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Criteria to Indicate Testosterone Administration

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Criteria to Indicate Testosterone Administration Br. J. Sp. Med; Vol 24, No. 4 Br J Sports Med: first published as 10.1136/bjsm.24.4.253 on 1 December 1990. Downloaded from Criteria to indicate testosterone administration A.T. Kicman1, R.V. Brooks2, S.C. Collyere, D.A. Cowan', M.N. Nanjee2, G.J. Southan2 and M.J. Wheelee 'Drug Control and Teaching Centre, King's College, London University 2Department of Chemical Pathology, UMDS, London University A detection method for testosterone administration was was found to increase after an intramuscular injection developed using radioimmunoassay to measure the of testosterone heptanoate in all ten normal males, urinary ratios of testosterone (T) to epitestosterone (E) and although there was an overlap between the basal to luteinizing hormone (LH). A comparative study of the range and that obtained three days after injection. effect on these ratios of a single intramuscular injection of testosterone heptanoate followed by stimulation with The method used was radioimmunoassay as protein human chorionic gonadotrophin (HCG) in three normal hormones cannot be measured by gas liquid chroma- men was undertaken. To allow immediate investigation, a tography-mass spectrometry (GLC-MS). commercially supplied epitestosterone antiserum was In 1983, the International Olympic Committee used. This study showed that both T/E and T/LH ratios (IOC) adopted the ratio of urinary testosterone to could be used to detect testosterone administration, the epitestosterone (T/E) as the sole test for testosterone latter also being an indicator of HCG use due to doping as both these hormones could be convenient- cross-reactivity with the LH antiserum. Subsequently, an ly measured by GLC-MS. epitestosterone antiserum of superior specificity was Epitestosterone is the inactive 17a-epimer of raised and used in a study to demonstrate the insignificant testosterone which is also secreted by the gonads. effect of exercise on these ratios. Finally, an intramuscular injection of a combined preparation of testosterone/ Male or female urine contains approximately equal epitestosterone heptanoates resulted in raised ratios of concentrations of testosterone and epitestosterone2. T/LH but not of T/E. This demonstrated the importance of Donike et al. showed that the average ratio of urinary the T/LH ratio in circumstances where the T/E ratio can be testosterone to epitestosterone in 50 normal males easily circumvented. was 1.13 ± 0.57 SD (range 0.12 to 4.44) and in 47 http://bjsm.bmj.com/ normal females was 1.29 ± 0.89 SD (range 0.26 to Keywords: Testosterone, epitestosterone, luteinizing hor- 2.90) using combined GLC-MS techniques3. As mone, human chorionic gonadotrophin epitestosterone is only a very minor product of the metabolism of testosterone, androstenedione or dehydroepiandrosterone', the detection of tes- Introduction tosterone doping can be determined by an increase in the urinary T/E ratio. Detection of administration of the naturally occurring The initial aim of this study was to develop a on September 29, 2021 by guest. Protected copyright. anabolic steroid testosterone presented an entirely radioimmunoassay as an additional rapid screening new problem for drug control in sport. Hitherto, only technique for assessing urinary T/E ratios in athletes. foreign substances had been banned and all that was Kits could then be supplied to laboratories in required to indicate use was unequivocal proof that a countries which did not have GLC-MS facilities. The banned substance and/or its metabolites were present methods for determining urinary T/LH ratios were in a urine sample. Since untimed urine samples are also improved for two reasons. First, the grounds for collected from athletes, which vary considerably in banning a sports individual using testosterone would concentration, it is not possible to set a limit for the be stronger if two relevant urinary indices are used normal level of excretion of urinary testosterone. In instead of one. Secondly, the use of the T/LH ratio for 1979 Brooks et al. investigated the possibility of screening for testosterone administration may be a detecting testosterone abuse by its feedback effect on better method for the future. Although the IOC the suppression of luteinizing hormone (LH)1. The adopted the use of the T/E ratio, it is feasible that this ratio of total testosterone to LH (T/LH) in the urine test may be circumvented. Since only approximately 1% of testosterone is excreted unchanged (apart from Address for correspondence: D.A. Cowan, Drug Control and being conjugated with glucuronic acid) compared Teaching Centre, King's College, Manresa Road, London SW3 with 30% of epitestosterone, an intramuscular 6LX, UK administration of these two respective hormones in a ©)1990 Butterworth-Heinemann Ltd ratio of approximately 30 to 1 may give a normal 0306-3674/90/040253-12 urinary ratio of 1. Alternatively, human chorionic Br. J. Sports Med., Vol 24, No. 4 253 Criteria to indicate testosterone administration: A.T. Kicman et al. gonadotrophin (HCG) could be administered to male Radiolabelled 115I-LH was prepared using pituitary athletes to stimulate endogenous testosterone and LH standard (2 Rg, IRP 2/69) by a modification of a Br J Sports Med: first published as 10.1136/bjsm.24.4.253 on 1 December 1990. Downloaded from epitestosterone production, thus raising 'beneficially' method for radiolabelling human growth hormone8. the blood testosterone concentration without altering the urinary T/E ratio. To allow immediate investigation, a commercial Assay buffer epitestosterone antiserum was purchased. The nor- The buffer used was NAFA as described by Brooks et mal ranges of T/E and T/LH ratios in male urine al.9 with bovine serum albumin (BSA, 0.1-2.5% w/v). together with cut-off points were established. The effect of a single dose of testosterone heptanoate followed by HCG stimulation on these urinary ratios Methodology and selected serum hormones was studied in three The measurement of total urinary testosterone and normal men. Urinary HCG was measured after HCG epitestosterone stimulation by means of a commercial enzymatic Urinary steroids were hydrolysed with glucuroni- immunoassay kit and a specific radioimmunoassay dase, extracted and measured by radioimmunoassay. (routinely performed by the Supraregional Assay Male urine (25 VI) was diluted with distilled water Laboratory at Charing Cross Hospital, London). (500 d) and 550 Fishman units of 3-glucuronidase Subsequently, a more specific epitestosterone enzyme in acetate buffer (50 sd, 1M, pH 4.5) was antiserum was raised. During this period the added. The tubes were sealed and incubated at 550C National Institute for Biological Standards and for one hour. Controls stopped supplying human urinary meno- The hydrolysed samples were then cooled in an ice pausal gonadotrophin standard and recommended bath. Extraction was performed by the addition of the use of pituitary LH standard for urinary LH dichloromethane (4ml) and shaking for 10 minutes assays. New normal ranges of T/E and T/LH ratios on a multivortex mixer. were evaluated. The effects of strenuous exercise on The tubes were then returned to the ice bath and these ratios were investigated to establish whether the upper aqueous layer removed by suction. any changes may give rise to false positives. Portions (1 ml) of the extracted samples were dis- Finally, a male volunteer who had participated in pensed into glass tubes and dried down under air at the previous experiment (testosterone/HCG adminis- 370C. tration) was injected intramuscularly with a com- The dried extracts were reconstituted with NAFA bined preparation of testosterone heptanoate with buffer (0.1% bovine serum albumin, 2.5ml). The epitestosterone heptanoate in a ratio of 28:1. The reconstituted sample extracts were used for both the effects on the urinary T/E and T/LH ratios were total urinary testosterone and epitestosterone in studied in the hope of illustrating the importance of separate radioimmunoassays. Up to 35 samples may the latter ratio where the use of the T/E ratio may be be analysed in one assay. Portions (100 d) of the negated. appropriate standards in assay buffer (0.1%BSA) were taken to give a calibration range of 0-200 pg/ http://bjsm.bmj.com/ tube for the testosterone and epitestosterone assays. Experimental materials Duplicate portions (100 id) of standards and recon- stituted sample extracts were dispensed into dispos- Antisera able polystyrene tubes (75mm x 12mm). Duplicate The testosterone antiserum produced in our labora- zero tubes (no standard present) and non-specific tory and the commercially supplied epitestosterone binding tubes (used to determine the fraction of antiserum (obtained from Wien Laboratories, Succa- labelled analyte which is bound to sites other than on sunna, NJ 07876, USA) were both raised to their the antiserum) were prepared using 100 id and 200 VI on September 29, 2021 by guest. Protected copyright. respective steroid-3-(0-carboxymethyl)oxime-protein assay buffer respectively. conjugate. The assays were incubated throughout at 40C, Epitestosterone antiserum was raised against adding precooled reagents with vortex mixing. epitestosterone-3-(0-carboxymethyl)oxime bound to The appropriate antiserum was diluted to a bovine serum albumin by the method of Erlanger et working concentration with assay buffer. Testoster- al.6. The immunogen was injected subcutaneously one antiserum was diluted 1:16K; the commercial into a 'New Zealand White' rabbit. The antiserum was epitestosterone antiserum was reconstituted with harvested after six months. 30 ml assay buffer and our own laboratory raised The LH antiserum was kindly supplied by Profes- epitestosterone antiserum diluted 1:16K. Portions sor W. Butt of the Birmingham and Midland Hospital (100 id) were added to all the tubes except the for Women, Birmingham. Gonadotrophin reference non-specific binding tubes. The assay was incubated preparations were kindly supplied by Dr P. Storring with antiserum for 30 minutes. of the National Institute for Biological Standards and The relevant radioiodinated labelled steroid was Controls, Hertfordshire.
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