Detection of dihydrotestosterone (DHT) doping: alterations in the steroid profile and reference ranges for DHT and its 5 alpha-metabolites.

Dihydrotestosterone (DHT), a biologically active metabolite of testosterone, may be misused in sports to benefit from its anabolic and psychotropic effects. After DHT application, a significant increase of the glucuronides of DHT and its metabolites can be expected for a certain time period depending upon dose, formulation, route of administration, and in case of percutaneous administration the chainlength of the ester. DHT and its metabolites can be monitored by gas-chromatography/mass spectrometry (GC/MS) after enzymatic hydrolysis and trimethylsilylation. To investigate the extent of the alteration of the urinary steroid profile after DHT application, timely controlled experiments have been performed with: a) oral application of [16,16,17-2H3]-DHT, and b) sublingual application of a 25 mg dose of DHT. In the experiment with [16,16,17-2H3]-DHT within 24 hours about 44% of the applied dose was recovered after hydrolysis with beta-glucuronidase from E. coli as di- or tri-deuterated 5 alpha-androstane glucuronides: androsterone (33.2%), 5 alpha-androsta-ne-3 alpha,17 beta-diol (2.5%), 5 alpha-androstane-3 beta, 17 beta-diol (0.9%), DHT (7.2%). Hydrolysis with beta-glucuronidase/arylsulfatase from Helix Pomatia resulted in a about 10% higher yield except for DHT. In the study with sublingual application of 25 mg of DHT the extent of the recovery of DHT and its metabolites was in the same range as for the deuterated DHT. The urinary glucuronide concentrations of DHT, androsterone (AND), 5 alpha-androstane-3 alpha, 17 beta-diol (5 alpha A3 alpha D) and 5 alpha-androstane-3 beta,17 beta-diol (5 alpha A3 beta D) and their ratios to etiocholanolone (ETIO), 5 beta-androstane-3 alpha, 17 beta-diol (5 beta A3 alpha D) and epitestosterone (EPIT) were increased for up to 48 hours after application. For doping control purposes concentrations of DHT, 5 alpha A3 alpha D, 5 alpha A3 beta D and ratios of 5 alpha-metabolites to non 5 alpha-metabolites such as DHT/ETIO, DHT/EPIT, 5 alpha A3 alpha D/5 beta A3 alpha D, 5 alpha A3 beta D/5 beta A3 alpha D, and AND/ETIO outside the reference ranges are a proof for DHT application. Reference ranges for Asian and Caucasian male and female athletes are calculated from data bases of the Asian Games 1994, the previous Asian Games 1990 and the routine doping control samples of Caucasian athletes measured in Cologne 1994. At the occasion of the 1994 Asian Games in Hiroshima alterations in the concentrations and ratios of the DHT depending parameters for outside there reference ranges have been found and have been sanctioned on this basis by the Medical Commission of the Organisation of Olympic Council of Asia (OCA).