Metabolism of methandrostenolone in the horse: a gas chromatographic-mass spectrometric investigation of phase I and phase II metabolism.

This research explored how a horse metabolizes an anabolic steroid called methandrostenolone, noting the main metabolic processes and the forms in which metabolic byproducts occur after stages of metabolism.

Research Methodology and Process

• The research involved giving an oral dose of an anabolic steroid, methandrostenolone, to a standardbred gelding (a type of horse). This provided the test subject for understanding how the medication is processed within the horse’s system.
• Two stages of metabolism, known as phase I and phase II, were examined rigorously.
• In the phase I study, they isolated metabolites from the horse’s urine using a method known as solid-phase extraction, which allows researchers to capture and analyze compounds.
• These metabolites were then deconjugated – that is, simplified or broken down – through a process called acid catalysed methanolysis.
• The deconjugated compounds were then converted into their O-methyloxime trimethylsilyl derivatives to help with further analysis.

Findings from Phase I Metabolism Study

• After conducting a Gas Chromatography-Mass Spectrometry (GC-MS) analysis, it was revealed that the major metabolic processes involved sequential reduction of the A-ring and hydroxylation at C6 and C16. Essentially, this means the steroid undergoes chemical transformations in the horse’s body, specifically changing shape and gaining additional elements.

Methodology and Findings from Phase II Study

• For the phase II study, the researchers looked at the remaining, altered metabolites that had not been conjugated (connected to another compound), were attached to beta-glucuronidated structures (a primary method the body uses to make a wide variety of substances more water-soluble), and those linked with sulfated compounds.
• These metabolites were separated out and deconjugated using several techniques, including liquid-liquid extraction, enzyme hydrolysis, solid-phase extraction and acid catalysed methanolysis.
• These samples were made into derivatives and analyzed using GC-MS.
• The findings showed that there was extensive connection to both glucuronic acids and sulfuric acids, which suggests that the steroid is primarily excreted after being transformed to become more water soluble (either as glucuronides or sulfates). There were only a small proportion of metabolites found in an unconjugated form, i.e., not attached, modified, or made water-soluble.