DHEA, 7-keto-DHEA and Arimistane: precursors or metabolites. Is IRMS necessary?

Code: 19A09XD

The relationship among the dehydroepiandrosterone (DHEA) and its metabolites 7-hydroxylated and 7-keto have been widely described leading to think that their presence in urine supposes a DHEA abuse. On the other hand, we have confirmed the 3-deoxylation forming structures androst-3,5-diene-7-keto under acidic conditions; in that sense, the synthesis of arimistane is possible from 7-keto-DHEA under the conditions of the common procedures applied to detect steroids in antidoping laboratories. 7-keto-DHEA is well known by having no influence on androgens or estrogens metabolism; and that the configuration androst-3,5-diene-7-keto is essential to inhibition of aromatase occurs.

Preliminary results (n=1) showed that after a single oral dose of 7-keto-DHEA no alterations of the endogenous steroid profile occurred, the presence of arimistane and its main metabolite (7β-hydroxilated metabolite), and finally we obtained negative results for pseudo-endogenous steroids (i.e. testosterone, its main metabolites and DHEA) after the analysis by GC/C/IRMS. Several reduced, oxidized metabolites and poly-hydroxylated metabolites were found using GC/QTOF, most of them were not present in negative urine. These poly-hydroxylated metabolites and other isomers founded have not been described for DHEA so, probably could be specific of the 7-keto-DHEA metabolism.

It is then necessary to increase the volunteers looking for the individual variability in order (1) to confirm these results by GC/C/IRMS, (2) to confirm the true origin of the arimistane after the administration of 7-keto-DHEA searching for a suitable assay, (3) to look for the specific metabolites that discriminates between DHEA and 7-keto-DHEA administration, and finally (4) to correctly assign 7-keto-DHEA on the WADA Prohibited List considering the antiestrogenic properties according its chemical structure.

Main Findings

Preliminary data showed some cross-metabolic findings among DHEA, arimistane and 7-keto-DHEA (three compounds belong to two different sections of the WADA Prohibited List), so it is necessary to ascertain specific metabolic findings that can be assigned to each administration. Arismistane, at a single dose, does not provoke evident alterations in the urinary endogenous steroid profile. Arimistane itself is only detectable in the sulfate fraction more as an artifact or degradation product than to an actual phase II metabolite. The analysis of the sulfate fraction has some limitations. Taking into account the C3 de-oxygenation of the androst-3,5-diene-7-oxo structures under acidic conditions, its presence in the sulfated fraction may be due to the C3 de-oxygenation of endogenous 7-keto-DHEA. Twelve arimistane metabolites were described after GC and LC analysis. The main metabolite 7β-hydroxy-arimistane showed the longest-term excretion. Contrary to GC, traces of arimistane PC were observed by LC-MS analysis. Hydroxylated metabolites of arimistane in C2 was proposed although additional spectrometric techniques must be applied to finally elucidate the structure. Alternatively, the chemical synthesis of the compound is needed. The presence of arimistane in concentrations higher than its 7β-hydroxy metabolite could be an adequate marker because, after the administration of arimistane, itself is almost undetectable in urine. The detection of 7-keto-DHEA administration based on the endogenous steroid profile of ABP and the consequent analysis by GC-C-IRMS failed. IRMS data supported the fact that there is no back-formation of DHEA from 7-keto-DHEA. Ten metabolites proposed by GC-MS and LC-MS analyses were proposed, in addition to 7-keto-DHEA itself. They showed considerable responses in both the free+glucuronated and sulfate fractions. Among them, there were four metabolites excreted for longer times and with higher responses. Additional spectrometric techniques or the syntheses of the proposed structures are needed for a definitive confirmation of the configurations. Although considered a degradation metabolite, arimistane was observed in samples post- 7-keto-DHEA administration. After the analysis of samples from 5 volunteers, no arimistane metabolite (7β-hydroxy) was found. The metabolites are specific enough to avoid GC-C-IRMS confirmations. Artifacts may be produced during sample preparation and instrumental analysis.

After the GC analysis of trimethylsylil derivatives of 7-keto-DHEA 7α-OH-DHEA, arimistane was identified. Arimistane signal was proportional to the concentration of derivatization mixture used. Meanwhile, 7β-hydroxy-DHEA showed no degradation. Analysis by LC instruments (avoiding derivatization and high temperatures of the injector port) showed that 7-keto-DHEA preparations in protic solvents as MeOH favor the dehydration of the molecule, forming arimistane. This not occurs with aprotic solvents as DMSO. Approaches based on enzymatic hydrolysis using β-glucuronidase (E.coli) or glucuronidase/arylsulfatase (H. pomatia) did not favor the formation of arimistane. Nevertheless, the hydrolysis under strongly acidic conditions favors the complete degradation of 7-keto-DHEA into arimistane.

The correct detection of 7-keto-DHEA by using a suitable analytical procedure could avoid the risk of false positive findings for aromatase inhibitor arimistane and, at the same time could avoid the unnecessary application of IRMS since specific metabolites could be found.