| Aromatase inhibitor is a substance that can suppress the conversion of androgens into estrogens. An increase in the serum testosterone concentration level has been observed after oral administration of aromatase inhibitor. Therefore, use of aromatase inhibitors was prohibited by the International Olympic Committee (IOC) and World Anti-doping Agency (WADA) for male and female athletes in September 2001 and January 2005, respectively Aminoglutethimide, Letrozole and Anastrozole are the most popular non-steroidal aromatase inhibitors in the market. Steroid profiling was introduced to determine endogenous steroid misuse in sports. Thus, screening for exogenous use of these prohibited substances can be established by monitoring a range of endogenous steroids, which constitute the steroid profile, and evaluate their concentrations and ratios against reference values. Steroid profile evaluation is currently based upon population statistics. As large inter-individual variations exist, Athlete Biological Passport (ABP) analysis is ongoing. This study aimed to identify new biomarker(s) for aromatase inhibitor detection in sports using statistical analysis, and adapt the model into ABP analysis. At the same time, indentify the metabolites of the three non-steroids aromatase inhibitors by UPLC-Q-Exactive Plus Orbitrap Quotation to improve the detection ability of routine anti-doping control.Forty-one Chinese non-athlete volunteers (21 males and 20 females) were administered three non-steroidal aromatase inhibitors (Aminoglutethimide, Letrozole and Anastrozole), separately. Group A received an oral dose (250 mg×2/day) of Aminoglutethimide a day and consecutively administered for 3 days. Group B and C took Letrozole (2.5 mg×1/day) and Anastrozole (1mg×1/day) for 5 consecutive days respectively. Urine samples were collected prior 7 days (blank) up to 28 days post administration. The urine samples were analyized by GC-MS and UPLC-Q-Exactive Plus Orbitrap Quotation. Statistical analysis was performed upon 16 steroid profile parameters. Mass Frontier 7.0 SR software was used to find out the main metabolites in urine samples. Rstudio Version 0.99.467 was used to analzy the steroids profile.Results:(a) 33 main possible metabolite was found (Aminoglutethimide 24, Letrozole 4 and Anastrozole 5),24 of them were firstly be reported in the human beings, (b) After administration, the concentrations of endogenous androgen biomarkers such as T, ET, AN, ETIO,5α-diol,5β-diol, and DHEA were increased, while estrogen amounts were decreased accordingly. They were all back to normal within one month. In females, the concentrations of endogenous biomarkers were affected by non-steroidal aromatase inhibitors, without a common trend among them, (c) The measurement of negative criteria with an appropriate level of confidence intervals (95% and 99%, separately) using blank urine samples was carried out, and a new evaluation model was established. By utilizing two different evaluation approaches (three new models and WADA ratio biomarkers), average suspicious sample rates were increased sharply and displayed as follows:AN/Estrone (57.86%), ETIO/Estrone (58.25%) and T/Estrone (33.86%) at the level of 95% confidence intervals; AN/Estrone (42.14%), ETIO/Estrone (45.30%) and T/Estrone (24.79%) at the level of 99% confidence intervals. In contrast, using WADA EAAS TD, average suspicious sample rates were T/ET (5.21%),5a-diol/5p-diol (5.49%), AN/T (10.34%), AN/ETIO (4.90%) and 5a-diol/ET (3.78%) at the level of 95% confidence intervals; the rates were T/ET (3.05%),5a-diol/5β-diol (0.30%), AN/T (5.86%), AN/ETIO (1.37%)and 5a-diol/ET (1.10%) at the level of 99% confidence intervals. The higher the rate of the suspicious samples, the longer detection window limit, (d) Three new endogenous biomarkers (AN/Estrone, ETIO/Estrone and T/Estrone) rose significantly after treatment. The three new models were more sensitive than the WADA ratio biomarkers. They were also effective in exponentially weighted moving average chart analysis, (e) To verify the validity of the model, another experiment was designed. Exemestane (25 mg,30 tab; Pfizer Italia s.r.l, USA) was administered at single dose of 25 mg to one male volunteer. After administration all urine for 3 days was collected. From the 4th to 15th day, morning urine samples were collected. Exemestane was previously monitored by its major metabolite for doping control purpose. Using the current method, the drug could not be detected after 25.5h. However, there was overt change in the EWMA of T/Estrone, with detection window increasing from 1 to 10 days. Verification experiment demonstrated that the biomarker T/Estrone was valid in judging steroidal aromatase inhibitor abuse.Conclsions:In this study 33 metablites were found. (24 of which were first reported in this study) this study indicated that use of non-steroidal aromatase inhibitors affects both ovarian and adrenal androgens. Three new bio-markers (AN/Estrone, ETIO/Estrone and T/Estrone) were found as the most accurate biomarkers for detecting non-steroidal aromatase inhibitors, and can be used to estimate abuse of steroidal aromatase inhibitors. A combination of the three new biomarkers with appropriate reference levels can increase detection accuracy and prolong detection time for steroidal aromatase inhibitors could play a great supplementary role for metabolite monitoring in doping control routine work. The new biomarkers can also be used for ABP purpose.
|
PDF Full Text Request