RESUMO
Estra-4,9-diene-3,17-dione (dienedione) is an anabolic androgenic steroid (AAS) sold as a bodybuilding supplement. It is prohibited in both human and equine sports. With no report of 4,9-diene configuration in endogenous steroids, dienedione has long been considered a synthetic AAS. Nevertheless, the reoccurring detection of dienedione in colt (entire male horse) urine samples lead to the investigation of its possible endogenous nature in horses. This paper describes (i) the detection of naturally occurring dienedione in colts, (ii) the conjugation study of dienedione and (iii) the population study of free and glucuronide-conjugated dienedione in colt urine. Qualitative and quantitative analyses of dienedione content in colt urine were performed, employing liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Qualitative analyses showed that dienedione was endogenous in colt urine and mainly in the form of glucuronide conjugates. Glucuronidation of dienedione was believed to happen at 3-enol leading to dienedione-3-glucuronide. Upon the population study of free and glucuronide-conjugated dienedione in colt urine samples (n = 175), the mean ± SD was determined to be 2.5 ± 3.5 ng/ml. The population data fitted a normal distribution after a fifth root transformation with the exclusion of one outlier by Grubb's test. A possible in-house threshold was proposed at 30 ng/ml of free and glucuronide-conjugated dienedione in colt urine associated with a risk factor of 1 in 14,269 (with a degree of freedom of 173). This is the first report of endogenous dienedione in entire male horses and the approach for controlling its potential misuse by using a threshold is also presented.
RESUMO
Estra-4,9-diene-3,17-dione (dienedione) is an anabolic-androgenic steroid (AAS) available on the market as a dietary supplement for bodybuilding. It is prohibited in both human and equine sports due to its potential performance-enhancing effect. With the rare presence of the 4,9-diene configuration in endogenous steroids, dienedione has been considered as a synthetic AAS. Nevertheless, the reoccurring detection of dienedione in entire male horse urine samples led to the investigation of its possible endogenous nature in horses, and its endogenous nature in entire male horses has been recently confirmed and reported by the authors' laboratory. While dienedione is not detected in castrated horses (geldings), it is essential to study its elimination and identify its metabolites for its effective control. To study the elimination and biotransformation of dienedione, administration experiments were performed by giving three castrated horses (geldings) each single oral dose of 1500 mg of dienedione powder for seven consecutive days. The postulated in vivo metabolites included 17-hydroxyestra-4,9-dien-3-one (M1a and M1b), hydroxylated dienedione (M2a, M2b, M3a, M3b, M4, M5) and hydroxylated M1 (M6a, M6b, M7a, M7b, M8a and M8b), formed from hydroxylation and reduction of dienedione. To control the misuse of dienedione in geldings, M3a and M3b are the potential targets that gave the longest detection time, which could be detected for up to 2-5 days in urine and 0.4-4 days in plasma.