In-vitro biosynthesis of C18 neutral steroids in horse testes.

The research investigated the in-vitro synthesis of C18 neutral steroids in horse testes using radio-labelled steroid substrates and gas chromatography-mass spectrometry for analysis.

Methodology

• The researchers used deuterium, 14C- and 3H-labelled steroid substrates for their experiments. Deuterium, 14C, and 3H are isotopes commonly used as tracers in various bio-organic studies as they can be detected with high sensitivity in the body of an organism.
• These substrates were incubated with finely chopped testicular tissue from stallions of varying ages. By using testes from multiple age groups, the researchers aimed to observe any effect of age on their results.

Extraction and Separation Process

• After the incubation, the sample was subjected to extraction and separation processes. These procedures are fundamental in isolating the target compounds from the complex matrix of the tissue sample.
• The extraction generated two distinct factions: neutral and phenolic. Neutral steroids, also known as free steroids, are those not bound to any carrier proteins. Phenolic steroids, on the other hand, include molecules that have a hydroxyl (-OH) group attached to an aromatic phenyl ring.

Analysis and Identification of Metabolites

• The extracted metabolites were then identified using gas chromatography-mass spectrometry (GC-MS). GC-MS is a widely used analytical technique that couples the separation power of gas chromatography with the identification ability of mass spectrometry. It makes it possible to identify different substances within a test sample.
• The researchers confirmed the presence of C19 neutral and C18 phenolic steroids, which were the expected metabolites from the incubation of the horse testes with the steroid substrates. This indicates that the horse testes are able to biosynthesize those specific steroids.
• An unexpected result was the identification of an isomer of 5(10)-oestrene-3,17-diol. Isomers are molecules with the same molecular formula but different structural arrangements. The identification of this compound suggests an alternative pathway in the biosynthesis of steroids in the horse testes that was not previously recognized.