Ex vivo spontaneous generation of 19-norandrostenedione and nandrolone detected in equine plasma and urine.

The research investigated whether the anabolic-androgenic steroids 19-Norandrostenedione (NAED) and nandrolone, detected in plasma and urine samples of stallions, were genuinely endogenous or artifacts generated during sample processing. It concluded that most of these steroids were not naturally occurring but generated during sample storage.

Study Background

• Anabolic-androgenic steroids (AASs) like 19-Norandrostenedione (NAED) and nandrolone have been identified in plasma and urine samples of stallions, raising questions whether these steroids are naturally occurring or artifacts generated during sample processing.
• In the 1980s, trace amounts of apparently endogenous nandrolone were detected in urine samples obtained from intact male horses (stallions). Its endogenous origin was later reported in boars and bulls.
• The doubt arises as it was recently suggested that NAED and nandrolone detected in urine samples from stallions are primarily artifacts due to the analytical procedure.

Research Methods

• To clarify this point, this research analysed fresh plasma and urine samples from ≥8 stallions for the two AASs, soon after collection, via liquid chromatography hyphenated to tandem mass spectrometry (LC-MS/MS).
• The samples were observed for evidence of NAED and nandrolone both before and after storage, with attention paid to changes in their levels.

Research Findings

• NAED and nandrolone were not detected in fresh plasma samples but only in the same samples after storage.
• Concentrations increased with storage time, with increases more significant at a higher storage temperature (37°C versus 4°C, and ambient temperature versus 4°C).
• Although NAED was detected in some fresh stallion urine samples, its concentration was far lower than that in the same samples after storage at ambient temperature for 15 days.
• Nandrolone was not found in most fresh urine samples but emerged in the same samples after storage.

Conclusion

• The findings indicate that the presence of NAED and nandrolone in stored plasma samples of stallions, and most in the stored urine samples, is not due to endogenous origins but rather spontaneous generation during sample storage.
• This spontaneous generation most likely stems from spontaneous decarboxylation of androstenedione-19-oic acid and testosterone-19-oic acid.
• This revelation is significant as it could have a profound impact on the regulation of these two steroids in horse racing.
• This is the first time that the presence of NAED and nandrolone in plasma and urine samples of stallions has been demonstrated to be mainly due to spontaneous generation during sample storage.