Studies related to the origin of C18 neutral steroids isolated from extracts of urine from the male horse: the identification of urinary 19-oic acids and their decarboxylation to produce estr-4-en-17beta-ol-3-one (19-nortestosterone) and estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) during sample processing.

This paper explores the origins of certain steroids found in male horse urine and investigates the effects of processing on these compounds, specifically looking at the creation of specific steroids through enzymatic hydrolysis and solvolysis.

Overview of Research

• The researchers have found that treating samples of male horse urine with Escherichia coli for enzymatic hydrolysis and then solvolysis (using ethyl acetate, methanol, and sulphuric acid) results in significantly detectable amounts of certain steroids. These are estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) and estr-4-en-17beta-ol-3-one (19-nortestosterone, nandrolone).
• These same steroids were also isolated from extracts of horse testicular tissue, inferring a link between the hormone production in the testes and their presence in the urine.
• The researchers provide evidence that 19-nortestosterone found in “normal” urine samples from male horses may not have its origins in the 17beta-sulphate conjugate, a specific form of the compound.
• However, when anabolic steroids based on 19-nortestosterone are administered to horses, the subsequent urinary excretion of 19-nortestosterone primarily takes the form of the 17beta-sulphate conjugate. This divergence suggests different metabolic processes when the hormone is endogenous versus when it is introduced externally.
• The medical and surveillance implications of this distinction are significant. If the 19-nortestosterone-17beta-sulphate conjugate only appears following administration, it could indicate that a horse has been treated with anabolic steroids, allowing for potential drug testing in competitive scenarios.

Identification of Precursors

• The researchers also theorized that the precursors for 19-nortestosterone and 19-norandrost-4-ene-3,17-dione in urine have the same underlying structure, differing only in the functionality at the 17-position (a particular location on the chemical structure).
• To investigate this, they used preparative high pressure liquid chromatography (LC) and LC fractionation to separate these precursors from the large amounts of oestrogenic sulphates (another type of steroid) found in standard male horse urine.
• Once purified, they used liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) to identify the precursors further, establishing a more defined understanding of the chemical pathways leading to these steroids in male horse urine.