Testosterone Glucuronide Research Articles

LC-MS/MS-Based Assay for Free and Deconjugated Testosterone and Epitestosterone in Rat Urine and Serum

Testosterone and epitestosterone are mainly excreted as glucuronides. The aim of this study was to develop and validate a method using liquid chromatography tandem mass spectrometry (LC-MS/MS) to analyse testosterone and epitestosterone in rat serum and urine to assist in vivo studies on steroid metabolism. The method was developed by spiking charcoal stripped rat plasma and urine with the analytes. The developed method was then applied to serum (n=6) and urine samples (n=6) from young male brown Norway rats to determine testosterone and epitestosterone concentrations. The assay showed linearity within quantification range coefficient (r2) values above 0.991. Optimum conditions were determined for the deconjugation of glucuronidated testosterone and epitestosterone along with the internal standard stanozolol D3. Accuracy, precision and extraction recovery for both compounds was satisfactory in both matrices. The method was capable of quantifying 0.250 ng/mL concentrations of testosterone and epitestosterone in 100 μL of serum and urine. The average concentrations of free and deconjugated testosterone and epitestosterone found in the rat samples were: urine–201.68 ± 90.16 ng/mL and 85.37 ± 21.20 ng/mL; serum– 363.40 ± 11.615 ng/mL and 1.75 ± 0.118 ng/mL, respectively. This method is sensitive, specific and reproducible for the determination of free and deconjugated testosterone and epitestosterone in rat serum and urine. The method can be used for in vivo analysis for further investigations of testosterone and epitestosterone concentrations in studies monitoring endocrine dysfunctions and doping.

Circadian Changes in Testosterone and Estradiol-17 Glucuronides and Sulfate Steroids during Spawning Phase in Fresh Water Fish Female Cirrhinus mrigala

The aim of this investigation was to know daily changes (circadian) in the blood plasma free (unconjugated) testosterone (TF) and estradiol-17β(E2F) and its conjugated testosterone glucuronide (TG) and testosterone sulfate (TS) as well as estradiol-17β glucuronide (E2G) and estradiol-17β sulfate (E2S) sex steroids in a mature female fish Cirrhinus mrigala (Ham.) during spawning phase using enzyme-linked immunosorbent assay (ELISA) method. The two sex steroids testosterone and estradiol-17β exhibited identical circadian rhythm: a major peak 29.02±3.58 ng/ml occurred at the onset of the dark phase and a minor peak 12.79±1.89 ng/ml was generally observed 4 hour after the onset of light phase . The TG peak (9.23±1.04 ng/ml was in the dark phase whereas TS during 12.00 PM of dark phase. The E2G 7.19±.76 ng/ml peak was also indicated high during 04.00 am of the dark phase. Result indicated that during the dark phase elevation of free and conjugated sex steroids have important role for spawning, and maintaining the equilibrium of free and conjugated steroids.

Androgen activity, ischaemic heart disease and risk factors among men in NHANES III

Observationally, low serum testosterone among men is associated with cardiovascular diseases and its risk factors, but it is unclear whether raising endogenous androgens would be protective. To clarify the role of androgens, the association of two different androgen biomarkers (serum testosterone and androstanediol glucuronide) with cardiovascular disease risk factors and mortality was examined in a nationally representative sample of US men. Multivariable linear and proportion hazards regression were used to examine the adjusted associations of serum testosterone and androstanediol glucuronide with cardiovascular disease risk factors and death from major cardiovascular diseases in 1460 men from NHANES III phase 1 (1988-1991) followed-up through 2006. Serum testosterone and androstanediol glucuronide were weakly correlated (0·13). Serum testosterone was associated with healthier values of most cardiovascular disease risk factors but not with death from ischaemic heart disease or stroke, adjusted for age, education, race/ethnicity, smoking and alcohol use. Similarly adjusted, androstanediol glucuronide was associated with unhealthier values of some cardiovascular risk factors and death from ischaemic heart disease (hazard ratio 1·16, 95% confidence interval 1·003-1·33 per standard deviation). Androgen biomarkers had inconsistent associations with cardiovascular disease risk factors and ischaemic heart disease. Androstanediol glucuronide, rather than serum testosterone, had associations with cardiovascular disease risk factors more similar to those seen in randomized controlled trials of testosterone therapy, with corresponding implications for raising androgens.

Androgen activity and markers of inflammation among men in NHANES III.

Inflammation contributes to chronic diseases. Lower serum testosterone among men is associated with less inflammation, yet immune defense is thought to trade-off against reproduction with androgens adversely affecting immune function. Anti-androgens are effective at castrate levels of serum testosterone, suggesting serum testosterone may not capture all androgen activity. The association of two androgen biomarkers with key markers of inflammation was examined. The adjusted association of serum testosterone and androstanediol glucuronide with C-reactive protein, white blood cell, granulocyte and lymphocyte count, fibrinogen, and hemoglobin, as a control outcome because testosterone administration raises hemoglobin, were examined in a nationally representative sample of 1,490 US men from the National Health and Nutrition Examination Survey III phase 1 (1988-1991) using multivariable linear regression. Serum testosterone and androstanediol glucuronide were weakly correlated (0.13). Serum testosterone was associated with lower white blood cell count [-0.26 × 10(-9) per standard deviation, 95% confidence interval (CI) -0.37 to -0.14] and granulocyte count (-0.21 × 10(-9) , 95% CI -0.29 to -0.13) but not with hemoglobin (0.02 g/l, 95% CI -0.89 to 0.92), adjusted for age, education, race/ethnicity, smoking, and alcohol. Similarly adjusted, androstanediol glucuronide was not associated with white blood cell count (0.10 × 10(-9) , 95% CI -0.05 to -0.25), granulocyte count (0.12 × 10(-9) , 95% CI -0.02 to 0.25), or fibrinogen (0.05 g/l, 95% CI -0.004 to 0.11), but was with hemoglobin (0.70 g/l, 95% CI 0.07 to 1.32). Different androgen biomarkers had different associations with inflammatory markers, highlighting the need to consider several androgen biomarkers. The possibility remains that androgens may generate inflammatory processes with implications for chronic diseases.

Modulation of UDP Glucuronosyltransferase 2B15 and 2B17 and Prostate Cancer Risk: Current Perspectives

UDP glucuronosyltransferases (UGTs) are a family of enzymes that glucuronidate a variety of exogenous and endogenous compounds, including androgens; which makes them more hydrophilic, changes biological activity, and aids biological excretion. UGT2B17 has been identified as the major enzyme for testosterone glucuronidation, where UGT2B15 has been suggested to play a minor role. A deletion polymorphism in the UGT2B17 gene and an amino acid change in the UGT2B15 gene at codon 85 (aspartate>tyrosine) has been described where the resulting reduced rate of glucuronidation was implicated in the increased risk of prostate cancer. This review aims to analyse the impact that UGT2B15 and UGT2B17 genetic polymorphisms have on the risk of prostate cancer, through the use of studies previously conducted assessing the level of expression of each independent UGT2B enzyme, whereby ethnicity was taken into account. Positive and negative outcomes of clinical studies have been outlined for both UGT2B15 and UGT2B17, where conflicting evidence has led to inconclusive results for determining the consequence of UGT2B enzymes in prostate cancer risk. Additionally, inhibitors of testosterone glucuronidation such as non-steroidal, antiinflammatory drugs (diclofenac and ibuprofen), tea and red wine extracts have been identified to potentially affect the level of circulating testosterone by inhibiting UGT2B enzymes, thus potentially exacerbating prostate cancer risk. Future trials would involve a wider examination of other food substances and pharmaceuticals that can be attributed to the inhibition of UGT2B enzymes.

Testosterone challenge and androgen receptor activity in relation to UGT2B17 genotypes

We investigated the androgen receptor (AR) bioluminescense response in serum and urine before and after testosterone challenge in different genotypes of the UGT2B17 enzyme, which catalyses testosterone glucuronidation. The androgen receptor activity was determined using a yeast-based bioluminescence assay. The androgens were analysed using LC-MS/MS, and the individuals were genotyped for UGT2B17 deletion polymorphism using real-time polymerase chain reaction. The serum concentrations of testosterone and dihydrotestosterone (DHT) were markedly elevated on days 2 and 4 and were still above baseline on day 15 after a dose of 500mg testosterone enanthate. The androgenic activity in serum increased in parallel and correlated with the hormone concentrations and remained above baseline on day 15. The urinary androgenic activity increased 4-5-fold and was closely related to the unconjugated testosterone and independent of the UGT2B17 genotype. The AR assay may serve as a complement to the urinary testosterone/epitestosterone (T/E) doping test, because this is profoundly influenced by the UGT2B17 deletion polymorphism. It may also be useful for detection of other illicit androgens in sports, or in the society, or for monitoring and diagnostics of androgen-related disorders.

Non-Steroidal Anti-Inflammatory Drugs Do Not Influence the Urinary Testosterone/Epitestosterone Glucuronide Ratio

The UDP Glucuronosyl Transferase (UGT) enzymes are important in the pharmacokinetics, and conjugation, of a variety of drugs including non-steroidal anti-inflammatory drugs (NSAIDs) as well as anabolic androgenic steroids (AAS). Testosterone glucuronidation capacity is strongly associated with a deletion polymorphism in the UGT2B17 gene. As the use of high doses of NSAIDs has been observed in athletes there is a risk for a drug–drug interaction that may influence the doping tests for AAS. In vitro studies show inhibitory potential on UGT2B7, 2B15, and 2B17 enzymes by NSAIDs. The aim of this study was to investigate if concomitant use of NSAIDs and a single dose of testosterone enanthate would affect the excretion rate of testosterone and epitestosterone glucuronide (TG and EG) as well as the T/E ratio, thereby affecting the outcome of the testosterone doping test. The study was designed as an open, randomized, cross-over study with subjects being their own control. The 23 male healthy volunteers, with either two, one or no allele (ins/ins, ins/del, or del/del) of the UGT2B17 gene, received the maximum recommended dose of NSAID (Ibuprofen or Diclofenac) for 6 days. On day three, 500 mg of testosterone enanthate was administered. Spot urine samples were collected for 17 days. After a wash-out period of 4 months the volunteers received 500 mg testosterone enanthate only, with subsequent spot urine collection for 14 days. The glucuronides of testosterone and epitestosterone were quantified. NSAIDs did not affect the excretion of TG or EG before the administration of testosterone. The concomitant use of NSAIDs and testosterone slightly increased the TG excretion while the EG excretion was less suppressed compared to testosterone use only. The effects of the NSAIDs on the TG and EG excretion did not differ between the UGT2B17 genotype groups. In conclusion, the outcome of testosterone doping tests does not seem to be affected by the use of NSAIDs.

UGT2B17 Genotype and the Pharmacokinetic Serum Profile of Testosterone during Substitution Therapy with Testosterone Undecanoate. A Retrospective Experience from 207 Men with Hypogonadism

Background: Testosterone (T) is mainly excreted in the urine as testosterone glucuronide (TG). This glucuronidation is partly dependent on the UGT2B17 genotype, and TG excretion is therefore lower in men having the UGT2B17 deletion. However, the possible influence of UGT2B17 genotype on serum T during androgen therapy is unknown. We retrospectively investigated the possible association between the UGT2B17 gene polymorphism and serum T levels in hypogonadal men during Testosterone undecanoate (TU) substitution therapy.Subjects and Methods: Two hundred and seven patients treated with TU (Nebido®) were genotyped by quantitative polymerase chain reaction for the UGT2B17 deletion polymorphism. All were given 1000 mg TU per injection at 0, 6, and 18 weeks. Blood samples were taken 2 and 6 weeks after the first and second injection, prior to the third injection, and after 2–3 years of treatment. We analyzed for the levels of T, luteinizing hormone (LH), sex-hormone-binding globulin, estradiol, prostate specific antigen, hematocrit, hemoglobin, and total cholesterol.Results: The UGT2B17 genotype frequency was: ins/ins: 42%, ins/del: 44%, and del/del: 14%. During the initial 18 weeks of TU treatment, large intra- and inter-individual variations in serum T levels were observed. Large peaks in T levels, ranging from 6.7 to 69.5 nmol/l, were noted 2 weeks after injections, regardless of the genotype. T levels did not differ between the three genotypes prior to the third injection, but the del/del group had significantly lower levels of LH. At follow-up after 2–3 years, the injection interval or daily T dosage was not dependent on the UGT2B17 genotype.Conclusion: In conclusion, we found large intra- and inter-individual variations in serum T during standard TU treatment regimen in hypogonadal men. Only subtle differences in serum T and LH were noted according to UGT2B17 genotype, which however suggest that the UGT2B17 genotype exert modest influence on the pharmacokinetic profile of T after TU treatment.

Effects of Dietary Components on Testosterone Metabolism via UDP-Glucuronosyltransferase

The potential interference in testosterone metabolism through ingested substances has ramifications for: (i) a range of pathologies such as prostate cancer, (ii) medication contra-indications, (iii) disruption to the endocrine system, and (iv) potential confounding effects on doping tests. Conjugation of anabolic steroids during phase II metabolism, mainly driven by UDP-glucuronosyltransferase (UGT) 2B7, 2B15, and 2B17, has been shown to be impaired in vitro by a range of compounds including xenobiotics and pharmaceuticals. Following early reports on the effects of a range of xenobiotics on UGT activity in vitro, the work was extended to reveal similar effects with common non-steroidal anti-inflammatory drugs. Notably, recent studies have evidenced inhibitory effects of the common foodstuffs green tea and red wine, along with their constituent flavonoids and catechins. This review amalgamates the existing evidence for the inhibitory effects of various pharmaceutical and dietary substances on the rate of UGT glucuronidation of testosterone; and evaluates the potential consequences for health linked to steroid levels, interaction with treatment drugs metabolized by the UGT enzyme and steroid abuse in sport.

Anti‐Dope Testing in Sport: The History and the Science

This summer, as the XXX modern Olympic Games and the XIV Summer Paralympic Games were held in London, millions watched magnificent efforts from the best athletes in the world. The ability of the athletes to produce their best personal performances in the spotlight and the pressure of the Olympic Games was a testament to the human spirit. But, because the margin between winning and losing is so small, one imprecisely placed foot or hand, one moment of indecision, one gust of wind at the wrong moment, and the gold medal is gone. Behind the scenes at these Olympics—properly so— was a scientific team performing under great pressure to ensure safe and fair competition by testing urine and blood for traces of performance-enhancing prohibited substances and methods. Their efforts were crucial, because, in partnership with the clean athletes, they support the integrity and the spirit of sport. More than 1000 samples were analyzed within a few days after each event for stimulants, steroids, masking agents, recombinant proteins like erythropoietin and growth hormone (GH), and other substances on the World AntiDoping Agency (WADA) Prohibited List (1). The List of Prohibited Substances and Methods is maintained by a committee of internationally recognized scientists and sport administrators. A revised list is released annually by the WADA. In deciding whether to add a compound or method to the list, the committee considers whether there is potential to enhance performance; a potential health risk for the athlete; or if use of the substance or method violates the spirit of sport. If two of the three criteria are met, the substance is added to the Prohibited List. The inclusion of the word “potential” recognizes that a substance may emerge for which there are no toxicological data. For example, the identification of the “designer” steroid tetrahydrogestrinone (THG) in a drop of fluid in a syringe turned in to the U.S. Anti-Doping Agency (USADA) in 2003 illustrates the problem. THG had been developed by Wyeth in the 1960s and taken to clinical trials as a potential anabolic agent, but never brought to market. It was impossible to locate any records from the clinical trials 40 years after the fact. THG had been shown to have anabolic activity (2). There were no publications about toxicological studies, but as a 17 -methyl steroid, comparison to similar structures strongly suggested the potential for some degree of hepatotoxicity. Based on this information, THG was added to the Prohibited List in 2006.

Red wine and component flavonoids inhibit UGT2B17 in vitro

BackgroundThe metabolism and excretion of the anabolic steroid testosterone occurs by glucuronidation to the conjugate testosterone glucuronide which is then excreted in urine. Alterations in UGT glucuronidation enzyme activity could alter the rate of testosterone excretion and thus its bioavailability. The aim of this study is to investigate if red wine, a common dietary substance, has an inhibitory effect on UGT2B17.MethodsTestosterone glucuronidation was assayed using human UGT2B17 supersomes with quantification of unglucuronidated testosterone over time using HPLC with DAD detection. The selected red wine was analyzed using HPLC; and the inhibitory effects of the wine and phenolic components were tested independently in a screening assay. Further analyses were conducted for the strongest inhibitors at physiologically relevant concentrations. Control experiments were conducted to determine the effects of the ethanol on UGT2B17.ResultsOver the concentration range of 2 to 8%, the red wine sample inhibited the glucuronidation of testosterone by up to 70% over 2 hours. The ethanol content had no significant effect. Three red wine phenolics, identified by HPLC analyses, also inhibited the enzyme by varying amounts in the order of quercetin (72%), caffeic acid (22%) and gallic acid (9%); using a ratio of phenolic:testosterone of 1:2.5. In contrast p-coumaric acid and chlorogenic acid had no effect on the UGT2B17. The most active phenolic was selected for a detailed study at physiologically relevant concentrations, and quercetin maintained inhibitory activity of 20% at 2 μM despite a ten-fold excess of testosterone.ConclusionThis study reports that in an in vitro supersome-based assay, the key steroid-metabolizing enzyme UGT2B17 is inhibited by a number of phenolic dietary substances and therefore may reduce the rate of testosterone glucuronidation in vivo. These results highlight the potential interactions of a number of common dietary compounds on testosterone metabolism. Considering the variety of foodstuffs that contain flavonoids, it is feasible that diet can elevate levels of circulating testosterone through reduction in urinary excretion. These results warrant further investigation and extension to a human trial to delineate the health implications.

UDP‐glucuronosyltransferase 2B17 genotyping in Japanese athletes and evaluation of the current sports drug testing for detecting testosterone misuse

Ethnicity has been found to influence urinary testosterone glucuronide to epitestosterone glucuronide (T/E) ratios among athletes. Uridine diphospho-glucuronosyltransferase 2B17 (UGT2B17) is the most active enzyme in testosterone glucuronidation. UGT2B17 polymorphism analysis is rarely performed in Japanese athletes, and the influence of testosterone administration on steroid profiles and carbon isotope ratios, according to gene polymorphisms, in Asians remains unknown. The prevalence of UGT2B17 genotypes and urinary androgenic steroid profiles, classified according to UGT2B17 genotypes, was investigated in Japanese athletes (255 male and 256 female). Testosterone enanthate (100 mg) was administered intramuscularly to Japanese female volunteers (del/del: n = 6, del/ins: n = 3, ins/ins: n = 1). The distribution rates of the UGT2B17 del/del genotype in Japanese male and female athletes were 74.5% and 60.2%, respectively. The ins/ins genotype was detected in only three male (1.2%) and seven female (2.7%) athletes. The prevalence of the UGT2B17 deletion genotype was extremely high in Japanese athletes. The T/E ratio in the del/del group was significantly lower than that in the other groups. After testosterone was administered to female volunteers, the T/E ratios for the del/del individuals failed to reach the positivity criterion of 4. By contrast, in all of the del/del subjects, the gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS) analysis successfully fulfilled the positivity criterion. The overall result has demonstrated the limited effectiveness of population-based T/E ratios in screening tests for testosterone use. Subject-based steroid profiling with UGT2B17 genotyping will be an effective strategy for detecting testosterone misuse.

A novel stacking method of repetitive large volume sample injection and sweeping MEKC for determination of androgenic steroids in urine

In this research, a novel stacking capillary electrophoresis method, repetitive large volume sample injection and sweeping MEKC (rLVSI-sweeping MEKC) were developed to analyze the presence of three androgenic steroids considered as sport doping drugs, testosterone (T), epitestosterone (E) and epitestosterone glucuronide (EG) in urine. This method provides better sensitivity enhancement than the traditional large volume sample stacking-sweeping strategies due to sensitivity enhancement by repetitive injections. This multiple sampling method enhances sensitivity of monitoring of urine samples by UV detection (254nm). Firstly, the phosphate buffer was filled into an uncoated fused silica capillary and the samples were injected into the capillary at 10psi for 20s, and then stacked at −10kV for 1min using phosphate buffer containing SDS. The above injecting and stacking steps were repeated five times. Finally, separation was performed at −20kV, using phosphate buffer containing methanol, SDS and (2-hydroxypropyl)-β-cyclodextrin. Method validation showed that calibration plots were linear (r≧0.997) over a range of 5–200ngmL−1 for T, 20–200ngmL−1 for E and 0.5–500ngmL−1 for EG. The limits of detection were 1.0ngmL−1 for T, 5.0ngmL−1 for E and 200.0pgmL−1 for EG. When evaluating precision and accuracy, values of RSD and RE in intra-day (n=3) and inter-day (n=5) analysis were found to be less than 10.0%. Compared with the simple LVSS-sweeping, which is also a stacking strategy, this method further improves sensitivity up to 25 folds (∼2500 folds with MEKC without preconcentration). This method was applied to monitor 10 athletes’ urine, and did not detect any analyte. The novel stacking method was feasible for monitoring of doping by sportsmen.

Androgens and doping tests: genetic variation and pit‐falls

The large variation in disposition known for most drugs is also true for anabolic androgenic steroids. Genetic factors are probably the single most important cause of this variation. Further, there are reasons to believe that there is a corresponding variation in efficacy of doping agents. Doped individuals employ a large variety of doping strategies in respect of choice of substance, dose, dose interval, duration of treatment and use of other drugs for enforcement of effects or correction of side effects. Metabolic steps up-stream and down-stream of testosterone are genetically variable and contribute substantially to the variation in disposition of testosterone, the most common doping agent in sports and in society. Large inter- and intra-ethnic variation in testosterone glucuronidation and excretion is described as well as the pit-falls in evaluation of testosterone doping test results. The hydrolysis and bioactivation of testosterone enanthate is also genetically variable yielding a 2-3 fold variation in excretion rate and serum concentration, thereby implicating a substantial variation in 'efficacy' of testosterone. Given this situation it is logical to adopt the new findings in the doping control programme. The population based cut-off level for the testosterone : epitestosterone ratio should be replaced by a Bayesian interpretation of consecutive tests in the same individual. When combined with the above genetic information the sensitivity of the test is considerably improved. The combination of the three approaches should reduce the rate of falsely negative or positive results and the number of expensive follow-up tests, stipulated by the World Anti-Doping Agency.

Doping and Genetic Testing: Sex Difference in UGT2B15 Expression, Testosterone Glucuronidation Activity and Urinary Testosterone/ Epitestosterone Glucuronide Ratio

Doping with testosterone is conventionally detected by urinary assay of the testosterone/epitestosterone (T/E) glucuronide ratio. This ratio is strongly associated with a deletion polymorphism in the UDP-glucuronosyltransferase (UGT) 2B17 gene. The metabolism of certain UGT specific drugs has been shown to be different in men and women. The objective of this study was to evaluate the impact of gender as well as human genetic variation on testosterone metabolism and the T/E ratio. We also carried out legal and policy analysis of the extant regulatory documents with a view to assess their preparedness for the introduction of genetic testing to the field of doping, and interpretation of doping related tests in the future. Testosterone glucuronidation rate was measured in female and male liver microsomes and analyzed with HPLC. UGT2B15 mRNA was measured in human liver samples using real-time PCR. Elite athletes participating in different endurance sports were genotyped for the UGT2B17 deletion polymorphism and compared with their urinary T/E ratio. The testosterone glucuronidation rate was 1.7 times higher in male than in female liver microsomes (p <0.05). Male del/del livers had significantly higher expression of UGT2B15 compared to female del/del livers (p <0.05). Men, but not women, seem to compensate for their lack of UGT2B17 activity with increased UGT2B15 expression. The T/E ratios in males, but not in females, are strongly associated with the UGT2B17 ins/ins and ins/del genotype (p <0.05).The results presented herein directly inform the ways in which doping tests may have to be designed and interpreted in the future so as to take into account host (athletes’) individual characteristics and gene-by-gender interactions. Additionally we suggest that the current legal and policy frameworks are in need of substantive amendment if the field of doping testing is to benefit from advances in genomics technologies and personalized medicine. Keywords: Androgens, doping and genetic testing convergence, sex difference, legal and technology policy analysis, sports medicine, T/E ratio, testosterone, UGT2B17

Dietary green and white teas suppress UDP-glucuronosyltransferase UGT2B17 mediated testosterone glucuronidation

The anabolic steroid testosterone can be used by athletes to enhance athletic performance and muscle growth. UDP-glucuronosyltransferase (UGT2B17) is the key enzyme involved in the glucuronidation of testosterone to testosterone glucuronide, which also serves as a marker for the testosterone/epitestosterone (T/E) ratio used to detect testosterone abuse in sport. Inhibitors of testosterone glucuronidation could have an impact on circulating testosterone levels, thus aiding performance, as well as potentially affecting the urinary T/E ratio and therefore masking testosterone abuse. Previous reports have revealed that non-steroidal, anti-inflammatory drugs, diclofenac and ibuprofen, inhibit the UGT2B17 enzyme. The aim of this study is to analyse dietary tea samples for inhibition of testosterone glucuronidation and, where inhibition is present, to identify the active compounds. Analysis of testosterone glucuronidation was conducted by performing UGT2B17 assays with detection of un-glucuronidated testosterone using high performance liquid chromatography. The results from this study showed that testosterone glucuronidation was inhibited by the green and white tea extracts, along with specific catechin compounds, notably: epicatechin, epigallocatechin gallate (EGCG) and catechin gallate. The IC50 inhibition value for EGCG was determined, using a Dixon plot, to be 64μM, equalling the most active NSAID inhibitor diclofenac. Thus, common foodstuffs and their constituents, for the first time, have been identified as inhibitors of a key enzyme involved in testosterone glucuronidation. Whilst these common compounds are not substrates of the UGT2B17 enzyme, we showed that they inhibit testosterone glucuronidation which may have implications on current doping control in sport.

Concerns About Serum Androgens Monitoring During Testosterone Replacement Treatments in Hypogonadal Male Athletes: A Pilot Study

A well-tailored testosterone replacement treatment (TRT) in male hypogonadal athletes plays a pivotal role to restore physiological performances, to reduce health risks, and to guarantee the ethic of competition. Few studies evaluated individual androgens profiles during TRT in trained individuals. The aim of this article was to verify the efficacy in restoring eugonadal serum and urinary androgens profiles after testosterone enanthate (TE) and gel (TG) administration. Ten male Caucasian-trained volunteers affected by severe hypotestosteronemia (<8 nmol/L) were included. Serum androgens and urinary testosterone metabolites were evaluated, in the same subjects, before and weekly for 5 weeks after both a single intramuscular TE injection (250 mg) and during a daily administration of TG (50 mg/die of testosterone), respectively. The main outcome measures of this article were serum total testosterone (TT), dihydrotestosterone (DHT), calculated free and bioavailable testosterone (cFT, cBioT), 17-β-estradiol, and urinary glucuronide testosterone metabolites. Supraphysiological TT concentrations were observed in 50% of our volunteers until 7 days after TE and in the 4% of total samples after TG. Serum DHT was high both after TE (all volunteers on day 7 and 50% on day 14) and during TG (32% of total samples). A relatively low number of samples showed normal cFT and cBioT both after TE and TG (20-44%, respectively). Urinary metabolites were related to the type of treatment and to serum androgens profile and resulted in the normal ranges from 15% to 60% of total samples. Besides well-known variations of mean serum TT, we showed a high percentage of serum and urinary samples with abnormal androgens, being TG safer than TE. We conclude that monitoring TRT with TT only may be inaccurate because of abnormal fluctuations of other circulating androgens. Further studies to identify the appropriate markers of eugonadism during TRT are highly warranted both in athletes and in non-athletes.

Glucuronide directed molecularly imprinted solid-phase extraction: isolation of testosterone glucuronide from its parent drug in urine

Two molecularly imprinted polymers (MIPs) that we recently described to be class-selective for glucuronides have been successfully exploited for the molecularly imprinted solid-phase extraction (MISPE) of testosterone glucuronide (TG) from its parent drug (T) in urine. Both sorbents targeted the glucuronate fragment but feature different functional groups for binding the carboxylate anion, MIP1, a neutral 1,3-diarylurea group, and MIP2, a cationic imidazolium functionality. MISPE-HPLC-UV methods developed using both sorbents allowed the extraction of TG from its parent compound in urine samples spiked at 150, 300 or 600 ng mL(-1) for TG and at 50 ng mL(-1) for T. By comparing the performance of the two sorbents it came out that MIP1 is a more suitable SPE packing than MIP2, since it isolated the glucuronide with a higher precision (RSD 2-5%, n = 3) and with an enhanced enrichment factor (EF = 4.2). On the basis of these results, the imprinted receptor MIP1 can be applied for the direct extraction of TG in doping and clinical analysis and to selectively capture any other relevant glucuronated metabolite avoiding tedious deconjugation steps prior to quantification.

Androgen Sulfation in Healthy UDP-Glucuronosyl Transferase 2B17 Enzyme-Deficient Men

The conspicuous interindividual differences in metabolism and urinary excretion of testosterone and its metabolites make it challenging to reveal testosterone doping. The variation in testosterone glucuronide excretion is strongly associated with a deletion polymorphism in the uridine diphosphate-glucuronosyltranferase (UGT) 2B17 gene. The objective of the study was to identify additional biomarkers to detect testosterone abuse and to elucidate alternative pathways for testosterone elimination in individuals devoid of the UGT2B17 enzyme. For this purpose a new ultraperformance liquid chromatographic tandem mass spectrometric method for simultaneous determination of 10 different sulfo- and glucuronide-conjugated steroids was developed. Fifty-four healthy male volunteers with two, one, or no allele (ins/ins, ins/del, or del/del) of the UGT2B17 gene participated in the study. Intervention included a single im dose of 500 mg testosterone enanthate. Urinary sulfo- and glucuronide-conjugated steroids were measured. Testosterone sulfate levels decreased in all individuals after the dose. The individual differences in the excretion of all sulfated metabolites were large. Thus, these metabolites will not serve as appropriate biomarkers for testosterone abuse. However, androsterone glucuronide excretion increased in all of our study subjects after the testosterone dose. Etiocholanolone sulfate was excreted at significantly higher levels in UGT2B17 del/del individuals. We propose that the androsterone glucuronide to epitestosterone glucuronide ratio may serve as a complementary biomarker to reveal testosterone abuse.

Determination of testosterone and epitestosterone glucuronides in urine by ultra performance liquid chromatography-ion mobility-mass spectrometry

UPLC-ion mobility spectrometry separations combined with mass spectrometry (UPLC-IM-MS) and tandem mass spectrometry (UPLC-IM-MS/MS) have been investigated for the simultaneous determination of testosterone and epitestosterone glucuronides in urine. The glucuronide epimers of testosterone and epitestosterone were separated by ion mobility spectrometry prior to mass analysis on the basis of differences in their collision cross sections, which have been measured in nitrogen. Combining ion mobility separation with UPLC/MS enhances the analysis of these low-abundance steroids in urine by selective interrogation of specific retention time, mass-to-charge and mobility regions. Detection limits for the UPLC-IM-MS/MS analysis of TG and ETG were 9.9 ng mL(-1) and 98 ng mL(-1) respectively, equivalent to 0.7 ng mL(-1) and 7.4 ng mL(-1) in urine, with linear dynamic ranges corresponding to 0.7-108 ng mL(-1) and 7.4-147 ng mL(-1) in urine. Repeatability (%RSD) for urine extracts was 0.64% and 2.31% for TG and ETG respectively.