356-63 (2006).
  116. Sottas, P.E. et al. Statistical classificiation of abnormal blood profiles inathletes. International Journal of Biostatistics 2, 3 (2006).
  117. WADA Athlete Biological Passport: Operating guidelines and compilation of required elements. Verson 3.1 2012 http://www.wada-ama.org/Documents/Science_Medicine/Athlete_Biological_Passport/WADA-ABP-Operating-Guidelines_v3.1-EN.pdf.
  118. Ekblom, B. & Berglund, B. Effect of erythropoeitin administration on maximal aerobic power in man. Scandanavian Journal of Medicine and Science in Sport 1, 88-93 (1991).
  119. Jelkmann, W. Biosimilar recombinant human erythropoietins ("epoetins") and future erythropoiesis-stimulating treatments. Expert Opin Biol Ther 12, 581-92 (2012).
  120. Catlin, D.H., Fitch, K.D. & Ljungqvist, A. Medicine and science in the fight against doping in sport. J Intern Med 264, 99-114 (2008).
  121. Lopez, B. The invention of a 'drug of mass destruction': deconstructing the EPO myth. Sport in History 31, 84-109 (2011).
  122. Lasne, F. & de Ceaurriz, J. Recombinant erythropoietin in urine. Nature 405, 635 (2000).
  123. Lasne, F., Martin, L., Crepin, N. & de Ceaurriz, J. Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones. Anal Biochem 311, 119-26 (2002).
  124. Lasne, F., Thioulouse, J., Martin, L. & de Ceaurriz, J. Detection of recombinant human erythropoietin in urine for doping analysis: interpretation of isoelectric profiles by discriminant analysis. Electrophoresis 28, 1875-81 (2007).
  125. Leuenberger, N., Reichel, C. & Lasne, F. Detection of erythropoiesis-stimulating agents in human anti-doping control: past, present and future. Bioanalysis 4, 1565-75 (2012).
  126. Breidbach, A. et al. Detection of recombinant human erythropoietin in urine by isoelectric focusing. Clin Chem 49, 901-7 (2003).
  127. Wickramasinghe, S. & Medrano, J.F. Primer on genes encoding enzymes in sialic acid metabolism in mammals. Biochimie 93, 1641-6 (2011).
  128. Beuck, S., Schanzer, W. & Thevis, M. Hypoxia-inducible factor stabilizers and other small-molecule erythropoiesis-stimulating agents in current and preventive doping analysis. Drug Test Anal 4, 830-45 (2012).
  129. La Merie Publishing Blockbuster Biologics 2012 2013 http://www.pipelinereview.com/.
  130. Imagawa, S. et al. Does k-11706 enhance performance and why? Int J Sports Med 28, 928-33 (2007).
  131. Richardson, R.S., Tagore, K., Haseler, L.J., Jordan, M. & Wagner, P.D. Increased VO2 max with right-shifted Hb-O2 dissociation curve at a constant O2 delivery in dog muscle in situ. J Appl Physiol (1985) 84, 995-1002 (1998).
  132. Pagel, P.S. et al. RSR13, a synthetic modifier of hemoglobin-oxygen affinity, enhances the recovery of stunned myocardium in anesthetized dogs. J Pharmacol Exp Ther 285, 1-8 (1998).
  133. Breidbach, A. & Catlin, D.H. RSR13, a potential athletic performance enhancement agent: detection in urine by gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom 15, 2379-82 (2001).
  134. Thevis, M., Krug, O. & Schanzer, W. Mass spectrometric characterization of efaproxiral (RSR13) and its implementation into doping controls using liquid chromatography-atmospheric pressure ionization-tandem mass spectrometry. J Mass Spectrom 41, 332-8 (2006).
  135. Casadevall, N. et al. Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. N Engl J Med 346, 469-75 (2002).
  136. Macdougall, I.C. et al. Antibody-mediated pure red cell aplasia in chronic kidney disease patients receiving erythropoiesis-stimulating agents: new insights. Kidney Int 81, 727-32 (2012).
  137. Unger, E.F., Thompson, A.M., Blank, M.J. & Temple, R. Erythropoiesis-stimulating agents--time for a reevaluation. N Engl J Med 362, 189-92 (2010).
  138. Bennett, C.L. et al. Venous thromboembolism and mortality associated with recombinant erythropoietin and darbepoetin administration for the treatment of cancer-associated anemia. JAMA 299, 914-24 (2008).
  139. Strippoli, G.F., Navaneethan, S.D. & Craig, J.C. Haemoglobin and haematocrit targets for the anaemia of chronic kidney disease. Cochrane Database Syst Rev, CD003967 (2006).
  140. Tonia, T. et al. Erythropoietin or darbepoetin for patients with cancer. Cochrane Database Syst Rev 12, CD003407 (2012).
  141. Rampling, M.W. Hyperviscosity as a complication in a variety of disorders. Semin Thromb Hemost 29, 459-65 (2003).
  142. Kovesdy, C.P. & Kalantar-Zadeh, K. Iron therapy in chronic kidney disease: current controversies. J Ren Care 35 Suppl 2, 14-24 (2009).
  143. Gagnon, D.R., Zhang, T.J., Brand, F.N. & Kannel, W.B. Hematocrit and the risk of cardiovascular disease--the Framingham study: a 34-year follow-up. Am Heart J 127, 674-82 (1994).
  144. Danesh, J., Collins, R., Peto, R. & Lowe, G.D. Haematocrit, viscosity, erythrocyte sedimentation rate: meta-analyses of prospective studies of coronary heart disease. Eur Heart J 21, 515-20 (2000).
  145. Braekkan, S.K., Mathiesen, E.B., Njolstad, I., Wilsgaard, T. & Hansen, J.B. Hematocrit and risk of venous thromboembolism in a general population. The Tromso study. Haematologica 95, 270-5 (2010).
  146. Holt, R.I., Erotokritou-Mulligan, I. & Sonksen, P.H. The history of doping and growth hormone abuse in sport. Growth Horm IGF Res 19, 320-6 (2009).
  147. Liu, H. et al. Systematic review: the effects of growth hormone on athletic performance. Ann Intern Med 148, 747-58 (2008).
  148. Birzniece, V., Nelson, A.E. & Ho, K.K. Growth hormone and physical performance. Trends Endocrinol Metab 22, 171-8 (2011).
  149. Baumann, G.P. Growth hormone doping in sports: a critical review of use and detection strategies. Endocr Rev 33, 155-86 (2012).
  150. Meinhardt, U. et al. The effects of growth hormone on body composition and physical performance in recreational athletes: a randomized trial. Ann Intern Med 152, 568-77 (2010).
  151. Berggren, A. et al. Short-term administration of supraphysiological recombinant human growth hormone (GH) does not increase maximum endurance exercise capacity in healthy, active young men and women with normal GH-insulin-like growth factor I axes. J Clin Endocrinol Metab 90, 3268-73 (2005).
  152. Irving, B.A. et al. The effects of time following acute growth hormone administration on metabolic and power output measures during acute exercise. J Clin Endocrinol Metab 89, 4298-305 (2004).
  153. Graham, M.R. et al. Short-term recombinant human growth hormone administration improves respiratory function in abstinent anabolic-androgenic steroid users. Growth Horm IGF Res 17, 328-35 (2007).
  154. Goto, K., Doessing, S., Nielsen, R.H., Flyvbjerg, A. & Kjaer, M. Growth hormone receptor antagonist treatment reduces exercise performance in young males. J Clin Endocrinol Metab 94, 3265-72 (2009).
  155. Healy, M.L. et al. High dose growth hormone exerts an anabolic effect at rest and during exercise in endurance-trained athletes. J Clin Endocrinol Metab 88, 5221-6 (2003).
  156. Healy, M.L. et al. Effects of high-dose growth hormone on glucose and glycerol metabolism at rest and during exercise in endurance-trained athletes. J Clin Endocrinol Metab 91, 320-7 (2006).
  157. Breederveld, R.S. & Tuinebreijer, W.E. Recombinant human growth hormone for treating burns and donor sites. Cochrane Database Syst Rev 12, CD008990 (2012).
  158. Takala, J. et al. Increased mortality associated with growth hormone treatment in critically ill adults New England Journal of Medicine 341, 785-92 (1999).
  159. Raschke, M. et al. Effects of growth hormone in patients with tibial fracture: a randomised, double-blind, placebo-controlled clinical trial. Eur J Endocrinol 156, 341-51 (2007).
  160. Sklar, C.A. et al. Risk of disease recurrence and second neoplasms in survivors of childhood cancer treated with growth hormone: a report from the Childhood Cancer Survivor Study. J Clin Endocrinol Metab 87, 3136-41 (2002).
  161. Ergun-Longmire, B. et al. Growth hormone treatment and risk of second neoplasms in the childhood cancer survivor. J Clin Endocrinol Metab 91, 3494-8 (2006).
  162. Carel, J.C. et al. Long-term mortality after recombinant growth hormone treatment for isolated growth hormone deficiency or childhood short stature: preliminary report of the French SAGhE study. J Clin Endocrinol Metab 97, 416-25 (2012).
  163. Mo, D., Hardin, D.S., Erfurth, E.M. & Melmed, S. Adult mortality or morbidity is not increased in childhood-onset growth hormone deficient patients who received pediatric GH treatment: an analysis of the Hypopituitary Control and Complications Study (HypoCCS). Pituitary (2013).
  164. Swerdlow, A.J., Higgins, C.D., Adlard, P. & Preece, M.A. Risk of cancer in patients treated with human pituitary growth hormone in the UK, 1959-85: a cohort study. Lancet 360, 273-7 (2002).
  165. Wilton, P., Mattsson, A.F. & Darendeliler, F. Growth hormone treatment in children is not associated with an increase in the incidence of cancer: experience from KIGS (Pfizer International Growth Database). J Pediatr 157, 265-70 (2010).
  166. Mackenzie, S. et al. Long-term safety of growth hormone replacement after CNS irradiation. J Clin Endocrinol Metab 96, 2756-61 (2011).
  167. Woodmansee, W.W. et al. Incidence of second neoplasm in childhood cancer survivors treated with GH: an analysis of GeNeSIS and HypoCCS. Eur J Endocrinol 168, 565-73 (2013).
  168. Jenkins, P.J., Mukherjee, A. & Shalet, S.M. Does growth hormone cause cancer? Clin Endocrinol (Oxf) 64, 115-21 (2006).
  169. Holly, J. & Perks, C. Growth hormone and cancer: are we asking the right questions?*. Clin Endocrinol (Oxf) 64, 122-4 (2006).
  170. Ho, K.K. & Nelson, A.E. Growth hormone in sports: detecting the doped or duped. Horm Res Paediatr 76 Suppl 1, 84-90 (2011).
  171. Hepner, F., Cszasar, E., Roitinger, E. & Lubec, G. Mass spectrometrical analysis of recombinant human growth hormone (Genotropin(R)) reveals amino acid substitutions in 2% of the expressed protein. Proteome Sci 3, 1 (2005).
  172. Hepner, F., Csaszar, E., Roitinger, E., Pollak, A. & Lubec, G. Mass spectrometrical analysis of recombinant human growth hormone Norditropin reveals amino acid exchange at M14_V14 rhGH. Proteomics 6, 775-84 (2006).
  173. Jiang, H., Wu, S.L., Karger, B.L. & Hancock, W.S. Mass spectrometric analysis of innovator, counterfeit, and follow-on recombinant human growth hormone. Biotechnol Prog 25, 207-18 (2009).
  174. Bidlingmaier, M. & Strasburger, C.J. Growth hormone. Handb Exp Pharmacol, 187-200 (2010).
  175. Bosch, J. et al. Analysis of urinary human growth hormone (hGH) using hydrogel nanoparticles and isoform differential immunoassays after short recombinant hGH treatment: preliminary results. J Pharm Biomed Anal 85, 194-7 (2013).
  176. Bidlingmaier, M. et al. High-sensitivity chemiluminescence immunoassays for detection of growth hormone doping in sports. Clin Chem 55, 445-53 (2009).
  177. Bidlingmaier, M. & Manolopoulou, J. Detecting growth hormone abuse in athletes. Endocrinol Metab Clin North Am 39, 25-32, vii (2010).
  178. Marks, V. Murder by insulin: suspected, purported and proven-a review. Drug Test Anal 1, 162-76 (2009).
  179. Will, R.G. Acquired prion disease: iatrogenic CJD, variant CJD, kuru. Br Med Bull 66, 255-65 (2003).
  180. Brown, P. et al. Iatrogenic Creutzfeldt-Jakob disease at the millennium. Neurology 55, 1075-81 (2000).
  181. Barroso, O., Schamasch, P. & Rabin, O. Detection of GH abuse in sport: Past, present and future. Growth Horm IGF Res 19, 369-74 (2009).
  182. Travis, J. Pharmacology. Growth hormone test finally nabs first doper. Science 327, 1185 (2010).
  183. Powrie, J.K. et al. Detection of growth hormone abuse in sport. Growth Horm IGF Res 17, 220-6 (2007).
  184. Nelson, A.E. et al. Pharmacodynamics of growth hormone abuse biomarkers and the influence of gender and testosterone: a randomized double-blind placebo-controlled study in young recreational athletes. Journal of Clinical Endocrinology and Metabolism 93, 2213-22 (2008).
  185. Krusenstjerna-Hafstrom, T. et al. Biochemical markers of bone turnover in tibia fracture patients randomly assigned to growth hormone (GH) or placebo injections: Implications for detection of GH abuse. Growth Horm IGF Res 21, 331-5 (2011).
  186. Barroso, O., Handelsman, D.J., Strasburger, C. & Thevis, M. Analytical challenges in the detection of peptide hormones for anti-doping purposes. Bioanalysis 4, 1577-90 (2012).
  187. Williams, R.M., McDonald, A., Savage, M.O. & Dunger, D.B. Mecasermin rinfabate: rhIGF-I/rhIGFBP-3 complex: iPLEX. Expert Opin Drug Metab Toxicol 4, 311-24 (2008).
  188. Guha, N. et al. IGF-I abuse in sport. Curr Drug Abuse Rev 2, 263-72 (2009).
  189. Ernst, S. & Simon, P. A quantitative approach for assessing significant improvements in elite sprint performance: has IGF-1 entered the arena? Drug Test Anal 5, 384-9 (2013).
  190. Thomas, A., Kohler, M., Schanzer, W., Delahaut, P. & Thevis, M. Determination of IGF-1 and IGF-2, their degradation products and synthetic analogues in urine by LC-MS/MS. Analyst 136, 1003-12 (2011).
  191. Hess, C. et al. Simultaneous determination and validated quantification of human insulin and its synthetic analogues in human blood serum by immunoaffinity purification and liquid chromatography-mass spectrometry. Anal Bioanal Chem 404, 1813-22 (2012).
  192. Guha, N. et al. Biochemical markers of recombinant human insulin-like growth factor-I (rhIGF-I)/rhIGF binding protein-3 (rhIGFBP-3) misuse in athletes. Drug Test Anal (2013).
  193. Guha, N., Cowan, D.A., Sonksen, P.H. & Holt, R.I. Insulin-like growth factor-I (IGF-I) misuse in athletes and potential methods for detection. Anal Bioanal Chem 405, 9669-83 (2013).
  194. Matheny, R.W., Jr., Nindl, B.C. & Adamo, M.L. Minireview: Mechano-growth factor: a putative product of IGF-I gene expression involved in tissue repair and regeneration. Endocrinology 151, 865-75 (2010).
  195. Esposito, S., Deventer, K. & Van Eenoo, P. Characterization and identification of a C-terminal amidated mechano growth factor (MGF) analogue in black market products. Rapid Commun Mass Spectrom 26, 686-92 (2012).
  196. Smith, R.G. Development of growth hormone secretagogues. Endocr Rev 26, 346-60 (2005).
  197. Hersch, E.C. & Merriam, G.R. Growth hormone (GH)-releasing hormone and GH secretagogues in normal aging: Fountain of Youth or Pool of Tantalus? Clin Interv Aging 3, 121-9 (2008).
  198. Thomas, A., Walpurgis, K., Krug, O., Schanzer, W. & Thevis, M. Determination of prohibited, small peptides in urine for sports drug testing by means of nano-liquid chromatography/benchtop quadrupole orbitrap tandem-mass spectrometry. J Chromatogr A 1259, 251-7 (2012).
  199. Thomas, A., Delahaut, P., Krug, O., Schanzer, W. & Thevis, M. Metabolism of growth hormone releasing peptides. Anal Chem 84, 10252-9 (2012).
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