Projets de recherche

Code: 10A24XD 

In extremely rare circumstances the presence of 19-norandrosterone (19-NA) in human urine can be explained by the “instability” or “activity” of the urine specimens. This is most likely due to the sample transportation and storage in the laboratory and is based on the 19- demethylation of abundant endogenous steroids in the urine samples.

Tests for the assessment of the activity of the urine samples have been established. The hypothesis is that 19-NA will be produced by 19- demethylation of androsterone, the main androgen metabolite present in the urine. Briefly, an aliquot of sample to be tested for activity will be incubated in the presence of deuterated androsterone. The formation of deuterated 19-NA will be the proof of urine activity.  

From the data collected until now, it appears that the instability of these urine samples is due to enzymatic activity expressed (from exogenous origin) in the sample. It seems unlikely that the 19-demethylation would be the result of a pure, unassisted chemical reaction. If this were the case, this phenomenon would be reproducible at any time, and is not.  The growth of microorganisms in the urine samples is not unlikely. It is reasonable to link this enzymatic activity to the presence of a microorganism growing in the urine.  The expression of aromatase is restricted to the gonads and brain in many vertebrates, from aquatic and avian species to mammals. The removal of the methyl group in the 19 position seems not linked to an aromatization process since no microorganism expresses such an enzyme.  An important demethylase enzyme present in some microorganisms is the 14-demethylase (CYP51A1). The activity of this enzyme is crucial to the life of these microorganisms since it is the responsible for the formation of ergosterol from lanosterol. The hypothesis is that CYP51 uses androsterone as “substrate” consequently producing 19-NA.

Main Findings: 

The present was focused on the hypothesis that the CYP51 (14α-demethylase) of fungal origin may be the cause of the alteration of the metabolic profile found in the so-called active urines. In these cases a concentration of 19-NA beyond the limits allowed, but with a ratio 19-NA/19-NE reversed compared to that A / E (usually the concentration of 19-NA in urine is greater) are observed.
To check if the CYP51 is able to demethylate androsterone and / or etiocholanolone, yeasts such as S. cerevisiae and C. albicans were chosen. As substrates of of the yeasts, androsterone, etiocholanolone and  androstenedione were selected as being the most probable substrates, based on their structure and on the amount present in routine anti-doping samples.
The following experiments were performed:
1. S. cerevisiae and C. albicans were incubated in the presence of androsterone, etiocholanolone, and androstenedione in culture medium and in synthetic urine (in sterile conditions). The products of fungal metabolism were found both in the supernatant and in cell lysate.
2. S. cerevisiae and C. albicans were incubated in human urine in non-sterile conditions. Alterations of the hormonal profile in urine were evaluated according to the protocols of the anti-doping laboratory of Rome, used in the screening of banned substances.
The main conclusions of the present project are:
· The demethylation process of steroids produced in the so-called active urines is not originated chemically, at least under the common laboratory conditions during the sample processing of the urine samples in doping control.
· The aromatization process that is the responsible of the in-vivo formation of 19-norandrosterone is not the responsible for the 19-demethylation ex-vivo during sample storage of the urine samples.
· In cultured C. albicans and S. cerevisiae conducted in SDB medium and in the presence of steroid hormones, any relevant reaction of demethylation was observed. By-products of fungal metabolism on these hormones have been detected.
· In human urine inoculated with the same microorganisms, changes in the hormonal profile were detected, that can be attributed to the activity of C. albicans. These observations were previously detected in culture medium under sterile conditions. Even in these experiments the formation of 19-narandrosterone and 19-noretiocholanolone was not detected.
· Although the formation of 19-norsteroids object of this study was not detected, the changes in urinary steroid profile as a result of contamination with fungi are considered to be relevant for the correct interpretation of the data in doping control.
Under the experimental conditions described, where the functional growth of fungi has been demonstrated, the transformation of androsterone and etiocolanolone to their respective 19-norderivatives, as happens in the case of active urine, was not observed.  With the current obtained knowledge, we can say that the ex-vivo process of demethylation is complex and involving several actors. The proposed test to demonstrate the “activity” of a particular sample provided that deuterated androsterone or etiocholanolone were transformed in their corresponding 19-nor deuterated products. This is not the case with the presence of the tested fungi alone. The process of demethylation implies the presence of an unsaturation in alpha with respect to the methyl group to be removed, absent in androsterone and etiocholanolone. Our experimental evidence supports the hypothesis that the process is a multistep process consisting in a first dehydrogenation of A ring, most probably in the C1 position,
followed by the multistep oxidation or the methyl group

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Dr. James Skinner, Griffith University, Australia

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Code: 10A1PV

The project aims to contribute to the development of the steroidal subunit of the Athlete’s Biological Passport (ABP) which is hitherto solely validated on a few markers, such as the T/E ratio.  

Recently, novel biomarkers were found that increase significantly the time detection window after administration of small doses of T, DHT and DHEA using the Adaptive Model of the ABP. These biomarkers consist of steroid ratios including minor metabolites sensitive to steroid administration. More data on intra-individual variation of the involved minor metabolites are nevertheless necessary to validate the proposed biomarkers. Therefore a large-scale investigation of long-term within-subject behaviour of an extended steroid profile will be conducted.

Data obtained on a larger cohort with comprehensive steroid profiling methods will allow the development of a multi-parametric marker of steroid doping that comprises the whole steroid profile. This model statistically classifies abnormal steroid profiles by outputting a single score. Longitudinal evaluation of this ‘Abnormal Steroid Profile Score’ (cfr. Abnormal Blood Profile Score in the Blood Passport) monitors any alteration in the steroid profile regardless to its cause. The goals are twofold. Firstly, when applied at the individual level, this model will allow the general screening of doping with endogenous steroids, food supplements and substances manipulating the steroid profile, such as after ethanol consumption. Secondly, and in contrary to blood doping and doping with growth hormone wherein markers having a detection time long enough to estimate the prevalence of doping already exist, this score might provide accurate estimates of the prevalence of steroid doping in elite sports when applied at the population level.

Moreover, the influence of genetic polymorphism on the new steroid profile parameters and an Abnormal Steroid Profile Score will be studied in order to increase the sensitivity of the model.

Main Findings:

A combination of the Support Vector Machine (SVM) algorithm with a comprehensive approach of steroid profiling resulted in a steroidomic model that enables to differentiate normal steroid profiles from abnormal ones. Theoretically, the SVM tool plots all monitored\steroids in a multi-dimensional hyperspace which makes the use of steroid ratios redundant to obtain a strategy with optimal detection sensitivity. Hence, the whole set of steroid profile values can be evaluated at once. In our model, however, the degree of abnormality was quantified by an Abnormal Steroid Profile Score (ASPS) for which values greater than 0.79 could be considered as deviating from normal.  
Since the introduction of the Athlete Biological Passport, the results of steroid profiling tests can be systematically stored in a central database enabling the estimation of the individual reference ranges. From such databases, longitudinal steroid profiling data can be made readily available to elaborate longitudinal strategies, thereby omitting a large contribution of the inter-individual variance. Similarly, the raw SVM model was improved by standardizing the training set using individual mean and standard deviation obtained with the adaptive model. The combination of the adaptive model and the SVM enhances the general performance accuracy of the raw SVM model from 62% to 84%, disregarding the kind of endogenous steroid administered. The diagnostic sensitivity of the resulting ASPS was 55% in a post-administration period of 7 days. Altered steroid profiles can be found until 5 days after ingesting a small single doses of T or DHEA or after topical application of T or DHT in therapeutically recommended doses. This drastic increase in sensitivity can be explained by the ability of the model to sensitively distinguish a prolonged recovery state of the steroid metabolism which is restoring the homeostasis of steroid profile to known basal levels.  
Since the model was trained on data obtained after T, DHT and DHEA administration, the model risked to be overfitted i.e. a specific detection tool for these steroids. This problem was addressed by leave-one-subject-out cross-validation and testing of the model on another volunteer, with another dose of DHEA and with other steroids. Testing of the excretion data from a 100mg dose of DHEA, 50mg Adion and 7-keto-DHEA ingested by another volunteer showed a clear response of the ASPSs. This indicates the polyvalent nature of the SVM model to detect any small disturbance of the steroid profile. Moreover, the high sensitivity of 97% obtained for this new test set illustrates the potential of the ASPS as a powerful biomarker for the general detection of misuse with endogenous steroids. Although, this single model shows excellent sensitivity for a wide range of administered steroids, it cannot specify which cause resulted in an aberrant steroid profile. For this information, specific metabolites should be evaluated separately. 
Despite the excellent preliminary results on low dose administration studies conducted on a limited study population - including subjects with atypical T/E’s that challenge the classification -, the applicability of this strategy will require further work and large scale validation procedure. In order to implement the ASPS in routine testing as a sensitive marker for of any misuse with endogenous steroids, the model should be tested on larger cohorts of data and external influences on the steroid profile that can alter the ASPS should be scrutinized in the future.  
In conclusion, a new strategy was developed that returns a single value ASPS as a denotation of the degree of abnormality of a steroid profile containing 24 steroid metabolites. With this strategy, the alteration of the steroid profile, caused by a variety of endogenous steroids, can be detected very sensitively. The longitudinal SVM model was shown to be a general model which can result in long detection of small doses of oral and topical steroid formulations up to 5 days. The overall model performance was very good, particularly when coupled with the longitudinal results from the adaptive Bayesian model. The combination of computer aided techniques as the Bayesian adaptive model and SVM algorithm provide a valuable steroidomic strategy for the long term detection of misuse with endogenous steroids in complement with current steroid profiling methods.