New markers of steroid profile in blood: Differentiating testosterone administration from (simultaneous) ethanol consumption (acronym: SPOLBlood)

Code: ISF19D02JP 

Ethanol affects the steroid profile in a way that may mask testosterone administration. Our group has shown that urinary ratios 6OHAndrosterone3G/Epitestosterone17G and 6OHEtiocholanolone3G/Epitestosterone17G increase after testosterone administration while preliminary results show they decrease after ethanol consumption. This behavious suggests that those two glucoronides may be useful to distinguish between changes in T/E due to ethanol consumption and those due to the combined administration of testosterone and ethanol.

A project to investogate those markers was approved in 2018 (ISF18D13OP). The clinical trial includes the administration of placebo, testosterone, alcohol and the combination of testosterone plus alcohol. Samples of urine, blood and saliva are collected. However, a budget reduction in the approved grant prevented investigating the new biomarkers not just in saliva, but even in blood.

The steroid profile in blood is very relevant. Previous attempts to develop a blood steroid profile lost the focus including a mixture of a few androgens, plus estrogens and corticoids. However, the key analytes in blood will also be steroid conjugates. The ratio of free to conjugated testosterone is known to change greatly after oral testosterone administration. Our primary results show how the new glucurono-conjugated biomarkers 6OH-A3G and 6OH-Etio-3G and others can be monitored in blood. The administration of ethanol affects phase II metabolism and therefore this specific blood steroid profile needs to be studied.

This project aims at studying the blood samples collected in project ISF18D13OP to study the behaviour of the new biomarkers 6OH-A-3G and 6OH-Etio-3G in blood as part of a more selective steroid profile, and the usefulness of the combination of phase I plus phase II metabolites in blood to differentiate between the consumption of alcohol alone and its consumption during testosterone administration.

Main Findings

The steroid profile is subject to several confounding factors. The use of alcohol is probably the most prevalent one. This projects aimed at finding new markers to tell the difference between the administration of testosterone and the administration of alcohol or the combination of both substances.

In the present study, urine and whlole blood samples (as for ABP hematological module) were collected from four volunteers after the administration of placebo, alcohol, testosterone transdermal or the combination alcohol plus testosterone.

The analysis of the plasma from blood samples collected as per ABP hematological module purposes, showed that from the many markers studied, androsterone glucuronide was the one showing a clearly different behaviour when administering alcohol (decrease) or testosterone (increase). This has been the key factor in the development of a new marker calculated as the product of the concentrations of testosterone glucuronide and androsterone glucuronide (T-G x AN-G) in plasma samples. This new marker has shown to be:

· sensitive to the administration of testosterone (transdermal)

· insensitive to the administration of alcohol

· still sensitive to testosterone when administered jointly with alcohol

Other markers, i.e. the recently proposed ratio testosterone over androstenedione (T/A4) have shown unable to discriminate between both substances.

The new marker (T-G x AN-G) has a great potential for the detection of testosterone use even when co-administered with alcohol. Furthermore, it can be readily implemented by accredited laboratories as these analytes are commercially available and laboratories have experience in the analysis of those substances. In order to confirm and validate this new marker, further studies wil be necessary increasing the cohort of subjects and to test its behaviour for other dosages and routes administration of testosterone, the impact of genetic/ethnic differences and other potential confounding factors.

Considering the latest publication of the WADA guideline Quantification of Endogenous Steroids in Blood for the Athlete Biological Passport (July 2023), analysis of these new markers in serum instead of plasma would be advisable for consistency with the approach followed by WADA.