Novel approaches to detection of the use of pseudo-endogenous anabolic-androgenic steroids in the horse

Abstract

The administration of anabolic-androgenic steroids is prohibited at all times in horseracing. Some prohibited steroids are present naturally and their use is monitored by international concentration-based thresholds or by the detection of synthetic steroid esters. However, these approaches have limitations, and thus, this research sought to address some of these issues.
This study established a concentration threshold for plasma testosterone in female horses. Post-race plasma samples (n=2,277) from six countries were analysed using a quantitatively validated liquid chromatography tandem mass spectrometric (LC-MS/MS) method, to establish an international reference range. The established threshold was shown to be effective in detecting the administration of testosterone esters.
The use of urinary steroid ratios was investigated as a novel approach to differentiate the use of testosterone and its pro-drugs from natural conditions that may result in atypical results in castrated male horses (geldings). Post-race urine samples (n=200) were analysed using another validated LC-MS/MS method for quantification of methyloxime derivatives of testosterone, epitestosterone and DHEA, to establish reference ranges for these steroids and their ratios. The administrations of testosterone esters and DHEA showed that the thresholds for steroid ratios were effective and confirmed that they are a valuable complementary tool to evaluate atypical testosterone concentrations. Other steroid biomarkers were monitored, but they did not offer any benefits over the steroid ratios and were not further pursued.
The formation of boldenone in horses of all genders was investigated to aid the development of the approaches to detect its administration. Boldenone was suspected to be a by-product of aromatisation in equine testes. Using in vitro incubations with isotope-labelled testosterone and androstenedione and analysis by LC-MS techniques, the testicular origin of boldenone and boldienone was confirmed for the first time.
The presence of boldenone in a urine sample could also be the result of microbial action, and to identify novel biomarkers. Investigation into the inadequate storage of urine samples and incubations with faeces identified Δ¹-progesterone as a novel potential biomarker, whereas its C20-reduced metabolites were identified in urine samples of horses fed with mouldy feed. Thus, it is postulated that Δ¹-progesterone could be used as a biomarker for microbial formation in voided urine, whilst these metabolites for microbial formations in either feed or gastrointestinal tract.
The findings presented in this thesis have improved the detection of testosterone misuse in female horses following an international adoption of the plasma threshold. The thesis also developed approaches to complement the existing testosterone threshold in gelding urine. Additionally, the better understanding of formation of boldenone in equine testis, and the identification of potential novel biomarkers (Δ¹-progesterone and metabolites) for microbial action will potentially help to distinguish the administration of boldenone related compounds in the future.

Date of Award	1 May 2020
Original language	English
Awarding Institution	King's College London
Supervisor	Kim Wolff (Supervisor)