Isolation and characterisation of a C(18) neutral steroid, oestra-5(10),7-diene-3,17-diol, from pregnant mare urine and allantoic fluid. Facile oxidation to yield oestra-5(10),6,8-triene-3, 17-diol (diol of Heard’s ketone).

The research article is centered around the isolation and characterization of two specific neutral steroids, derived from the urine and allantoic fluid of pregnant mares, as well as investigations into their behaviours under variable conditions.

Extraction and Purification

• The researchers extracted the steroids oestradiene-3,17-diol and oestratriene-3,17-diol on a large scale from samples of pregnant mares’ urine and allantoic fluid.
• This extraction began with an initial purification using column chromatography.
• Further purification efforts utilised high-performance liquid chromatography and silver nitrate thin layer chromatography to achieve the desired concentrations of the steroids.

Characterization and Analysis

• The scientists characterized the purified steroids using gas chromatography-mass spectrometry.
• They inferred the positions of double bonds in the Ring B structure of oestradienediol based on results from ultraviolet (UV) and nuclear magnetic resonance (NMR) spectroscopy, as well as hydrogenation and incubation studies with a specific enzyme.
• Comparison with a reference steroid, 7-dehydrocholesterol, indicated significant behaviour differences due to the absence of a conjugated system in oestradienediol.

Instability and Oxidation

• The researchers noticed that the instability of the dienediol became apparent when it was converted to its bis-trimethylsilyl (TMS) ether derivative.
• The resulting instability was magnified when derivatisation took place at higher temperatures or if the dienediol-containing fraction was stored at lower temperatures (4 degrees Celsius) before the derivatisation procedure.
• The oxidation product was shown to be consistent with the conversion of oestradienediol to oestratrienediol, leading to the supposition that these two steroids are related and the sites of unsaturation are located in the B ring of the structure.

Conclusions and Proposals

• The authors conclude that the steroids oestratrienediol (the diol of Heard’s ketone) and, by implication, Heard’s ketone itself, are created during the isolation procedures used in the study.
• The scientists propose a possible biosynthesis mechanism for the creation of the 5, 7-oestradienediol from an unsaturated C(19) compound, proposing a process that involves C(19) demethylation without aromatisation.