Dc\/IC\lf ADTI^ IC Sporti Mad 2008 38 («: 505^6KtV l tW MKI IL -LC oii2-iw2/08/ooo6-0505/Sde.oo/a

© 2008 Ads Doto Informotlon BV, All rights

Anabolic Steroid UsePatterns of Use and Detection of Doping

Michael R. Graham,^ Bruce Davies,^ Tergal M. Grace} Andrew Kicman^ andJuUen S. Baker^

1 Department of Health and Exercise Science, School of Applied Science, University ofGlamorgan, Pontypridd, UK

2 Drug Control Centre, King's College, London, UK

ContentsAbstract 5051. Background 506

1.1 What are Ariabollc-Androgenlc Steroids (AAS)? 5061.2 Classes of AAS Preparations 5071.3 Therapeutic Use of AAS 5111.4 Non-Therapeutic Use of AAS 5121.5 The Dangers of AAS Use 5121.6 The Prevalence of AAS Use 513.,7 Medicai Support for AAS Users 514

2. Methods of Using AAS by Athletes 5143. Dosages of AAS Used by Bodybuiiders 5154. Detection of AAS 515

4.1 Sample Coiiection from Athietes 5154.2 Analysis 515

5. Non-Steroidai Hormones, Human Chorionic Gonadotrophin and Human Growth Hormone 5206. Recommendations in Detection Procedures 5217. Current Worid Anti-Doping Agency Out-of-Competitlon Testing Programme 5218. Conciusions 522

Abstract Anabolic-androgenic steroids (AAS) were the first identified doping agentsthat have ergogenic effects and are being used to increase muscle mass andstrength in adult males. Consequently, athletes are still using them to increasephysical performance and bodybuilders are using them to improve size andcosmetic appearance. The prevalence of AAS use has risen dramatically over thelast two decades and filtered into all aspects of society. Support for AAS users hasincreased, but not by the medical profession, who will not accept that AAS usedependency is a psychiatric condition. The adverse effects and potential dangersof AAS use have been well documented. AAS are used in sport by individualswho have acquired knowledge of the half-lives of specific drugs and the dosagesand cycles required to avoid detection. Conversely, they are used by bodybuildersin extreme dosages with the intention of gaining muscle mass and size, with littleor no regard for the consequences. Polypharmacy by self-prescription is prevalentin this sector. Most recently, AAS use has filtered through to 'recreational street

506 Graham et al.

drug' users and is the largest growth of drugs in this subdivision. They are taken tocounteract the anorexic and cachectic effects of the illegal psychotropic streetdrugs. Screening procedures for AAS in World Anti-Doping Agency accreditedlaboratories are comprehensive and sensitive and are based mainly on gas chroma-tography-mass spectrometry, although liquid chromatography-mass spectrometryis becoming increasingly more valuable. The use of carbon isotope mass spec-trometry is also of increasing importance in the detection of natural androgenadministration, particularly to detect testosterone administration. There is a degreeof contentiousness in the scenario of AAS drug use, both within and outside sport.AAS and associated doping agents are not illegal per se. Possession is not anoffence, despite contravening sporting regulations and moral codes. Until AASare classified in the same capacity as street drugs in the UK. where possessionbecomes a criminal offence, they will continue to attract those who want to win atany cost. The knowledge acquired by such work can only assist in the education ofindividuals who use such doping agents, with a view to minimizing health risksand hopefully once again create a level playing field in sport.

1. Background '

This article summarizes the classification of ana-bolic-androgenic steroids (AAS) or anabolic ster-oids and differentiates between their therapeutic andnon-therapeutic use. The distinction between AASuse wilhiti and outside sport has been highlighted.This article also discusses the different types ofAAS used, the methods of administration and thecurrent strategies for detection procedures employedby authorities, intent on harm reduction.

A literature search was conducted using EM-BASE, Entrez-PubMed and MEDLINE. Articlesand abstracts have been cited and referenced from1935 until the present day. The authors have includ-ed their own published research and personal com-munications, and made reference to unpublisheddata, which are in press.

1.1 What are Anabolic-AndrogenicSteroids (AAS)?

AAS are a group of synthetic compounds similarin chemical structure to the natural anabolic steroidtestosterone (see figure l).''"^^ Testosterone, the pre-dominant circulating testicular androgen, is both anactive hormone and a prohormone for the formationof a more active androgen, the 5a-reduced steroiddihydrotestosterone (DHT). Physiological studies of

steroid homione metabolism in the postnatal statedemonstrated that DHT is formed in target tissuesfrom circulating testosterone and is a more potentandrogen than testosterone in several bioassay sys-

Genetic evidence indicates that these two andro-gens work via a common intracellular receptor. Theandrogen receptor (AR) is an intracellular ligand-dependent protein that modulates the expression ofgenes and mediates biological actions of physiologi-cal androgens (testosterone and 5a-DHT) in a cell-specific manner.'^!

During embryonic life, androgens cause the for-mation of the male urogenital tract and hence areresponsible for development of the tissues that serveas the major sites of androgen action in postnatallife.

It has been generally assumed that androgensvirilize the male foetus by the same mechanisms asin the adult, namely by the conversion of circulatingtestosterone to DHT in target tissues.

A role for steroid 5a-reduction in androgen ac-tion became apparent with the findings in 1968 thatDHT, the 5a-reduced derivative of testosterone, isformed in many androgen target tissues where itbinds to the AR.l*'l

DHT binds to the AR more tightly than testoster-one, primarily as a result of stabilization of the AR

© 2008 Adis Data Information BV. All rights reserved. Sports Med 2008: 38 (6)

\nabolic Steroid Use 507

complex, and at low concentrations is as effective astestosterone at high concentrations in enhancing thetranscription of one response element.f ' This find-ing clearly indicated that some effects of DHT arethe result of amplification of the testosterone signal.

Loss of function mutations of the steroid 5a-reductase 2 gene impairs virilization of the urogeni-tai sinus and external genitalia in males.' ^

In summary, DHT formation acts both as a gener-al amplifier of androgen action and conveys specificfunction to the androgen-AR complex. The mecha-nism by which this specific function is mediated isunknown.

The enzyme aromatase controls the androgen/estrogen ratio by catalyzing the conversion of testos-terone into estradiol (E2). Therefore, the regulationt)f E2 synthesis by aromatase is thought to be criticalIn sexual development and differentiation.^^'

The synthetic version of the testosterone mole-cule was originally synthesized from cholesterol bythe scientist Ruckzika in I935J""

Testosterone is synthesized by the interstitialLeydig cells of the testes, which are primarily underthe control of the gonadotrophins, secreted by thepituitary gland. Approximately 95% of circulatingtestosterone, originates directly from testicular se-

cretion.f'"' Following secretion, testosterone is thentransported via the blood to target organs and specif-ic receptor sites. The bodily functions that are underdirect control of testosterone and that have relevanceto the athlete can be divided into two broad classifi-cations: (i) androgenic functions - male hormonaleffects (male-producing); and (ii) anabolic functions- constructive or muscle building.

The clinical advantages of a pure anabolic agentwere recognized many years ago and work wasundertaken by a number of groups and drug compa-nies to modify the testosterone molecule with a viewto maximizing the anabolic effect and minimizingthe androgenic activity. Some of the structural mod-ifications of testosterone to dissociate the anabolicfrom the androgenic effects are shown in figure 1.The extent of the dissociation differs depending onthe modification, but there is no AAS that has ananabolic effect in an athlete without an androgeniceffect.l"i

1.2 Classes of AAS Preparations

There are three major classes of AAS used byathletes, based on the route of administration by theathlete or the carrier solvent:I. Oral AAS preparations.

Introduction of double bond

Removal of Iheangular methyl group

Attachment ot variousgroups at C-2

Esterjflcatlon confers depotactivity for IM administration

Attachment of 17a-alky1group confers oral activity

Attachment of pyrazolering to the A-ring

Attachment of 7a-methylgroup

Attachment of chlorine orhydroxyl group

Fig. 1. The structure of testosterone, and structural modifications to the A- and B-rings of this steroid that increase anabolic activity;substitution al C-17 confers oral or depot activity (reproduced from Kicman and Gower,'^' with permission from the Royal Society ofMedicine). IM = intramuscular.

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508 Graham et al.

2. injectable oil-based AAS preparations.

3. Injectable water-based AAS preparations.

Oral AAS are synthesized in order to offer pro-tection to the molecule when it becomes exposed tothe .strong acid solutions found in the stomach, andwhen it contacts the enzymic mechanisms of theliver. Oral activity is conferred by the substitution ofa methyl (CH3) or ethyl (C2H5) group for the Hattached to the carhon atom (C) on the cyclopentanering structure, in position 17 (as identified in figureI). The 17a-alkylated steroids prevent deactivationby the first-pass metabolism by sterically hinderingoxidation of the 17p-hydroxyl group. Liver dysfunc-tion has been recorded as a consequence of long-term (>6 months) abuse, but probably in excessivedosages.'"^ The treatment of hereditary angioedemapatients with therapeutic oral stanozolol or danazoldid not cause adverse hepatic changes.^'-' Oral ac-tivity can also be conferred by attachment of amethyl group at C-1 as in methenolone or mester-olone, but the potency of the steroid is far weaker.

There are weaker formulations of anabolic agentsthat have the classification of 'nutritional' or 'diet-ary' supplements and are marketed as 'pro-hor-mones' , particularly dehydroepiandrosterone(DHEA), androstenedione, 19-norandrostenedione,androstenediol and 19-norandrostenedioi.i'^' Thesesteroids lack the 17a-alkyl moiety and followingoral administration are extensively metabolized onfirst pass through the liver. DHEA and androstene-dione bind to the AR with such a weak affinity that itis barely activated, but enzymes convert a propor-tion to testosterone. Supplements of DHEA or an-drostenedione may be of little or no benefit tohealthy young men who wish to improve theirstrength and sporting performance if. as would beexpected, any anabolic effect is primarily mitigatedthrough peripheral conversion to testosterone. Anycontribution from exogenous DHEA or androstene-dione will be largely moderated by the large amountof testosterone contributed by the testis. In theyoung adult female, an increase in performance maybe possible following ingestion of these supple-ments, as circulating testosterone would be expectedto increase. The plasma concentration of testoster-

one in females (0.7-2.6 nmoI/L) is approximatelyone-tenth that found in men and the proportionarising from peripheral conversion is much greater.Even though only 12-14% of androstenedione isconverted peripherally to testosterone, this concen-tration accounts for about one-half of the circulatingtestosterone in the female.''•'•'''I As the peripheralcontribution to blood testosterone is far greater inthe young adult female than the male, ingestion ofmodest amounts of DHEA, androstenediol or an-drostenedione is likely to raise circulating testoster-one. There are a few studies describing a modest tolarge increase in circulating testosterone followingadministration to women."^"'^' An investigationwhere the androstenedione was mixed and groundwith lactose to aid dispersion in the gut showed thatthe plasma testosterone concentrations increasedfrom -1 nmol/L to a maximum mean of 25.1 nmol/Lat 75 minutes and remained significantly differentfrom control values between 30 minutes and 8 hourspost-administration.'"'' The mean exposure to tes-tosterone (a.s determined by tbe area under the plas-ma concentration-time curve) was greater than anorder of magnitude compared with the controlperiod and the plasma concentrations observed weresimilar to those encountered in abuse of testosteronefor anabolic purposes. A similar profile would beexpected with long-term administration, but the riskof virilization precludes such a study.

Androstenediol (the deIta-5 form) has beenshown to activate AR target genes in the presence ofAR,'-°' but this hormone also binds with strongaffmity to the estrogen receptor (it is not knownwhether the delta-4 isomer is a potent estrogen, butit is a distinct possibility). Little research has beendone on 19-norandrostenedione and 19-norandros-tenediol, but as a consequence of high-profile drugdoping offences, in athletes, 19-norandrostenedionewas made a controlled drug in the UK (table I).

Oral preparations are characterized by the fol-lowing:

• They have a structure that acidic gastric secre-tions of the stomach will not render ineffective bydegradation.

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Xnabolic Steroid Use 509

Table I. UK/European and US generic and trade names" of oral anabolic steroids (reproduced from Kicman and Gower,'^' witti permissionfrom the Royal Society of Medicine)

Generic name UK/Europe trade name US trade name

Methyliestosterone

Testosterone undecanoate

M eta ndie none

Fluoxymesterone

Oxymethoione

Stanozotol

Elhylestrenol

Norethandrolone

Metenolone

Mesteroione

Methyltestosterone

Restandol, Andriol, Undestor, Panlestone

Dianabol

Ultandfen

Anapolon, Adroyd

Stromba, Winstrol

Nilevar

Primoboian® Depot

Pro-Viron

Android, Metandren, Arcosterone

DJanabol, Dialone

Android-F, Halotestin®

Anadroi 50*

Winstroi*^

Maxibolin

Nilevar

a The use of trade names is for product identification purposes only and does not imply endorsement.

• They have the capability to be absorbed into thegastrointestinal tract, usually the stomach or theproxitnal stnall bowel.

• They are able to withstand total degradation bythe liver enzymes.

• They have a short half-life. In order to maintainthe appropriate blood concentration the drugmust be taken several times a day.

• Following the initial pass through the liver thedrug must still retain the capacity to bind with theAR sites present in skeletal muscle.Parenteral preparations do not require a 17a-

alkyl group, but the 17p-hydroxyl group is esterifiedwith an acid moiety to prevent rapid absorption fromthe oily vehicle, which is usually arachis oil andbenzyl alcohol.f^''

Injectable oil-based preparations are character-ized by the following:• They have a much longer half-life than oral or

water-based injectable steroids, usually in theorder of 1-4 weeks.

• They are normally comprised of a mixture ofarachis/sesame seed oil and alcohol, which formsthe basis of the oil-based carrier.

• The concentration of AAS esters range from 25to 250 mg/mL, per injection dosage.

• They have a degree of pain at the injection site.• They have a slow absorption rate into the blood

stream, so that the liver experiences a low con-centration of the drug compared with substancestaken orally. This may be associated with less

incidence of liver disorder than that associatedwith oral preparations.^^ '

• Basic alteration of the steroid ring at the 17p, willprolong the effect of the drug.Non-pharmaceutical water-based testosterone

suspensions for injection are advertised onbodybuilding websites and cheats in sport may findthese attractive as. in theory, they should be relative-ly short acting. Non-pharmaceutical-based prepara-tions, whether oil or water based, may be a particularhazard to health as the contents may not have beenprepared under sterile conditions. Injectable water-based steroids are characterized by the following:

• They have a half-life of 1-2 weeks, thereforethey require more frequent injections.

• They have less discomfort at the injection sitebecause of a lower viscosity compared with thesame oil-based anabolic agent.

• They have a molecular structure that is in mostcases identical to oil-based preparations.

• They have the ability to mix with other water-based anabolic steroids or water-based vitamins,e.g. vitamin B12.'"'Typical injectable anabolic steroids available in

the UK, Europe and the US, are presented in table II.Transdermal formulations are invariably testos-

terone based, legitimately designed for replacementtherapy, and include the patch and hydroalcoholicgels, to be applied on a daily basis. Other short-acting testosterone preparations include those de-signed to be administered by the sublingual or buc-

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510 Graham et al

Table II. UK/European and US generic and trade names^ of injectable anabolic steroids

Generic name UK/Europe trade name US trade nameBoldenone undecylenate

Drostanolone propionate

Nandrolone decanoale

Nandrolone phenylpropionate

Stanozolol

Testosterone enanthate

Testosterone cypionate

Vebonol

Masteron, Masteril, Metormon, Permastril

Deca-Duraboiln(^

Durabolin®

Stromba

Primoteston'-Depot, Testoviron®-Depol

Depo-Testosterone

Testosterone propionate, phenylpropionate, Sustanonisocaproate, decanoate

Testosterone propionate Testex Leo, Virormone

Trenbolone acetate Finaject. Rnajet

Equipoises-

Drolban

Androlone-D 200, Deca-Durabolin®, Hybolin

Oecanoate, Nandroboiic LA

Anabolin, Androlone, Durabolrn®

Winstrol^' V

Andro LA 200, Andryl 200. Delatestryl

Andro-cyp. Andronaq LA, Andronate,Depotest

Androian, Testex

Finajet, Finaplix-H®a The use of trade names is for product identification purposes only and does not imply endorsement.

cal route. Such short-acting formulations are of par-ticular concern in sports subject to anti-doping tests.

AAS are abused by athletes during training andare therefore usually not taken during the actualcompetitive period, in an attempt to avoid detection.Being aware of the pharmacokinetics of a widevariety of preparations, knowledge of a drug's half-life and detection methods has made it previouslypossible for some athletes to 'pass the test'. ' ' Sincetransdermal, sublingual and buccal preparations,even in large doses, can be cleared from the body inless than a week following withdrawal, oral prepara-tions between 2-14 days, and water soluble 'inject-ables' after 4 weeks, it is possible to use these agentsduring periods of intensive training and test nega-tive.

According to the World Anti-Doping Agency(WADA) statistics (2005), AAS are the most fre-quent adverse analytical findings in- and out-of-competition. Increased out-of-competition testinghelps to combat the cheat who is using short-actingpreparations and ceasing administration prior tocompetition in anticipation of testing.

Finally, there are designer steroids. In the field ofdrug control in sport, designer drugs can be consid-ered as ones that are manufactured specifically tocircumvent the doping tests, i.e, they are supplied inclandestine fashion and are not compounds that areadvertised for the bodybuilding market. The at-tempted use of such has become a covert science in

direct competition with advances in detection meth-ods. This indicates a deliberate involvement of qua-si-medical and even governmental agencies, in thepromotion of drug abuse in sport. With respect toanabolic steroids, there are few known examples todraw on. Classified documents saved after the col-lapse of the German Democratic Republic revealedthat since 1983 a pharmaceutical company had pro-duced parenterai preparations of epitestosteronepropionate exclusively for the governmental dopingprogramme.'-'*! Epitestosterone is a steroid with noanabolic activity, but its administration with testos-terone simultaneously or sequentially enables anathlete to manipulate the test for testosterone admin-istration if the test is based solely on determinationof a raised testosterone/epitestosterone (T/E) ratio(see section 4). One percent of testosterone is excret-ed unchanged, apart from conjugation to glucuronicacid, compared with -30% of epitestosterone. andthe T/E ratio approximates unity normally, but israised in testosterone users. However, administra-tion of these steroids in a ratio of -30 : 1; T/E, e.g.as parenterai or oral (undecanoate ester) prepara-tions will elevate plasma testosterone, but will notaugment the T/E ratio, although the urinary T/lutein-izing hormone (LH) ratio will be raised followingtestosterone administration.'"''"-^! More crudely,epitestosterone could simply be swallowed in antici-pation of a drug test or even attempts be made tourinate over a finger that surreptitiously bas epites-

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Anabolic Steroid Use 511

losterone residue on the surface. In an effort tocounter such strategies, WADA have set a urinarythreshold of 200 )J.g/L for epitestosterone.

More recently, the Bay Area Laboratory Co-operative (BALCO) affair, in California, USA, at-tracted media attention due to the high profile of theathletes involved, not least because of a transdermalpreparation (The Cream') was supplied containingtestosterone and epitestosterone. as well as a sublin-gual preparation of a new anabolic steroid te-trahydrogestrinone (THG), coded as "The Clear'.i **lUnderground chemists recently appear also to beaccessing information concerning other steroids thatwere synthesized several decades ago by pharma-ceutical companies, but were never marketed. Suchsteroids that have been detected to date arenorbolethonef-" ^ and madol'^"' (madol is also re-ferred to as desoxymethyltestosterone by theWADA-accredited laboratory in Montreal, who de-lected the administration of this steroid around thesame time as the laboratory at the University ofCalifornia, Los Angeles [UCLA]).

1.3 Therapeutic Use of AAS

Testosterone has potent androgenic as well asanabolic properties, therefore chemical modificationof the basic testosterone molecule has formed thebasis for the clinical application of synthetic AASfor anabolic purposes. Pharmaceutical companiesinitially developed these synthetic analogues of tes-tosterone in order to treat catabolic medical condi-tions. The intention was to alter the chemical struc-ture to maximize the anabolic and minimize theandrogenic effect, to avoid virillzing side effects inboth women and children in therapeutic doses. Nan-drolone (19-nortestosterone) [figure 2] was the firstsynthetic analogue of testosterone to show a favou-rable degree of anabolic-androgenic dissociation Inanimal experiments to allow it to gain a licence foruse in catabolic medical conditions.'-^'' In the UK.nandrolone was originally licensed for use In osteo-porosis in post-menopausal women, aplastic anae-mia and disseminated carcinoma of the breast. Theclinical usefulness of the many synthetic anabolicsteroids that were subsequently developed in revers-

Nandrolone

4-ene-3-oxo reduction. 17p-hydroxy oxidation

HO' HO"

Fig. 2. Structures of the metabolites ol nandrolone: (a) 19-norandrosterone (3a-hydroxy-5a-estran-17-one): (b) 3a-hydroxy-50-estra-n-17-one; and (c) 19-norepiandrosterone (3p-hydroxy-5a-eslran-17-one) [reproduced from Kicman and Gower,'^' with permission from theRoyal Society of Medicine].

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512 Graham et al.

ing the catabolic state of patients, such as those withsevere bums or wasting diseases, has not been real-ized based on the conclusions of previous reports.As a result, many anabolic steroids developed in thelast century have been withdrawn as licensed prod-ucts in the UK and numerous countries worldwide.Exceptions within the UK are:

• danazol and gestrinone (progestogens with ana-bolic-androgenlc properties), which are usedmainly for the treatment of endometriosis;

• oxymethoione, which has beneficial effects ondamaged myocardium;' - '

• stanozolol, used in the treatment of aplastic anae-mia;

• testosterone preparations (and also mesterelonebut seldom used, if at all) for hormone therapy inmale hypogonadism and castrates;'-'- '

• nandrolone decanoate for osteoporosis (but nownot an advocated treatment).f^ '

However, consideration of their therapeutic effi-cacy for anabolic purposes may need to be revisitedbased on recent reports, especially for the treatmentof sarcopenia (loss of muscle mass and strength).Testosterone and synthetic anabolic steroids, such asoxymetholone, appear to be extremely useful in thetreatment of HIV-related muscle wasting.'""^^l Nan-drolone decanoate has been demonstrated to be ef-fective in countering sarcopenia in patients receiv-ing dialysis.'^"'*"' Trestolone (7a-methyl-19-norT)may be a promising new androgen therapy, forexample in age-related sarcopenia.^'"•'''' AAS havealso been used in catabolic states such as chronicobstructive pulmonary disease'"^ ' and bums.I**'

Observational studies show that blood testoster-one concentrations are consistently lower amongmen with cardiovascular disease, suggesting a poss-ible preventive role for testosterone therapy. Short-term interventional studies show that testosteroneproduces a modest, but consistent, improvement incardiac ischaemia comparable with the effects ofexisting anti-anginal drugs.'^''' Such data would indi-cate that it is the level of androgen present thatcorrelates with the presence of cardiovascular dis-ease, as anabolic steroid abusers who administerexcessive amounts over long periods of time have

altered serum lipoprotein profiles, as found ingroups with an increased risk of developing corona-ry artery disease (see section 1.5).

1.4 Non-Therapeutic Use of AAS

AAS increase muscle mass and strength.''' ^ How-ever, no synthetic steroid has completely eliminatedthe androgenic effect, this is partly due to the factthat the androgenic and anabolic effects differ onlyin location and not in the mechanism of the steroidhormone action. Also, there is a body of evidence tosuggest that there is only one type of active AR.''' ^The steroids possessing the most potent anaboliceffects are those with the greatest androgenic ef-fects, such as nandrolone. metandienone and sta-nozolo!.'"^ ' A synthetic steroid may differ from thenatural androgenic steroid testosterone, by altera-tions in its basic structure. These alterations includethe addition of ethyl, methyl, hydroxyl or benzyl atone or more sites along the synthetic steroid struc-ture.l"*"! The International Olympic Committee(IOC) Medical Commission was established in1961, in an attempt to eradicate the use of drugs insport.l^"l In 1974, AAS became a banned class ofcompounds. It has subsequently been suggested thatUS track-and-field athletes were abusing AAS atthis time.'-''" Following the collapse of the SovietUnion and the defection of scientists to the West andwith the acquisition of clandestine German Demo-cratic Republic (GDR) government documents, aspreviously discussed in section 1.2. the GDR hadestablished a systematic doping programme for

thousands of athletes from the early 1960s until1990.124)

1.5 The Dangers of AAS Use

Psychological effects appear to be the only ad-verse consequence to an acute overdose of AAS, butlong-term administration leads to disturbance in thehypothalamic-pituitary-gonada! axis and the sup-pression of LH and follicle-stimulating hormone.This can result in infertility, testicular atrophy (inmales) and disturbances of the menstrual cycle andsecondary amenorrhoea (in females). The severityof adverse effects depends on which steroid or com-

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Anabolic Steroid Use 513

binatlon of steroids are being abused, the dosage andduration of administration. The adverse effects canbe divided into which end organ is affected; thebrain and therefore the psyche,'^--''^' the skin (cysticacne), the liver (adenoma, carcinoma, peliosishepatis, cholestatic jaundice), the cardiovascularsystem (atrial fibrillation and alteration in lipid pro-file and arterial structure and function)'^''"-''' and thegonadal systems, including the prostate and testes inmales and the ovaries in females.'"^

The main physiological side effects reported byAAS users are presented in figure 3. The mainpsychological side effects reported by AAS usersare presented in figure 4.

In 1994, recreational bodybuilders attending aWelsh needle-exchange clinic completed the 'Buss-Durke Inventory' on feelings of hostility/aggressionquestionnaire. Between AAS cycles they were AASfree. Subjects reported significantly higher feelingsof aggression towards objects, verbal aggression andaggression during training (but not physical aggres-sion towards people), during the 6- to 14-week AASperiods. Other changes during AAS administrationperiods included significantly higher feelings ofaleiiness. irritability, anxiety, suspiciousness andnegativism.'''"^

1.6 The Prevalence of AAS Use

A questionnaire study conducted in 1992 in theSouth Wales area of the UK'* '! found that 39% of160 respondents were regular AAS users.

Hair loss

Nose bleeds

(B Acne

o Aggression

E Testicuiar atrophy

^ insomnia

Water retention

Loss of libido

More powerfui

More satisfied body imageMore confident

Increased libidoMood changes

Unintended aggressionMore relaxed

EuphoriaSocial consequences

Urge to harm othersDecreased libido

DepressionParanoia

0 5 10 15 20 25 30 35 40 45 50

Percentage of AAS users {n = 42)

Fig. 3. Physiological side effects reported by anaboiic-androgenicsteroid (AAS) users (reproduced from Grace et ai.,'^*' with permis-sion from Tayior & Francis Ltd, http://www.tandf.co.uk/journals).

0 10 20 30 40 50 60

Percentage of AAS users (n = 48)

Fig. 4. Psychological side effects reported by anaboiic-androgenicsteroid (AAS) users (reproduced from Graham et ai..'^' witti per-mission).

In 1993, a report investigating use of anabolicsteroids in 21 gymnasiums in England, Scotland andWales found that 119 (9.1%) of the 1310 malerespondents to the questionnaire and eight (2.3%) ofthe 349 female respondents had taken anabolic ster-oids. The youngest user was aged 16 years. Theprevalence of use of anabolic steroids in the gymna-siums ranged from zero (in three gymnasiums) to46% (28 of 61 respondents). The response rate to thequestionnaire was 59% (1677 of 2834).'"'

In the South Wales area in 1996,1* 1 AAS use wasreported in 176 users (171 men and 5 women) andhighlighted that 37% of respondents indicated aneed for more knowledge among drug workers and aless prejudiced attitude from general practitioners.

In 1996. the Canadian Center for Drug-free Sportestimated that >83 000 11 - to 18-year-old Canadians(2.8% of the respondents) were estimated to haveused AAS in the year before the survey to improvesport performance. Twenty nine percent reportedthat they injected them and 29.2% reported shar-

In another questionnaire study conducted in 1997in the South Wales area,! ^ 100 AAS-using athletesreported high rates of polypharmacy (80%) with awide array of drug use amongst this sample group.In the South Wales area in 2001,69% of 107 respon-dents of hardcore gym weight-lifters, were identi-fied as abusing AAS, highlighting that AAS abuse

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514 Graham et al.

was certainly not on the decline.''^' Recent surveysconducted in 2005 in the South Wales area^^ ' and2006 online in AAS popular websites,'^'' estimatethat steroids are being abused by >1 million UKcitizens and >3 million Americans, with significantincreases in female users.

1.7 Medical Support for AAS UsersI

Needle-exchange clinics have been establishedfor AAS users, distinct from conventional drug ad-dicts, in an attempt to educate individuals hygienicuse of such drugs. Drugs in Sport Clinic and User'sSupport (DISCUS) was established by a UK generalpractitioner, but attracted opposition from themedical authorities who believed such drug use wasbeing condoned, rather than the promotion of harmminimization. AAS are now the third most com-monly offered drugs to children in the UK, follow-ing cannabis and amphetamines.' **'

Clinics have also been established, outside sport,for drug addicts, who are using AAS more frequent-ly. In Massachusetts, USA, prior AAS use appearsto be common, but under-recognized, among menentering inpatient substance abuse treatment, espe-cially those with opioid dependence. AAS use mayserve as a 'gateway' to opioid abuse in some casesand may also eause morbidity in its own right.''^'''

The 'Kaleidoscope project', Wales, UK, underthe auspices ofthe Gwent Specialist Substance Mis-use Service (GSSMS), UK, has identified an enor-mous increase in AAS use in drug addicts, who haveturned to AAS as a quick fix for their symptoms ofanorexia, following recreational drug use, From theperiod 2005-6, 700 clients attended from 3000 cli-ents registered with the clinic. Sixty eight percent(478 clients) agreed to answer a questionnaire oninjecting AAS and other ergogenic aids. Eighteenpercent (185 clients) admitted to injecting AAS.Twelve percent (55 clients) admitted to injectingAAS and growth hormone. The youngest AAS userwas 18 and the oldest AAS user was 59 years of age.In this time period, they were supplied with 256 579needles and syringes, equating to one needle persyringe per day.

2. Methods of Using AAS by Athletes

Personal discussions with users and articles inbodybuilding magazines has highlighted the follow-ing non-scientific methods of use:

1. Stacking/blending/shotgunning: using more thanone drug at the same time. Individuals frequentlyuse several anabolic steroids simultaneously, mix-ing oral and/or injectable types, sometimes usingdrugs such as stimulants or painkillers. The rationalefor stacking is a belief, which has not been testedscientifically, that different drugs have a synergisticeffect on muscle size.

2. Tapering: gradually decreasing intake.

3. Plateauing: when a drug becomes ineffective at aparticular level another drug is taken.

4. Cycling: using different drugs for a fixed periodof usually 6-12 weeks, stop administration for thesame period of time, and then repeat the cycle.

5. Pyramiding: maximizing dosage within a fixedspace of time and then minimizing the drug in thesame time frame.

The stacking of AAS preparations has been themost commonly used method by bodybuilders. Thisconcept of using smaller doses of different drugswith similar actions has been well established in themedical field. The overall idea has been to minimizethe potential side effects and maximize the effec-tiveness of the regimes. Taking smaller dosages ofmultiple drugs may reduce the chance of liver ab-normalities when compared with huge dosages of asingle drug.I'1

There is also evidence to suggest that there maybe an increased liver tolerance to a smaller dose ofmultiple drugs compared with a large dose of asingle anabolic agent. This increased tolerancewould allow the liver to increase its degradation ofone particular drug, in much lower concentrations.This may also facilitate the administration of multi-ple anabolic agents for longer periods, minimizingthe plateauing effect.f -'

© 2008 Aais Data Information BV, All rights reserved. Sports Med 200B; 38 C6)

Anabolic Steroid Use

Table HI. Typical anaboiic-androgenrc steroid regime (number of doses) of a first-time user

Drug

Sustanon

Metandienone

Nandrolone decanoate

Stanozolol suspension

Dosage (route of

administration)

250 mg/mLJwk (IM)

5 mg tablet/d (PO)

lOOmg/mL/wk (IM)

50 mg/mtVwk (Ifuf)

tM = intramuscular (parenterat); PO = oral.

Week

1

1

6

2

1

6

3

1

6

4

1

6

2

5

6

2

6

6

2

7

CO

C

M

8

6

2

9

6

3

10

CO

C

O

11

6

3

515

12

6

3

3. Dosages of AAS Usedby Bodybuilders

Bodybuilders are known to misuse enormousdosages of AAS., which have contributed to dysH-poproteinaemia. hyperhomocysteinaemia and pre-mature death.'™' Table III and table IV list exanaplesof the illogical cocktails and current dosages that arebeing promoted, equating to excessive weekly dosesof thousands of milligrams.

4. Detection of AAS

4.1 Sample Collection from Athletes

tjrine is the preferred biological fluid for detec-tion of drugs of abuse. Independent sampling of-ficers in the UK must witness a urine sample beingdelivered into a collection vessel. The sample kitsand the chain-of-custody documentation must beable to withstand legal challenges. The athlete mustpass urine equally into two coded glass bottles, eachassigned a unique code. The samples are designated'A-sample' approximately 70 mL and 'B-sample'approximately 30 mL for confirmatory analysis. Thebottles are sealed using tamper-proof lids and thensent to the laboratory within a sealed shippingcontainer. The independent sampling officer is alsorequired to measure the pH and specific gravity ofthe urine.

When the samples reach the laboratory, the A-sample seal is broken and the urine analysed. If theA-sample fails a drug test, the B-sample seal isbroken at a later date and the analysis repeated. Thetailed drug sample athlete or sports person has theoption to witness this procedure with an independentscientific expert and a legally qualified representa-tive.

Urine specimens are collected from individuals atmultiple locations within a country, therefore trans-port difficulties may lead to delays of several days.Storage of samples in IOC-accredited laboratories isat +4°C or -20°C. In the UK, samples are stored at-20°C. AAS are not thermally labile, but there hasbeen concern about the possibility of microbial pro-duction of testosterone at temperatures that can leadto urine degradation causing positive urinary re-sults.'•"' Markers of degradation include a pH >8.3and/or a high level of 5a-androstenedione and freesteroids that were originally glucuronidated (andro-sterone; etiocholanolone). In 2002, it was demon-strated that testosterone can be elevated by inocula-tion of urine with Candida albicans, but the increasewas minor and of little evidential value, i.e. sampleswould not test positive.' ^l It has been argued thatadding a preservative to urine samples may compro-mise the test as an adverse finding may be chal-lenged on the basis that failure of the test wasbecause of adulteration with a foreign material. Inour opinion, this argument appears to be weak,given the WADA protocol for blood collection,where blood can be drawn into a tube containing aserum separator gel and a clotting activation factor.

4.2 Analysis

In human sports, the IOC Medical Commissionintroduced anabolic steroids as a banned class inApril 1974 following the development of a screenfor the 17a-alkylated orally active drugs. The nameof this banned class was amended to anabolic agentsin the 1990s to incorporate out-of-competition test-ing for clenbuterol and other P2-agonists, which arealso considered to have anabolic activity. In 1999.the WADA was set up as a foundation under theinitiative of the IOC with the support and participa-

> 2006 Adis Dc3ta Irtformatlon BV. All tights reserved. Sports Med 2006; 36 {6)

516 Graham et at.

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© 2006 Adis Data Information 8V. All rights reserved. Sports Med 2008; 38 (6)

Anabolic Steroid Use 517

tion of intergovernmental organizations, govern-ments, public authorities and other public and pri-vate bodies fighting against doping in human sport.Under WADA, the rules and technical documentsconcerning anabolic steroids (and other drugs) areconstantly evolving and for up-to-date informationthe render is strongly advised to access the WADAweb site (www.wada-ama.org). '

In 1969, the first application of radioimmunoas-say (RIA) for the measurement of steroids in biolog-ical fluids was published.'^^i At that time there were14 licensed orally active AAS. These steroids had acommon I7a-alkyl substituent (12 with a 17a-me-thyl group and two with a 17a-ethyl group). Themethod of detection used was to raise immu-noglobulins that could target these two alky! func-tions.' '*' Any presumptive positive samples couldtben be analysed by ga.s chromatography-mass spec-trometry (GC-MS) for confirmatory identifica-tion. ''''l A trial test targeting the orally active alkylat-ed steroids was introduced at the CommonwealthGames in New Zealand in February 1974. Nine of55 samples failed the immunoassay screen andseven samples eonfirmed positive by GC-MS. InApril 1974, the IOC Medical Commission intro-duced AAS as a banned class of compounds in theAnti-Doping Code. In 1979, RIA screens were de-veloped to detect the presence of nandrolone inurine, this AAS being manufactured for intramuscu-lar injection.'''^' Subsequently, RIAs were developedfor nandrolone metabolites.'^^'

In the early 1980s, improvements in the MSallowed IOC-accredited laboratories to develop spe-cific and comprehensive screens able to detect <1[ig/L of an AAS and its metabolite in urine.' '*' Theradioimmunoassay screening procedures to detectadministration of orally active and injectable ana-bolic steroids were replaced in a few years by ana-lysis employing GC-MS. The introduction of abenchtop quadrupole gas chromatograph-massspectrometer offered specificity, sensitivity and ex-cellent data handling, together with a reduction incost compared with previous MS instruments. In-creased chromatographic resolution was obtainedwith the use of superior capillary columns, and

increased sensitivity was achieved by using electronimpact MS in the selected-ion monitoring mode. Inaddition, with automated sample injection and shortchromatographic run times (typically 20-30 minutesbecause of oven temperature programming), largesample throughput made GC-MS the preferred ana-lytical tool. Since AAS are often metabolized exten-sively, with little parent steroid being excreted intothe urine, identification of metabolites for drugmonitoring purposes is required. It is important tonote that the named compounds listed under adversefindings reflect interpretation by the sporting au-thority WADA, ofthe steroid that had been probablyadministered rather than what the laboratory de-clares, which is often a diagnostic metabolite, e.g.19-norandrosterone is the chosen diagnostic metab-olite of nandroione (figure 2). For many steroids,there is more than one diagnostic metabolite. Themetabolism of many AAS in humans, the chemicalsynthesis of the major metabolites and their GCretention times and characteristic mass spectrums.was reviewed in 1993 by Schanzer and

GC-MS continues to be the predominant analyti-cal approach adopted by WADA-accredited labora-tories, screening for anabolic steroids it is supple-mented by the application of more sophisticated MS(currently using magnetic sector instruments) orMS/MS,' ' ' and more recently still by liquid chro-matography-MS/MS (LC-MS/MS). The develop-ments in LC-MS/MS are encouraging and the use ofthis technique is proven as a powerful aid to analysefor anabolic steroids, for example, for the potentialscreening of future unknown designer steroids.'**'' In2004, the designer steroid THG was identified byCatlin et al.,' **' ofthe WADA accredited laboratorywithin UCLA, and several international athletestested positive for this drug and were banned fromcompetition. The American nutritional company'BALCO* has been prosecuted for federal offencesfor the manufacture of the drug and the illegalsupply to athletes. WADA-accredited laboratoriesrapidly screen for the presence of THG in urine byLC-MS/MS. Even so, currently there is no LC-MS/MS procedure that has been developed to date thatcan match that of GC-MS for the comprehensive

S) 2006 Adls Oata Information BV, All rights reserved. Sports Med 2008; 38 (6)

518 Graham et al

screening of the numerous anabolic steroids andtheir metabolites in urine. Primarily, this is becausethe chromatographic resolution of high-performanceliquid chromatography columns is far inferior to thatof GC columns. The development of ultra-peri^orm-ance liquid chromatography-MS (UPLC-MS) maypossibly help to address this issue, offering thepotential advantage of a screening procedure thatavoids time-consuming extraction, glucuronide hy-drolysis and derivatization steps necessary for ana-lysis by GC-MS.

Effective tests for detection of synthetic (foreign)AAS resulted in a large increase in the use of thehormone testosterone over the last 20 years or so, onthe assumption that a test could not be produced todetect a substance that the body produces natural-ly I821 despite its unfavourable androgenic potencycompared with the synthetic drugs. A test based ondetermining whether a urine concentration of testos-terone exceeds the upper limit of a reference rangewould be insensitive because of the wide variabilityin excretion associated with a single-pass urine col-lection. To overcome the problem, in 1979. Brookset al.,''^' in anticipation of athletes switching to theuse of testosterone to evade detection, introducedthe concept of the hormone ratio. The use of a ratiowas considered to be independent of urinary fiowrates. The ratio of testosterone to LH (T/LH) wasoriginally proposed, but this necessitates an immu-noassay procedure for LH and, in retrospect, immu-noprocedures are generally accepted as not havingthe discriminatory power of MS for evidential ana-lysis,' -*' although it is useful as an ancillary test. Inaddition, LH secretion will be suppressed in womenusing oral contraceptives, and therefore the mea-surement of urinary T/LH is only applicable to men.In 1982, the test adopted by the IOC for detection oftestosterone administration was based on the GC-MS determination of the ratio of testosterone to its17a-epimer, epitestosterone, following glucuronidehydrolysis (often referred to as the T/E ratio).'*'''l TheT/E decision limit was derived empirically from anobserved distribution of measurements in speeimenscollected from a large number of individuals. Inhealthy men and women, the median T/E ratio ap-

proximates unity, but supra-physiological doses oftestosterone cause an increase in the ratio as a resultof increased excretion of testosterone, the laboratoryreporting threshold chosen being recently loweredby WADA from a T/E ratio of 6 to a T/E ratio of 4.

In the case of a T/E ratio >4, a reliable method ofdetection (e.g. isotope ratio mass spectrometryIIRMS]) has not determined the exogenous sourceof the substance, further investigations may be con-ducted to ascertain whether a doping offence hasoccurred. Usually it is concluded that surreptitioustestosterone administration has happened, but occa-sionally the athlete may have a physiologically in-creased ratio, being a 'natural biological outli-er' .[85-90] ju addition, the possibility of a pathologicalcondition, e.g. a T-secreting tumour accounting foran augmented ratio in a sports competitor must notbe neglected, although there is no such case reportdescribed in the scientific literature (possibly be-cause such tumours are most likely to be of testicularorigin and that these also secrete epitestosterone).

With an adverse finding, investigating the T/Eresults from previous and subsequent tests, i.e. as-sessing the T/E intra-individual (within-subject)variability, is useful in determining whether an of-fence has occurred. However, to date, there are verylimited data on intra-individual variation of T/Eratios presented in the peer-reviewed literature. Intheir article on detection of testosterone and xenobi-otics, Catlin et al.,'^'' have reviewed the data onintra-individual variability. They present their crite-ria for determining whether testosterone doping hasoccurred in men, based on T/E ratio data from drug-free males who showed an intra-individual co-efficient of variation (CV) of <60% (variation fromthe collection of three or more samples of urinetaken at monthly or greater intervals). In contrast,they report an example of a case of an athlete withan initial T/E ratio of 8.2, and after being sampledfour times had a CV of 114%, indicating that testos-terone administration had occurred. This pattern wasconsidered to be typical of an individual who iscaught and then discontinues testosterone adminis-tration. In these authors' experience, most testoster-one users who provide three or more urine samples

© 2008 Adls Data Information BV. All rights reserved. Sports Med 200B; 38 (6)

\nabolic Steroid Use 519

have a CV of >60%. In 1997, individuals with a CV<60% and a T/E ratio between 6 and 10, weretentatively classified as 'naturally increased''*^'' (ther/E threshold was 6:1) . WADA in their TechnicalDocument (TD2004EAAS) state that "normal varia-tion of up to 60% may be expected" and that "usingappropriate statistical evaluation is found to be sig-nificantly different, that will constitute a proof of theadministration of a source of testosterone." In theevent that previous T/E results are not available.three further unannounced tests should be carried(lut, preferably within a 3-month period followingihe report of the suspicious analytical result.

WADA have also adopted the isotope ratio ap-proach as an important tool to aid the determinationof natural androgen administration, particularly tes-tosterone.'^- '^-' The natural abundance of '^C is-1.11%, the human diet consisting of plant andanimal sources, with varying '- C isotope contentrelative to '^C due to isotopic fractionation in bio-logical systems.''"'' Endogenously produced ster-oids should thus have a '-^C/'^C that refiects anaverage of that in the carbon sources ingested (fig-ure 5). Testosterone used in pharmaceutical formu-lations is now generally synthesized from soya-beanstigmasterol, which has a smaller '^C content. De-tection of misuse of testosterone and related andro-

Low High

Vegetable material

\

Animalconsumption

Humanconsumption

Pharmaceuticalsteroids

Endogenouslyproduced steroids

Fig. 5. Endogenously produced steroids have a '^C/'^C contentthat reflects an average of that in the carbon sources ingested,whereas testosterone in pharmaceutical formulations are synthe-sized from soy, which has a smaller 13C content (a more negativedelta value). The dark arrow represents the direct route to pharma-ceutical steroids containing low levels of ' ^c (reproduced fromKicman and Gower.Pl with permission from the Royal Society ofl^edicine).

gens can therefore be based on assessing whetherthere is a reduction in the relative carbon isotopecontent of targeted urinary steroids. The analyticalprobe used is GC/combustion/IRMS (GC/C/IRMS),with a number of recent articles'* '**' reporting itspotential for detecting testosterone administration inhuman sport. This states that the isotopic ratios O^C/'2C) of the relevant metabolites of such androgensshould whenever possible be measured each time anelevated parameter of the steroid profile is estimatedfrom the Screening Procedure or Confirmation Pro-cedure and reported to the Testing Authority ashaving been determined.''"^' The results of theIRMS analysis and/or of the steroid profile mea-sured by GC-MS shall be used to draw conclusionsas to whether a doping violation may have beencommitted. WADA recommend IRMS analysis on aurine sample with a T/E ratio of >4. or whereconcentrations of the following androgens exceedconcentration limits (in parentheses): testosterone orepitestosterone (200 ng/mL), DHEA (100 ng/mL),androsterone or etiocholanolone (10 000 ng/mL).

The lowering of the threshold for the T/E ratiofrom 6 to 4 has led to intense discussion with theaccredited laboratories and raised questions. Ac-cording to the International Federation of Associa-tion Football (FIFA) database 2005, none of thesamples with elevated ratios between 4 and 6 show-ed evidence of exogenous intake in the GC-IRMStests. Legal difficulties arise in cases where the T/Eratio is between 4 and 6, but GC-IRMS does notverify exogenous intake.'""' The most likely reasonfor this disparity is because IRMS demands a largeramount of analyte injected eompared with standardGC-MS and for this reason metabolites of testoster-one are usually targeted for isotopic analysis ratherthan testosterone itself. Metabolism from an exoge-nous source of testosterone appears to be similar tothat of endogenous testosterone, where a large pro-portion (-30%) is converted to androsterone andetiocholanolone (figure 2) with only a very minorproportion (-1%) being excreted as testosterone(mainly as glucuronide conjugate). Following glu-curonide hydrolysis and extraction procedures, anathlete will have failed a dope test if the GC/C/

2008 Adis Doto Information BV. All rights reserved. Sports Med 2008; 38 (6)

520 Graham et al.

IRMS values of androsterone and/or etiochola-noione in a sample are more negative than a chosencut-off value. Alternatively, a value that i.s suspi-ciously negative, but more positive than the choseticut-off, might require further samples collected onseparate occasions also to be measured by IRMS todetermine the 'usual' delta value for that individual.The problem with the approach of measuring andro-sterone and etiocholanolone is that the exogenousportion of these 17-oxosteroid metabolites is dilutedby an endogenous source that is not suppressed withtestosterone administration. It is well recognizedthat the clinical assay used for urinary 17-oxoster-oids was a poor index of androgenic status, sinceabout two-thirds originates from adrenal steroidmetabolism.!'"'''*! Xhg adrenal steroids secretedthat are metabolized to urinary androsterone andetiocholanolone glucuronides are DHEA sulphateand DHEA (which can be peripherally intercon-verted), and androstenedione. This adrenal contribu-tion will attenuate the sensitivity of the IRMS testfor etiochoianolone and androsterone, especiallywhere smaller doses of testosterone are being ad-ministered or when the sample collected is on thetail end of the elimination of testosterone. To ad-dress this problem, a number of laboratories are nowdeveloping methods so that the parent steroid, tes-tosterone ilself, can be analysed.

5. Non-Steroidat Hormones, HumanChorionic Gonadotrophin and HumanGrowth Hormone

Athletes abusing drugs up to the time of competi-tion have used human chorionic gonadotrophin(hCG). hCG is a polypeptide glycoprotein hormoneproduced by the female placenta, having the actionof pituitary LH and has been used by bodybuildersin an attempt to prevent testicular atrophy duringprolonged administration of AAS. In 1987, identifi-cation of hCG in sports samples led to its banning bythe IOC Medical Commission.""""

The use of hCG results in an elevation of testos-terone and epitestosterone. since the testis contrib-utes approximately 95% of the pool of urinary tes-tosterone and epitestosterone glucuronide in

eugonadal males. Consequently, there is an equalstimulation of urinary testosterone and epitestoster-one and the T/E ratio remains relatively unal-tered.'"'^' The rise in serum testosterone is modest,being only 2- to 3-fold in healthy eugonadal men.More insidiously, hCG co-administered with testos-terone can suppress the urinary T/E ratio elevationby stimulating epitestosterone production and mayconfound the testing procedure in comparison totaking testosterone alone.''"''^' A validated immu-noassay is required to detect and quantitate hCG.For confirmation, a second different immunoassayof hCG is required, which may lead to variance inresults due to difference in immunoglobuiin speci-ficity between kit manufacturers. A recommendeddetection limit of 10 TU/L in ultrafiltered urine sam-ples, above which a sample is positive was recom-mended using the Serono MAIAclone assay.!''"^Currently there is no reporting threshold set byWADA, but there is a required performance limitwhere all the accredited laboratories must be able todetect a minimum of 5 lU/L. If methods based onMS for endpoint measurement are to achieve thesame sensitivity associated with immunoassays forprotein hormones, then they may be adopted forconfirmatory analysis.!""! The diagnostic capabilityof MS has been shown by the enzymal lysis of hCG,using matrix-assisted laser desorption ionizationtime ofnight(MALDI-TOF)/MS.!"" Subsequently,methods for the detection of low concentrations ofurinary hCG by LC-MS have been developed basedon iinalysis of trypsin digests of extracts from immu-noaffmity trapping and analysis.!"^! Following onfrom this work,!"^! a limit of detection correspond-ing to that of 5 IU/L of urinary hCG has beenachieved. The approach of immunoaffinity trappingand concentration of digested sample extract prior toMS analysis can demonstrate similar analytical sen-sitivity to that of immunoassay. Although such anapproach confers the huge advantage of informativedata, a major disadvantage is that it is relatively slowand labour intensive.

Blood testing is required to identify the abuse ofhuman growth hormone (hGH), which is a proteinhormone with anabolic properties. In growth hor-

© 2006 Adis Data infofmation BV. All rights reserved. Sports Med 2008; 38 (6)

Anabolic Steroid Use 521

mone deficiency, recombinant hGH is a potent ana-holic homione that stimulates the intracellular trans-port of amino acids and causes nitrogen retentionand anabolism.'"''' In fit young men and women,however, current research has demonstrated no sta-tistically significant increase in muscle size orstrength with recombinant hGH administra-(JQjj [115,1 i6] ij jg possible that larger doses over alonger time may achieve the 'desired effect', but it ismuch more likely that such a regime would bedetrimental to health and athletic performance be-cause of the manifestation of symptoms expectedwith 'iatrogenic' acromegaly. To date, rhGH is themost difficult drug to detect using established druglesting protocols,'"^' despite using sophisticatedlogarithmic calculations of insulin-like growth fac-lor-I and N-terminal extension peptide of procol-iagen type III.'"**! both of which are increased mark-edly in response to growth hormone administrationand are claiming 86% chance of success in malesand 60% in females. However, this is assumingindividuals are using supraphysiological doses ofgrowth hormone, leaving a minimum window ofopportunity of 14%.

6. Recommendations inDetection Procedures

The existing arrangements have proven success-ful, some offenders have been deterred, and prose-cuted, but there is still potential for evasion withsome anabolic agents.'""*' In 1991, Voy''^"' suggest-ed that the following principles could be incorporat-ed into a more sophisticated drug testing regime:

• Testing must be independent, sampling must becarried out by the appropriate officers, indepen-dent of governing bodies and trained in IOCprocedures. This would ensure that sample col-lections are above suspicion.

• Testing should be more effective, rigorous andentirely random. The selection of individuals tobe tested must be varied and unpredictable, in-cluding the possibility of 100% testing duringcompetition. During the training phase., athletesshould be regularly called upon for testing. The

system used should be effective and efficient,inspiring confidence and respect.

• Competitors in all sports must be required tomake a personal declaration of willingness toundertake tests during training and competition.Athletes who fail to comply should not receivesupport or grant aid from the Sports Council, theSports Aid Foundation, the British Olympic As-sociation and the National Coaching Foundation.In addition, drug users should not be allowed torepresent their country.

• Penalties for taking drugs should be effective andconsistent.

• The role of the drug advisory groups should beenhanced.

• There should be wide publicity relating to legalactions taken against offenders, and greater infor-mation available outlining the different offencesthat are committed by athletes. Education andprevention may be key strategies.

7. Current World Anti-Doping AgencyOut-of-Competition Testing Programme

WADA tested 3114 athletes in 2005, comparedwith 1848 athletes in 2004. They conducted bloodand urine testing according to the 2005 list. Inaddition. World Championships in sports such asathletics, aquatics and weight-lifting meant that test-ing was also increased in these sports compared withprevious years.

In 2005, there were a total of 61 adverse analyti-cal findings (AAFs) and two other anti-doping ruleviolations (ADRVs), as compared with 19 AAFsand four other ADRVs in 2004.

The 2005 figures include several elevated T/Eratios of >4. which were not reported in previousyears when the threshold was 6, partially accountingfor the increased number of AAFs in 2005. How-ever, there is a significant increase in other AAFs,such as those for steroids. WADA's primary goalsare to establish a level playing field for athletesworldwide, to ensure that all athletes are subject tothe same anti-doping protocols.

Rather than using random selection to pick allathletes to be tested, WADA has adopted a scientific

' 2008 Adis Data information 8V. All rlgt ts reserved. Spoils Med 2008; 38 (6)

522 Graham et ai

approach and selects a significant proportion of ath-letes based on several key factors, including theirrecent performance, history of doping and vulnera-bility to the temptation to take performance-enhanc-ing substances. WADA does not have its own sam-ple collection personnel, but works in partnershipwith selected sample collection authorities world-wide. Samples are always sent to WADA-accreditedlaboratories. In 2005. WADA out-of-competitiontesting covered 40 sports. Athletes from 119 coun-tries were tested, and testing was conducted in 70countries.f'^'l

8. Conclusions

Implementing testing procedures is difficultsince few national sporting bodies have the financialresources available to make the programmes effec-tive. It has not extended into competitive bodybuild-ing, which has remained largely immune comparedwith sports such as athletics, in which the sportscouncil has spent significant sums on testing pro-grammes.

It is possible that past surveys have been skewedby the concentration on hard-core gymnasia. Alter-natively, with the prosecution of high-profile ath-letes and the administration of lighter sentences toobtain co-operation in identifying sources of druguse and drug users, the extent of such abuse is onlyjust becoming apparent.

A controversial solution that has been called for;the introduction of a chemical level playing field,such as an Olympic Games where athletes use dop-ing agents freely, under medical supervision. Such acontrolled environment could contribute to scien-tific research and minimize the risks to the ath-letes, t'=2-123]

This has been fiercely contested by anti-dopingsupporters, citing that the main stakeholders, i.e. thepharmaceutical companies, appear to be indifferentto the misuse of their performance-enhancing prod-ucts by athletes for non-medical purposes.•'^''i

The war against drug abuse within sport contin-ues.

Acknowledgements

No sources of funding were used to assist in the prepara-tion of this manuscript. The authors have no conflicts ofinterest that are directly relevant to the content of this manu-script. The authors would like to thank Mr Gary Biddiscombefrom the Kaleidoscope Project. Wales, UK, and Mr MikeMallett from the Gwent Specialist Substance Misuse Service(GSSMS), Wales. UK, for their data on AAS use by drugaddicts.

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Correspondence: Prof, julien S. Baker, Department ofHealth and Exercise Science, School of Applied Science,University of Glamorgan, CF37 lDL Pontypridd, Wales.E-mail: jsbaker@glam.ac.uk

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