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Br J Sp Med 1991; 25(2)

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Human chorionic gonadotrophin and sportA. T. Kicman, R. V. Brooks and D. A. CowanDrug Control Centre, King's College, London, UK

Human chorionic gonadotrophin (hCG) is a glycoproteinhormone which is produced in large amounts duringpregnancy and also by certain types of tumour. Thebiological action of hCG is identical to that of luteinizinghormone, although the former has a much longer plasmahalf-life. Some male athletes use pharmaceutical prepara-tions of hCG to stimulate testosterone production beforecompetition and/or to prevent testicular shutdown andatrophy during and after prolonged courses of androgenadministration. Testosterone administration can be de-tected by measuring the ratio of concentrations oftestosterone to epitestosterone (TIE). An athlete is oftenconsidered to have failed a drug test if the urinary T/E ratiois greater than 6. In contrast, hCG administrationstimulates the endogenous production of both testosteroneand epitestosterone without increasing the urinary T/Eratio above normal values. Although the administration ofhCG was banned by the International Olympic Committee(IOC) in 1987, no definitive test for hCG has beenapproved by the IOC. Currently, the only way ofmeasuring small concentrations of hCG is by immunoas-say, and this does not have a discriminating power as greatas gas-liquid chromatography with mass-spectrometrywhich is necessary to satisfy IOC requirements. Extractionprocedures and chromatographic steps could be intro-duced before using a selected immunoassay for hCG tomeet these requirements.

Keywords: hCG, sport, immunoassay, detection, doping

With the introduction of 'peptide hormones and theiranalogues' as a new doping class by the InternationalOlympic Committee (IOC), attention has focused onthe problem of human chorionic gonadotrophin(hCG) administration. Although there is a vastamount of literature on hCG, very little to date hasbeen published in relation to hCG and sport. Thisreview attempts to address this problem by giving aconcise but general account on hCG with particularemphasis on its detection and misuse in sport.

Origin and functionHuman chorionic gonadotrophin is a glycoproteinwhich is produced in large amounts during preg-nancy and also by certain types of tumour. It is also

secreted in very small amounts, probably by thepituitarT gland, in normal men and non-pregnantwomen. Small concentrations of hCG have beenmeasured in the serum of normal subjects (less than5 IU/1), and hCG or hCG-like material has been foundin tissues, pituitary and urine extracts2-4.Immunoassay for hCG is generally used as a

diagnostic tool for pregnancy, trophoblastic tumour(hydatidiform mole, choriocarcinoma and germ celltumour) and non-trophoblastic tumour (commonsites being the testis, ovary, stomach and pancreas).

In pregnancy, hCG is secreted into the maternalblood shortly after conception (within 9 days) by thefused outer layer of cells surrounding the implantedblastocyst called the syncytiotrophoblast cells (syn,together; kytos, cell; trephein, to nourish; blastos,germ). The rate of secretion of hCG from thedeveloping placenta, which is called the chorion(trophoblast cells with a lining of mesoderm), risesrapidly. Maximum concentration is reached in mater-nal blood and urine at approximately 8-10 weeksgestation (100 kIU/I). This is followed by a gradualdecline to a nadir at 17-20 weeks, which ismaintained at approximately 20 kIU/l, followed by asecondary much smaller peak around weeks 32-33.The major role of hCG in pregnancy is considered

to be the maintenance of the corpus luteum for theproduction of progesterone and to a lesser extent ofoestradiol. It may also play an important part in malesexual development, by stimulating fetal Leydig cells,as there is a fall of fetal testosterone productionduring the second and third trimester correspondingto that of maternal hCG.Hydatidiform mole is the end stage of a degener-

ating pregnancy, in which the trophoblastic tissue ofthe chorion has undergone hypertrophy, leading toabsorption of the fetus. The villi of the mole maybecome locally invasive with some metastasis. Alter-natively, the mole could progress to choriocarcinomawhich is widely metastatic.

In the USA, the incidence of testicular tumour is2-3 per 100000 men. Nevertheless, it is the secondmost common malignancy after leukaemia in 20-35-year-old men5. Non-trophoblastic tumours of thetestes are far rarer than germ cell tumours (which areof trophoblastic origin), accounting for only 1-2% oftesticular neoplasms. Trophoblastic tumours can leadto the urinary excretion of several million internation-al units of hCG per day, whereas non-trophoblastictumours do not generally exceed a few hundredinternational units per day6.

Br J Sp Med 1991; 25(2) 73

Address for correspondence: D. A. Cowan, Drug Control Centre,King's College, Manresa Rd, London SW3 6LX, UK) 1991 Butterworth-Heinemann Ltd.0306-3674/91/020073-08

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Human chorionic gonadotrophin and sport: A. T. Kicman et al.

In the male, hCG-secreting tumours can give rise toelevations in testosterone and oestradiol secretionbecause of the action of hCG in stimulating normalsteroidogenesis.

StructureThe structure and properties of hCG have beenreviewed extensively by Birken and Canfield7 andPierce and Parsons8.

Thyroid-stimulating hormone (TSH), together withthe gonadotrophins, luteinizing hormone (LH), folli-cle-stimulating hormone (FSH) and hCG, forms afamily of glycoprotein hormones which are com-posed of two dissimilar non-covalently linked sub-units. The subunits, designated ax and f3, are linkedby electrostatic and hydrophobic interactions. Thestrength of this binding is demonstrated by the factthat the subunits are not easily separated in a highconcentration of urea (1OM) at neutral pH but areseparated in acidified urea (0.001 M HCl, 40'C, 1 h).

In humans, the approximate molecular weights ofhCG, FSH, LH and TSH are 37000, 33 000, 28000 and28 000 respectively. The a-subunit is very similar inprimary structure within this family but the P-subunits differ. The hCG a-subunit consists of 92amino acids and has two carbohydrate moietieswhich are branch chained to asparagine residues9,giving an average total molecular weight of 14500.Although there is some homology between the

various 3-subunits, in particular between LH andhCG, there are variant amino acid sequences whichaccount for different target receptor recognition. FSHand TSH have very different variant sequences incomparison to hCG and LH. The homology betweenhCG and LH accounts for their identical target cellreceptor recognition and action in stimulating ster-oidogenesis.

Importantly, the ,-subunit of hCG consists of 145amino acid residues, having 30 additional residues atthe C-terminal region compared with the n-subunit ofLH. It is within this region that a specific antigenicdeterminant is located which allows for immuno-logical distinction as opposed to biological distinctionbetween hCG and LH.

Attached to the P-hCG subunit are six carbohydratemoieties'0, giving an average total molecular weightof 22000. Two branch chain moieties are linked toasparagines and four are linked to serines within theunique C-terminal peptide region (Figure 1).

a - subunitCHO CHO

* ~ ~~~IH2N - ALA - PRO- ASP- VAL --- ASN--- ASN--- SER - COOH

2 3 4 52 78 92

, -subunitCHO CHO CHO CHO CHO CHO

I ItH2N - SER --- ASN --- ASN--- SER-- SER--- SER --- SER---GLN - COOH

I, 13 30 121 127 132 138 145

Figure 1. Position of carbohydrate (CHO) attachments tothe a- and ,-subunits of hCG. The a-subunit N-terminus isheterogeneous and begins at one of the three amino acidsunderlined (summarized from figures by Birken andCanfield')

COOH

H

H HHOH2C------ C -

HO HO

*

Figure 2. Structure of N-acetyineuraminic acid. Theasterisk indicates the point of attachment to the rest of thecarbohydrate moiety (2--3 linkage)

The studies by Kessler et al.9"0 showed that thecomplete hCG molecule has eight carbohydratemoieties in total, making it approximately 30%carbohydrate content by weight. Each moiety con-tains two attached N-acetylneuraminic acid groups,which is a sialic acid (Figure 2), giving rise to 16 sialicacids per hCG molecule. However, as there ismicroheterogeneity in the carbohydrate content ofthe gonadotrophins, and as these forms have not yetbeen fully characterized, it is important to appreciatethat the total number of sialic acid groups per hCGmolecule will only represent an average amount andthis number will vary between reported studies. Thegreater number of sialic acid groups on hCG leads toa greater ionization constant and hence lower pIvalue when compared with the other gonado-trophins. The pI of hCG is about 3.5, FSH about 4.5and that of LH about 5.5.

Differences in hCG carbohydrate content lead toobservable differences in electrophoresis and isoelec-tric focusing. These differences are probably due tolosses in isolation procedures as well as biologicalvariation, together with very minor N-terminalheterogeneity in the or-subunit amino acid sequence.The higher carbohydrate content of hCG leads to a

larger Stokes' radius compared to FSH, LH and TSH(Stokes' radius is defined as the radius of a perfectunhydrated sphere having the same rate of passagethrough a size-exclusion column as the unknownprotein in question). This causes the hormone toelute in the 65-70000 MW region instead of its trueweight of 37000.

International Standards and unitsThe first International Standard (IS) for hCG wasestablished in 1938 having a potency of only 10lIU/mgof crude fraction isolated. Using hCG isolated frompregnant female urine, a second IS (code 61/6) wasestablished in 1964". Its potency was assessed interms of bioassay as compared with the first IS.Amongst the bioassays applied were increases in theweight of the rat or mouse ventral prostate andseminal vesicles as a result of hCG stimulation oftestosterone production (interstitial cell stimulatingactivity). Even though the second IS was consider-ably purer than the first, there was only approximate-

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ly 20% (w/w) of pure hCG present in the materialisolated.With the introduction of radioimmunoassays for

hCG in the 1970s, it became obvious that the secondIS was not suitable for immunoassays because of itsimpurity and heterogeneity. This led to the prepara-tion by the Center for Population Research, Washing-ton DC, of a new reference compound specifically forradioimmunoassay. Part of this material, codedCR119, was used by the World Health Organization(WHO) to prepare an International Reference Prepa-ration (IRP) for immunoassay12'13.The IRP (code 75/537) was very pure, having less

than 1% (w/w) of contaminants. Its unitage wasexpressed in terms of the second IS using bioassaycalibration. This was necessary to allow continuity inclinical laboratories' results. Each ampoule suppliedby WHO contained 650 IU of hCG with 1 IU beingequivalent to 108 ng of hCG.When the supplies of the second IS ran out, WHO

logically renamed the pure IRP, calling it the third IS,but kept the designated code number of 75/537.Although the third IS is very pure, quantities ofstandard are normally expressed in terms of interna-tional units rather than weight. This is because hCGis microheterogeneous in structure and new biologic-al standards are rarely identical to the ones that theyreplace.

Standards of the a- and P-subunits of hCG are alsoexpressed in international units, where 1 IU = 1 ,ug.Here the unitage is defined in terms of mass alone asthe isolated subunits of hCG have no biologicalactivity.

Biological action at the cellular levelThe biological action of hCG at the cellular level isidentical to that of LH. A full account of the receptorinteraction and transducing systems involved isbeyond the scope of this brief review and for furtherdetailed information the review by Cooke andRomerts14 is recommended.

In the male, LH and hCG interact with specifictarget receptors on the surface of Leydig cells in thetestes. The hormone-bound receptors activate cyclicAMP and calcium ion secondary messenger systems,which in turn activate various protein kinases, finallystimulating steroidogenesis and protein synthesis.There is evidence to suggest that phospholipidmetabolites, in particular the leukotrienes, may alsoact as secondary messengers in LH/hCG inducedsteroidogenesis.

Initially, the interaction of hCG with the receptor isby low affinity binding with the ,-subunit. This leadsto a second and much higher affinity interaction withthe ox-subunit'5 16. The binding of LH and hCG to thereceptors is not completely reversible, as someinternalization and degradation of the receptor-bound complex occurs

Stimulation of testicular steroidogenesis by hCG inhealthy adult men is very rapid. A 50% increase inplasma testosterone concentration has been mea-sured 2 h after an intramuscular injection of 6000 IU ofhCG"8. However, there is no direct correlationbetween plasma concentrations of hCG and testoster-

E

a

0

O

0

0

E0

a-

a

75n.IX.

To

b25 50 70 100 150

Time (h) after hCG injection 6000 IU intramuscularly

Figure 3. Effects of a single injection of 6000 IU hCGintramuscularly on plasma levels of hCG (a) and testoster-one (b). Values are the mean (s.e.m.) of the three subjects(hCG) or seven subjects (testosterone). (Reproduced withpermission from Saez JM, Forest MG. Kinetics of humanchorionic gonadotropin-induced steroidogenic responseof the human testis. 1. Plasma testosterone: implicationsfor hCG stimulation test. J Clin Endocrinol Metab 1979; 49:278-83)

one, the rise in plasma testosterone being biphasic(Figure 3). The favoured hypothesis for the biphasicresponse is that oestradiol plays an intratesticular rolein androgen production1. A big dose of hCG isknown to induce Leydig cell aromatase which causesa large increase in testicular oestradiol secretion.The rising oestradiol partially suppresses the

cytochrome P450c17* enzyme20 activities leading to adampening effect on the hCG-induced increase intestosterone synthesis. Importantly, the increase inaromatase activity would not significantly deplete thetestosterone formed in the testes as the proportionconverted by aromatization to oestradiol would stillbe very low. As the plasma hCG concentration beginsto fall there is an accompanying decrease inaromatase activity and hence oestradiol synthesis,which relieves the partial enzymatic block. Amaximum in plasma testosterone concentration isthen attained around 3-4 days after hCG administra-tion.

*Prenvously known as C17,20-1yase and 17a-hydroxylase (seeMiller20)

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In adult men with hypogonadotrophic hypogona-dism due to the administration of anabolic-androgenicsteroids, the increase in plasma testosterone isattenuated following hCG stimulation and there is nosignificant change in plasma oestradiol concen-trations. The steroidogenic enzymes cannot be fullyreactivated with a single dose of hCG21. However, ifchronic administration of hCG is continued (e.g.5000IU, 3 times per week) during and/or after theadministration of supraphysiological doses of tes-tosterone, then testosterone secretion and sperma-togenesis can be stimulated and maintained.

Evidence suggests that hCG binds with low affinityto FSH receptors in rodents and has an intrinsicallyweak follicle-stimulating activity and thyroid-stimu-lating activity due to its general structural homologywith FSH and TSH23 24. This secondary biologicalactivity is borne out in humans by the ability of largeplasma concentrations of hCG to stimulate andmaintain spermatogenesis in hypogonadotrophichypogonadism and by the hypothesis that thethyrotoxicosis, which is associated in patients withtrophoblastic diseases, is caused by the exceedinglyhigh concentrations of serum hCG.

Metabolic clearance rate and half-lifeAbout 20-30% of hCG administered intravenously orintramuscularly is excreted in the urine during thesucceeding 5-6 days25 26. The metabolic clearancerate (MCR) is 3.4 and 3.9 ml/min in men and womenrespectively.Although the MCR of the 13-subunit of hCG is

about tenfold greater than that of hCG, less than 1%of the 13-subunit is excreted in urine26. Evidencesuggests that 13-hCG is metabolized in the kidney to asmallercomponent, ofmolecularweight 12 000-17 500.This 13-core component is devoid of the carboxy-terminal peptide immunological determinant whichis also necessary for biological activity. The 13-corefragment is present in large quantities in the urine ofpregnant women27 and crude commercial hCGpreparations24.The difference in the degree of sialylation between

the gonadotrophins (hCG > FSH > LH) influenceshepatic uptake and hence MCR and plasma half-life.A comparative study of half-lives of LH followingcomplete hypophysectomy and hCG after removal ofthe placenta showed the distribution of both hor-mones to be in at least two compartments. There wasa fast initial process of disappearance of t1/2 ofapproximately 21 min for LH compared with 11 h forhCG and a slower process of disappearance of t1/2 ofapproximately 235 min for LH compared with 23 h forhCG28. The plasma half-life values quoted for hCGdiffer somewhat from those found by Nisula andWehmann26 but nevertheless the comparative studydemonstrates more than adequately the markeddifference in the disappearance rates of LH and hCG.Administrations of hCG injected intravenously andintramuscularly have demonstrated that there is aslightly longer half-life of the hormone for the latterroute, probably because of the slower release into thecirculation29. Desialylated hCG has a very shortplasma half-life, due to the exposure of free galactose

groups within the carbohydrate moieties. Thesegroups are specifically recognized by hepatic recep-tors leading to the uptake, internalization anddestruction of asialo-hCG.

Renal clearanceThe renal clearance of hCG approximates 1 ml/min26.This means that in 1 min the equivalent mass of hCGin 1 ml of plasma is secreted into the bladder from thevolume of glomerular filtrate formed (average rate is125 ml/min).The renal clearance (CR) of hCG may be defined by

the equation:

CR = [U]V/[P]where [U] is the concentration of hCG in urine; V isthe volume (ml) of urine secreted per minute, and [P]is the concentration of hCG in plasma.As the normal volume of urine secreted into the

bladder approximates 1 ml/min over a 24 h period andthe clearance rate of hCG approximates to 1 ml/min, itfollows that plasma and 24 h urinary concentrationsof hCG are of similar nature. Measurements of hCGconcentrations in first morning-voided urine samplescan also give an approximation of hCG plasmaconcentrations simply because of less fluctuation inthe renal clearance of hCG during the sleeping hours(Table 1). However, a number of assumptions havebeen made in all of the above and there is a need tocharacterize more fully the possible relationshipsbetween urinary filtration rate and hCG disposition.

Immunoassay and detectionAn excellent general introduction to the principles ofimmunoassay techniques has been written by Ed-wards30.

Initially, polyclonal antibodies for use in hCGimmunoassays were raised by immunization with theintact molecule. With the postulate of conformationalsimilarities between the native hormones31, therewas up to 100% cross-reaction of these antisera withLH. This problem was overcome to a large degree bythe introduction of antibodies raised to antigenic siteson either the intact 13-subunit of hCG32 or itscarboxy-terminal region. These antibodies normallyrecognize the corresponding antigenic sites on thewhole hCG molecule as well as the free 13-subunit.

Specificity was further improved by the introduc-tion on a commercial scale of monoclonal antibodiesraised to hCG and/or the free 13-subunit. Typicalcross-reactions of monoclonal antibodies with thestructurally related glycoprotein hormones are muchless than 1%33-35Although the problem of immuno-crossreactivity

between the gonadotrophins has been solved, thereremains the general problem of measuring anypolypeptide or protein analyte with a microhetero-geneous structure36' 37. The heterogeneity of thegonadotrophins has been studied by Stockell Hartreeet al.38 using ion-exchange chromatography. Theelution pattern of these hormones showed that therewas no separation of these components as sharppeaks, which indicates considerable heterogeneity.

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Table 1. Concentrations of hCG in early morning plasma and first morning-voided urine after intramuscular injection of hCG (5000 IU) onday 1. Measurements were performed using immunoradiometric (Maiaclone) and immunoenzymetric (Serozyme) kits54 (SeronoDiagnostics Ltd, Windsor, UK)

hCG (IUII)

Serum Urine

Subject Day Maiaclone Serozyme Maiaclone Serozyme

1 Pre-injection <0.1 <1.0 2.2 <1.03 164 152 149 845 55 43 146 1218 18 10 49 28

2 Pre-injection <0.1 <1.0 0.8 <1.03 164 145 330 2475 51 38 57 478 13 9 20 1210 5 3 14 7

3 Pre-injection <0.1 <1.0 2.6 <1.02 244 223 251 2164 83 74 77 758 13 11 30 21

10 6 5 3 2

Such heterogeneity could be due to artefacts gener-ated mainly by desialylation and other carbohydratelosses during the isolation procedures used, althoughundoubtedly a number of naturally occurring isoformsdo exist. Evidence suggests that the carbohydratemoieties influence the structural conformation of theglycoprotein hormones39. Heterogeneity of gonado-trophin standards and immunogens is generallyaccepted as accounting for the discrepancies inresults between different competitive immunoassaysfor hCG and also between those methods and'two-site' immunometric methods. These differentresults are apparent in the UK external qualityassessment scheme. In the future, it may be possibleto resolve this problem by characterizing the differ-ences in structures between the isoforms and only touse monoclonal antibodies with particular epitopespecificity.At the Drug Control Centre, hCG administration in

sportsmen is currently detected by analysing un-timed urine samples using immunometric assayswhich incorporate highly specific monoclonal anti-bodies. The principle of the immunometric assay isthat labelled antibody is added in excessive amountsto the antigen being measured. With 'two-site'immunometric assays the antigen-antibody complexis then coupled to another antibody which is linked toa solid phase. The solid-phase linked antibody is alsoadded in excess and the overall antigen-antibodycomplex is commonly called a 'sandwich'. The waythe sandwich is then isolated depends on the type ofsolid phase used, e.g. precipitation or magneticseparation. End-point measurement depends on thetype of label being used, e.g. radioactive, enzymatic,fluorometric.There are two limitations with the two-site im-

munometric assay. With very large concentrations ofhCG such as found with trophoblastic tumours, abiphasic response known as the 'hook effect' can beelicited (Figure 4). This occurs with certain methods

because the labelled antibody and solid-phase linkedantibody are present together in a milieu of antigenexcess. Increasing amounts of antigen excess resultsin decreasing values of analyte measured because ofthe decreasing amounts of antigen-antibody sand-wiches formed. In these cases, results due to a smallconcentration of analyte measured may not bedistinguished from those of very large concentration.

Certain commercial assay kits which are used toquantitate hCG show 'hook effect' characteristicswith concentrations in excess of 500 000-1 000 000IU/1, far higher concentrations than could ever beachieved in the urine as a result of hCG administra-tion. Other immunometric assay procedures over-come the problems of a biphasic response byincorporating a wash step between the addition ofthe solid-phase linked antibody and the labelledantibody. Hence, any unbound antigen is removedbefore the addition of labelled antibody. This will

c

Ca

ct

Assay calibration range

Value interpolatedConcentration of antigen

Excess antigen

Figure 4. Biphasic response ('hook effect') in the two-siteimmunometric assay

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result in concentrations of antigen greater than thelargest calibrant concentration appropriate for thatassay, giving results equal to or greater than thelargest calibrant but smaller than their true value.A second limitation is due to the presence of

antibodies in the blood of certain individuals, whichare directed against the same immunoglobulins asthose which are used in the assay, e.g. sheep, rabbitor mouse4o. In the two-site immunometric assays,this results in cross-linking between the labelledantibody and the solid-phase linked antibody, givingrise to false positives. There is no known presence ofthese interfering antibodies in urine. In clinicallaboratories which analyse blood samples for hCG byusing immunometric assays, cases of false positiveshave arisen due to the presence of these heterophilicantibodies4-43. It follows that caution must beexercised, if in the future blood samples are to beused for drug testing in sport.

Misuse of hCG in sportThere is little conclusive evidence from the manyscientific studies performed that the use of syntheticanabolic-androgenic steroids in physiological orsupraphysiological concentrations compared withthat of testosterone, has any appreciable directanabolic effect on skeletal muscle growth (hyperpla-sia and/or hypertrophy) in healthy adult males '45.Nevertheless, there is some evidence to suggest thatany enhancement in athletic performance may be dueto the androgenic effect of these steroids upon thecentral nervous system resulting in increased aggres-sion and competitiveness46-48. The experienced maleathlete may gain an anabolic effect, albeit indirectly,by knowing how to capitalize on the behaviouraleffects resulting from androgen administration toenable him to train harder. The improvement inperformance may be so small as to be virtuallyimpossible to prove with statistical significance.However, a tiny improvement in performance maybe all the sportsman requires to win a better place in aclosely fought competition. In any case, anabolicsteroids continue to constitute the greatest percen-tage of total positive samples detected by the IOC-accredited laboratories year after year (e.g. 49% in1989).Other possible mechanisms by which the use of

anabolic steroids may enhance performance are bystimulating the production of erythropoietin and alsoby direct action on the stem cells in the bonemarrow49. This would cause an increase in erythro-cyte formation and therefore an increase in oxygenuptake, resulting in a possible improvement inendurance capacity. Also some athletes claim that theadministration of synthetic anabolic steroids enablesfaster recovery from fatigue during training. Thismay be due to a general anticatabolic effect onsubjects with large concentrations of endogenouscorticosteroids due to the stress of severe trainingschedulesm.

Consequently it is possible that, if the action ofanabolic and androgenic steroids is by the effectsdiscussed rather than by any direct myotrophic actionthen there may be no advantage in taking synthetic

anabolic steroids in preference to testosterone.Indeed, the many undesirable effects of syntheticanabolic steroids, in particular the risks of hepatoxic-ity, hepatoma and prostatic carcinoma associatedwith 17x-alkylated steroids, combined with theknowledge of efficient gas-liquid chromatographywith mass-spectrometry (GC-MS) as a detectionsystem for these drugs, may prompt the abuse oftestosterone as the steroid of choice. Alternatively,some athletes may switch from a course of syntheticanabolic steroids to administering testosterone inanticipation of testing. Now that testing is beingintroduced at training periods as well as at competi-tions in the UK and a number of other countries, thelogical temptation of administering testosterone asthe chosen steroid of abuse may be very strong.Indeed in 1989, the IOC-accredited laboratories foundthat testosterone was the second most commonanabolic steroid detected after nandrolone.

Pharmaceutical preparations of hCG can be used tostimulate testosterone production in men beforecompetition. In addition, it may be used to preventtesticular shutdown and atrophy during and/or afterprolonged courses of natural or synthetic androgenadministration. The alternative use of LH prepara-tions for these purposes is not practical due to thesmall amount supplied per ampoule (e.g. 75 IU) andits much shorter plasma half-life compared to hCG. Apossible undesirable effect of continued hCG admini-stration in the healthy adult male is that ofgynaecomastia, probably as a result of raised oestro-gen secretion from the testes. This effect can becounteracted by concomitant testosterone admini-stration22.

In non-pregnant women, it is difficult to see anyadvantages to be gained in athletic performance fromthe administration of hCG. It may be that timed hCGstimulation could be used to mimic early pregnancyby prolonging the luteal phase of the menstrual cycle,thus enabling the administration of a course ofanabolic-androgenic steroids without accompanyingovarian atrophy. Nevertheless, as it is currently notpossible to distinguish between hCG administrationand hCG production in early pregnancy, female urinesamples are not analysed for hCG.

In 1979, a prototype assay for the detection oftestosterone administration using the ratio of urinaryconcentrations of testosterone to LH was pub-lished51. Only untimed urines are collected for druganalysis, so this ratio was used because it isindependent of the volume of water excreted.However, this method was not fully explored byother countries, who were very reliant on GC-MStechniques which cannot measure protein hormonessuch as LH.

In 1982, an alternative method, which has beenadopted by the IOC, using the ratio of urinary totaltestosterone to the 17x-isomer, epitestosterone, wasdeveloped by the IOC-accredited laboratory inCologne52. As epitestosterone is only a very minorproduct of testosterone metabolism (less than 1%),the detection of testosterone administration can bedetermined by an increase in the ratio of urinarytestosterone to epitestosterone (T/E). This evidenceled to a ban based on the TIE ratio by the IOC in 1983.

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Action is often taken against an athlete if the urinaryT/E ratio exceeds 6.The administration of hCG to normal adult males is

known to stimulate endogenous epitestosteronesecretion as well as testosterone secretion, thusincreasing the blood testosterone concentration with-out altering the urinary T/E ratio53. Initial results fromthis work led to the testing of 740 samples from maleathletes for hCG. Of these samples, 21 were found tohave unnaturally large concentrations of urinaryhCG'. These results led to a ban on hCG administra-tion by the IOC in November 1987.However, the only practical way for testing hCG is

by immunoassay. Although there are a number ofcommercial immunoassay kits on the market whichuse highly specific monoclonal antibodies to detectthe presence of hCG in urine, the singular use ofimmunoassay is currently not an acceptable definitivetest for hCG by the IOC Medical Commission.Although polypeptide and protein hormone adminis-tration is banned under the doping class of 'peptidehormones' there are no IOC-approved tests for them.The rationale for this unacceptability is that theseassays do not have the discriminating power,equivalent to GC-MS, sufficient to satisfy the IOCrequirements.With GC-MS, the banned drug is separated from

other potentially interfering substances in the urineby high-resolution chromatography before beingidentified by the mass-spectrometer. A similar sort ofapproach could be taken with testing for hCGadministration where it is separated from otherpossible interfering solutes present in the urinebefore confirmation by immunoassay. Extractionprocedures and/or size-exclusion chromatographycould be employed for this purpose.The problem of microheterogeneity together with

possible immunoreactive fragments could be over-come by standardization, using either one particularcommercially supplied hCG immunoassay kit orusing a number of kits which give concordant results.An external quality assessment scheme has beensuggested between the IOC-accredited laboratories tomonitor consistencies in results55. A normal range forhealthy adult men, together with a suitable cut-offpoint for the determination of hCG administration,would have to be established. Of course, the failureof an hCG drug test due to an hCG-secreting tumourwould be a piece of singular good fortune in causingthe athlete to seek medical advice.Another challenging problem to be overcome is the

effect of possible changes in the renal clearance rateof hCG such as might result from prolonged vigorousexercise. As only single untimed urine samples arecollected from athletes, a proposed cut-off levelwould have to be well in excess of normal values forurinary hCG obtained from individuals after exercise.In addition, an hCG to LH ratio would givesupplementary information which might not besubject to the effects of changes in renal clearancerate.

ConclusionDrug control in sport has evolved from the compara-tively simple task of detecting foreign substances in

urine. Now that 'peptide' hormones have been addedas a doping class, all the IOC-accredited laboratorieshave a responsibility to implement suitable detectionmethods for hCG administration.

AcknowledgementWe would like to thank the Sports Council for their continuedsupport.

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SeppalA M. Serum levels of human chorionic gonadotropin innonpregnant women and men are modulated by gonadotro-pin-releasing hormone and sex steroids. I Clin EndocrinolMetab 1987; 64: 730-6.

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