Potentially harmful advantage to athletes: a putative connection between UGT2B17 gene deletion polymorphism and renal disorders with prolonged use of anabolic androgenic steroids

ABSTRACT: BACKGROUND AND OBJECTIVE: With prolonged use of anabolic androgenic steroids (AAS), occasional incidents of renal disorders have been observed. Independently, it has also been established that there are considerable inter-individual and inter-ethnic differences, in particular with reference to the uridine diphosphate-glucuronosyltransferase 2B17 (UGT2B17) gene, in metabolising these compounds. This report postulates the association of deletion polymorphism in the UGT2B17 gene with the occurrence of renal disorders on chronic exposure to AAS. PRESENTATION OF THE HYPOTHESIS: The major deactivation and elimination pathway of AASs is through glucuronide conjugation, chiefly catalyzed by the UGT2B17 enzyme, followed by excretion in urine. Excretion of steroids is affected in individuals with a deletion mutation in the UGT2B17 gene. We hypothesize that UGT2B17 deficient individuals are more vulnerable to developing renal disorders with prolonged use of AAS owing to increases in body mass index and possible direct toxic effects of steroids on the kidneys. Elevated serum levels of biologically active steroids due to inadequate elimination can lead to prolonged muscle build up. An increase in body mass index may cause renal injuries due to sustained elevated glomerular pressure and flow rate. TESTING THE HYPOTHESIS: In the absence of controlled clinical trials in humans, observational studies can be carried out. Real time PCR with allelic discrimination should be employed to examine the prevalence of different UGT2B17 genotypes in patients with impaired renal function and AAS abuse. In individuals with the UGT2B17 deletion polymorphism, blood tests, biofluid analyses, urinalysis, and hair analyses following the administration of an anabolic steroid can be used to determine the fate of the substance once in the body. IMPLICATIONS OF THE HYPOTHESIS: If the hypothesis is upheld, anabolic steroid users with a deletion mutation in the UGT2B17 gene may be exposed to an increased risk of developing renal disorders. In the current detecting - sanctioning anti-doping system, athletes motivated by the potential to evade detection owing to their unique genetic make-up could subject themselves to a serious health consequence. More research on AAS metabolism in the presence of UGT2B17 gene deletion is required. Benefit - harm evaluations in therapeutic use of anabolic steroids should also consider this potential link between UGT2B17 gene deletion polymorphism and renal disorders

Development and application of novel methods for the detection of anabolic androgenic steroids in hair and other matrices using liquid chromatography tandem mass spectrometry

Owing to an increase in the number of doping cases with performance enhanc-ing drugs (PEDs), mainly anabolic androgenic steroids (AASs), and the limitations of urinalysis and self-reports, there is an ever increasing need to develop new methods for detecting doping. This thesis reports the development and application of a series of nov¬el analytical methods for the detection of frequently used AASs in hair and other matri¬ces using liquid-chromatography tandem mass-spectrometry (LC-MS/MS). Assays capable of detecting 0.5 pg/mg stanozolol and 3.0 pg/mg nandralane (with 20 mg hair) were developed. Hair samples from 180 subjects (108 males, 72 fe-males) were screened using ELISA, which revealed 16 subjects to be positive for stanozolol and 3 for nandrolone. LC-MS/MS analysis confirmed that only 11 subjects were positive for stanozolol (5.0 pg/mg to 86.3 pg/mg) and just 1 for nandralane (14.0 pg/mg), thus showing the inaccuracy of using ELISA screening alone. The analytical fmdings were successfully employed to verify self-reported drug use data. An assay capable of detecting 0.1 pg/mg testosterone (T) and 0.25 pg/mg epitestosterone (E) (with 50 mg hair) was developed. Seventy-five hair samples were collected from healthy volunteers (49 males, 26 females), with the natural levels of T 0.7-11.81 pg/mg and 0.33-6.05 pg/mg and the natural levels ofE 0.63-8.27 pg/mg and 0.52-3.88 pg/mg, in males and in females respectively. This thesis reports for the first time the TÆ ratio in hair, 0.5-3.37 in males and 0.56-1.81 in females. Assays capable of detecting 0.125 pg/mg stanozolol/0.25 pg/mg 3'-hydroxystano-zolol (with 50 mg rat hair) and 0.063 ng/mL stanozolol/0.125 ng/mL 3'-hydroxystanozolol (with 100 uL of rat urine or serum) were developed. Diclofenac was found to significantly reduce the urinary excretion of 3'-hydroxystanozolol, but did not influence the concentration of both compounds in hair. This is the first in vivo study to report this effect. Assays capable of detecting 0.25 pg/mL stanozolol and 0.5 pg/mL of 3'-hydroxystanozolol in 5 mL aqueous matrices were developed in order to investigate en-vironmental contamination. Three out of six samples from the River Danube, collected from December '09 to November '10, were found to contain stanozolol, (upto 1.82 pg/mL). In contrast, stanozolol was detected only once (1.19 pg/mL) in tap water from Budapest city

Detection of testosterone and epitestosterone in human hair using liquid chromatography tandem mass spectrometry

The feasibility of using hair analysis as a complimentary test in doping control has received increased attention in the scientific community. The aim of the study was to take a step forward to this goal and develop a method that, for the first time, is able to detect testosterone (T) and epitestosterone (E) in human hair, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and alkali digestion followed by extraction using pentane. The method was linear within the quantification range of 0.25-100pg/mg for T and 0.5-100pg/mg for E, with determination coefficient (r(2)) values >0.9987. The limits of detection for T and E were 0.1pg/mg and 0.25pg/mg respectively. The accuracy, precision and extraction recovery of the assay were satisfactory for the detection of T and E when ca. 50mg hair was processed. The validated method was successfully applied for the analysis of 75 hair samples collected from healthy volunteers (65.3% males), with the concentration of T between 0.7-11.81pg/mg and 0.33-6.05pg/mg and the concentration of E between 0.63-8.27pg/mg and 0.52-3.88pg/mg in males and in females respectively. In males, the T levels were significantly higher (p=0.020) but there was no difference in the E levels (p=0.359). However, E was not detectable in 34 samples (of which 19 were females). The T and E levels showed linear correlation (r=0.698, p<0.001) with average T/E ratio of 1.32±0.7. The newly developed analytical method was rapid, facile, sensitive, selective, reproducible and reliable for determining the levels of T and E in hair and thus for calculating the T/E ratio in hair

Incongruence in doping related attitudes, beliefs and opinions in the context of discordant behavioural data: in which measure do we trust?

BACKGROUND: Social psychology research on doping and outcome based evaluation of primary anti-doping prevention and intervention programmes have been dominated by self-reports. Having confidence in the validity and reliability of such data is vital. METHODOLOGY/PRINCIPAL FINDINGS: The sample of 82 athletes from 30 sports (52.4% female, mean age: 21.48±2.86 years) was split into quasi-experimental groups based on i) self-admitted previous experience with prohibited performance enhancing drugs (PED) and ii) the presence of at least one prohibited PED in hair covering up to 6 months prior to data collection. Participants responded to questionnaires assessing a range of social cognitive determinants of doping via self-reports; and completed a modified version of the Brief Implicit Association Test (BIAT) assessing implicit attitudes to doping relative to the acceptable nutritional supplements (NS). Social projection regarding NS was used as control. PEDs were detected in hair samples from 10 athletes (12% prevalence), none of whom admitted doping use. This group of 'deniers' was characterised by a dissociation between explicit (verbal declarations) and implicit (BIAT) responding, while convergence was observed in the 'clean' athlete group. This dissociation, if replicated, may act as a cognitive marker of the denier group, with promising applications of the combined explicit-implicit cognitive protocol as a proxy in lieu of biochemical detection methods in social science research. Overall, discrepancies in the relationship between declared doping-related opinion and implicit doping attitudes were observed between the groups, with control measures remaining unaffected. Questionnaire responses showed a pattern consistent with self-reported doping use. CONCLUSIONS/SIGNIFICANCE: Following our preliminary work, this study provides further evidence that both self-reports on behaviour and social cognitive measures could be affected by some form of response bias. This can question the validity of self-reports, with reliability remaining unaffected. Triangulation of various assessment methods is recommended

Virtue or pretense? Looking behind self-declared innocence in doping

BACKGROUND: Social science studies of doping practices in sport rely predominantly on self-reports. Studies of psychoactive drug use indicate that self-reporting is characterised by under-reporting. Likewise doping practice is likely to be equally under-reported, if not more so. This calls for more sophisticated methods for such reporting and for independent, objective validation of its results. The aims of this study were: i) to contrast self-reported doping use with objective results from chemical hair analysis and ii) to investigate the influence of the discrepancy on doping attitudes, social projection, descriptive norms and perceived pressure to use doping. METHODOLOGY/PRINCIPAL FINDINGS: A doping attitudes questionnaire was developed and combined with a response latency-based implicit association test and hair sample analysis for key doping substances in 14 athletes selected from a larger sample (N = 82) to form contrast comparison groups. Results indicate that patterns of group differences in social projection, explicit attitude about and perceived pressure to use doping, vary depending on whether the user and non-user groups are defined by self-report or objectively verified through hair analysis. Thus, self-confessed users scored higher on social projection, explicit attitude to doping and perceived pressure. However, when a doping substance was detected in the hair of an athlete who denied doping use, their self-report evidenced extreme social desirability (negative attitude, low projection and low perceived pressure) and contrasted sharply with a more positive estimate of their implicit doping attitude. CONCLUSIONS/SIGNIFICANCE: Hair analysis for performance enhancing substances has shown considerable potential in validating athletes' doping attitude estimations and admissions of use. Results not only confirm the need for improved self-report methodology for future research in socially-sensitive domains but also indicate where the improvements are likely to come from: as chemical validation remains expensive, a more realistic promise for large scale studies and online data collection efforts is held by measures of implicit social cognition

Analysis of anabolic steroids in human hair using LC-MS/MS

New highly sensitive, specific, reliable, reproducible and robust LC-MS/MS methods were developed to detect the anabolic steroids, nandrolone and stanozolol, in human hair for the first time. Hair samples from 180 participants (108 males, 72 females, 62% athletes) were screened using ELISA which revealed 16 athletes as positive for stanozolol and 3 for nandrolone. Positive samples were confirmed on LC-MS/MS in selective reaction monitoring (SRM) mode. The assays for stanozolol and nandrolone showed good linearity in the range 1 to 400 pg/mg and 5 to 400 pg/mg respectively. The methods were validated for LLOD, interday precision, intraday precision, specificity, extraction recovery and accuracy. The assays were capable of detecting 0.5 pg stanozolol and 3.0 pg nandrolone per mg of hair, when approximately 20mg of hair were processed. Analysis using LC-MS/MS confirmed eleven athletes' positive for stanozolol (5.0 pg/mg to 86.3 pg/mg) and 1 for nandrolone (14.0 pg/mg) thus avoiding false results from ELISA screening. The results obtained demonstrate the application of these hair analysis methods to detect both steroids at low concentrations, hence reducing the amount of hair required significantly. The new methods complement urinalysis or blood testing and facilitate improved doping testing regimes. Hair analysis benefits from non invasiveness, negligible risk of infection and facile sample storage and collection, whilst reducing risks of tampering and cross contamination. Owing to the wide detection window, this approach may also offer an alternative approach for out-of-competition testing

A review of methods for the simultaneous detection of illegal ingredients in food supplements

Food supplements are at risk from contamination with illegal ingredients on a global scale. To date, the official food control laboratory system in the UK does not appear to have been particularly active in the analytical control of illegal ingredients in food supplements. From a survey of notifications (2009 to 2016) to the European Union rapid alert system for food and feed (RASFF) food supplements are shown to be adulterated with a complex range of compounds and substances. These include permitted food additives in excess of their limits, contaminants, unauthorised novel food ingredients, unauthorised nutritionally-related compounds, excess vitamins, controlled drugs, and one instance of the poison strychnine. However, arguably, the most important factors that jeopardise the safety of food supplements are their adulteration with synthetic pharmaceutical drugs. In order to assist official control and trade analysts to regulate the safety of food supplements with regard to the presence of illicit pharmacologically active ingredients or contaminants the RASFF database, 2009 to 2016, was surveyed for such compounds. The most frequently notified were sildenafil and its analogues, sibutramine and derivatives, 1,3-dimethylamylamine (DMAA), yohimbine and tadalafil. Their toxicology and methods for their detection and determination have been reviewed in this paper. Method details are tabulated with references and general conditions have been suggested for a first choice liquid chromatography-mass spectrometry (LC-MS) screening method for the compounds sildenafil, tadalafil, vardenafil and yohimbine. If high field NMR is available this would appear to be an excellent first-line method of control for herbal food supplements. It would be helpful that when the suggested method(s) are assessed and validated in Association of Public Analyst member laboratories against a matrix of food supplements incurred or spiked with the target illegal ingredients that the data be reported herein

Detection of stanozolol in environmental waters using liquid chromatography tandem mass spectrometry

Background Owing to frequent administration of a wide range of pharmaceutical products, various environmental waters have been found to be contaminated with pharmacologically active substances. For example, stanozolol, a synthetic anabolic steroid, is frequently misused for performance enhancement as well as for illegal growth promoting purposes in veterinary practice. Previously we reported stanozolol in hair samples collected from subjects living in Budapest. For this reason we initiated this study to explore possible environmental sources of steroid contamination. The aim of this study was to develop a method to monitor stanozolol in aqueous matrices using liquid chromatography tandem mass spectrometry (LC-MS/MS). Results Liquid-liquid extraction using pentane was found to be an efficient method for the extraction of stanozolol from water samples. This was followed by direct detection using LC-MS/MS. The method was capable of detecting 0.25 pg/mL stanozolol when only 5 mL water was processed in the presence of stanozolol D3 as internal standard. Fifteen bottled waters analysed were found to be negative for stanozolol. However, three out of six samples from the Danube river, collected from December '09 to November '10, were found to contain stanozolol at concentrations up to 1.82 pg/mL. In contrast, only one sample (out of six) of urban tap water from Budapest city was found to contain stanozolol, at a concentration of 1.19 pg/mL. Conclusion The method developed is efficient, rapid, reproducible, sensitive and robust for the detection of stanozolol in aqueous matrices