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Drug testing and analysis2024; 17(1); 75-87; doi: 10.1002/dta.3685

Endogenous nature of estra-4,9-diene-3,17-dione in entire male horses.

Abstract: 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.
© 2024 John Wiley & Sons Ltd.
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Publication Date: 2024-03-26 PubMed ID: 38532598DOI: 10.1002/dta.3685Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study reports the natural occurrence of estra-4,9-diene-3,17-dione (dienedione), an anabolic androgenic steroid, in urine of entire male horses (colts), a finding that challenges the previous assumption that dienedione is solely synthetic.
  • The research investigates how dienedione appears in horse urine, its biochemical conjugation, and proposes a threshold for distinguishing natural presence from misuse in sports doping controls.

Background

  • Esra-4,9-diene-3,17-dione (dienedione) is an anabolic androgenic steroid (AAS) often used as a bodybuilding supplement.
  • It is banned in both human and equine sports due to its performance-enhancing effects.
  • Until this study, dienedione was regarded as entirely synthetic because no endogenous (naturally produced) 4,9-diene steroids had been identified in biological samples.
  • However, repeated detection of dienedione in urine from entire male horses (colts) suggested the possibility that it might be naturally produced in these animals.

Objectives of the Study

  • To detect whether dienedione occurs naturally in colt urine.
  • To understand the form in which dienedione exists in urine, focusing on conjugation (binding) with glucuronic acid.
  • To analyze a population of colt urine samples to quantify dienedione levels and to propose a threshold that distinguishes natural occurrence from doping.

Methods

  • Qualitative and quantitative analyses of dienedione were conducted using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), a sensitive and specific analytical technique.
  • The study included investigation of glucuronide conjugates, as steroids are commonly metabolized and excreted in conjugated forms, particularly through glucuronidation.
  • A large sample size of 175 colt urine samples was analyzed to understand the natural range of dienedione concentrations.
  • Statistical methods such as the Fisher normality test (fifth root transformation) and Grubb’s test for outliers were applied to the data.

Key Findings

  • Dienedione was detected endogenously in the urine of entire male horses (colts), confirming it is not exclusively synthetic in this species.
  • The predominant form of dienedione in urine was identified as glucuronide-conjugated at the 3-enol position, specifically dienedione-3-glucuronide.
  • The mean concentration of total dienedione (free and glucuronide-conjugated) in colt urine was 2.5 ± 3.5 ng/ml.
  • The distribution of dienedione across the population fit a normal distribution after appropriate statistical transformation, with one outlier removed.

Implications for Sports Doping Control

  • Because dienedione is naturally present in entire male horses, its detection alone cannot serve as definitive evidence of doping.
  • The researchers proposed an in-house threshold of 30 ng/ml for free and glucuronide-conjugated dienedione in colt urine to distinguish natural levels from potential misuse.
  • This threshold corresponds to a risk factor of 1 in 14,269 for false positive doping accusations, providing a statistical basis for its use in regulation.
  • This approach allows regulatory agencies to control potential anabolic steroid misuse while accounting for the steroid’s endogenous presence in colts.

Significance and Novelty

  • This is the first scientific evidence documenting the endogenous presence of dienedione in entire male horses.
  • The study challenges the previous understanding that dienedione is only synthetic, contributing important knowledge about steroid metabolism in equine biology.
  • The establishment of a concentration threshold provides a practical tool for doping control laboratories to evaluate dienedione findings in horse urine accurately.
  • The findings have direct implications for equine sports, ensuring fair play and better interpretation of doping test results.

Summary

  • Dienedione, previously known only as a synthetic AAS, naturally occurs in entire male horses mainly as a glucuronide conjugate.
  • New detection and quantification methods confirm this endogenous status, supported by a population study of 175 colts.
  • A recommended threshold of 30 ng/ml for total dienedione can help differentiate natural presence from illicit use.
  • This research advances equine anti-doping science by providing a clear framework to interpret dienedione test results responsibly.

Cite This Article

APA
Ho HSM, Ho ENM, Wong WT. (2024). Endogenous nature of estra-4,9-diene-3,17-dione in entire male horses. Drug Test Anal, 17(1), 75-87. https://doi.org/10.1002/dta.3685

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 17
Issue: 1
Pages: 75-87

Researcher Affiliations

Ho, Helen S M
  • Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hong Kong, China.
Ho, Emmie N M
  • Racing Laboratory, The Hong Kong Jockey Club, Hong Kong, China.
Wong, Wing-Tak
  • Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hong Kong, China.

MeSH Terms

  • Animals
  • Horses / urine
  • Male
  • Tandem Mass Spectrometry / methods
  • Tandem Mass Spectrometry / veterinary
  • Doping in Sports
  • Substance Abuse Detection / methods
  • Substance Abuse Detection / veterinary
  • Anabolic Agents / urine
  • Chromatography, High Pressure Liquid / methods
  • Glucuronides / urine
  • Chromatography, Liquid / methods

Grant Funding

  • Racing Laboratory
  • The Hong Kong Jockey Club
  • Hong Kong Polytechnic University

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