Epitestosterone Glucuronide Research Articles

Human efflux transport of testosterone, epitestosterone and other androgen glucuronides

Several drug-metabolizing enzymes are known to control androgen homeostasis in humans. UDP-glucuronosyltransferases convert androgens to glucuronide conjugates in the liver and intestine, which enables subsequent elimination of these conjugated androgens via urine. The most important androgen is testosterone, while others are the testosterone metabolites androsterone and etiocholanolone, and the testosterone precursor dehydroepiandrosterone. Epitestosterone is another endogenous androgen, which is included as a crucial marker in urine doping tests. Since glucuronide conjugates are hydrophilic, efflux transporters mediate their excretion from tissues. In this study, we employed the membrane vesicle assay to identify the efflux transporters for glucuronides of androsterone, dehydroepiandrosterone, epitestosterone, etiocholanolone and testosterone. The human hepatic and intestinal transporters MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), BCRP (ABCG2) and MDR1 (ABCB1) were studied in vitro. Of these transporters, only MRP2 and MRP3 transported the androgen glucuronides investigated. In kinetic analyses, MRP3 transported glucuronides of androsterone, epitestosterone and etiocholanolone at low Km values, between 0.4 and 4 μM, while the Km values for glucuronides of testosterone and dehydroepiandrosterone were 14 and 51 μM, respectively. MRP2 transported the glucuronides at lower affinity, as indicated by Km values over 100 μM. Interestingly, the MRP2-mediated transport of androsterone and epitestosterone glucuronides was best described by sigmoidal kinetics. The inability of BCRP to transport any of the androgen glucuronides investigated is drastically different from its highly active transport of several estrogen conjugates. Our results explain the transporter-mediated disposition of androgen glucuronides in humans, and shed light on differences between the human efflux transporters MRP2, MRP3, MRP4, BCRP and MDR1.

Effects of transdermal administration of testosterone gel on the urinary steroid profile in hypogonadal men: Implications in antidoping analysis

Testosterone is one of the most abused pseudo-endogenous anabolic steroids in sport doping. The current method adopted to detect the abuse of testosterone and other pseudo-endogenous steroids (endogenous steroids when administered exogenously) is first based on the longitudinal monitoring of several urinary biomarkers, which constitute the so called “steroidal module” of the Athlete Biological Passport (ABP): atypical samples undergo a confirmation analysis based on the measurement of the 13C/12C isotopic ratio of selected target compounds, to distinguish their endogenous or exogenous origin. At the same time, testosterone administration can be allowed in athletes diagnosed with hypogonadism, provided they are granted a therapeutic use exemption by the relevant medical authority.In this pilot study we have investigated whether the approach based on the preliminary determination of the urinary steroid profile, in the format considered in the steroidal module of the ABP, also integrated with the inclusion of the sulfo-conjugates and of additional target steroids, can retain its validity also in the case of hypogonadal athletes. We have studied the effects of a single low dose (40 mg) of testosterone gel (T-gel) on the urinary concentration of the markers of steroidal module of the ABP, as well as on some additional steroid markers. The study was based on the analysis of urinary samples from 19 non-hospitalized hypogonadal men, 10 of them with late-onset hypogonadism (LOH), collected before, after 4 h and after 24 h the transdermal self-administration of 40 mg of T-gel. None of the patient had any co-morbidities possibly affecting the urinary excretion of the steroidal markers. The steroidal markers were quantified by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) after the enzymatic hydrolysis of the respective glucuro-conjugates and the chemical hydrolysis of the respective sulfo-conjugates. Targeted GC-MS/MS analysis was carried out operating in electron impact (EI) ionization mode, with acquisition in multiple reaction monitoring (MRM) mode.Our preliminary results show that, as expected, the treatment with T-gel leads, in all hypogonadal men, to an increase of the urinary concentration of the glucuro-conjugate metabolites of testosterone and its main metabolites, with special relevance to those with 5α-reduction. Furthermore, samples collected from non-LOH hypogonadal men showed an increase also in the levels of epitestosterone glucuronide, testosterone sulfate and epitestosterone sulfate. Apart from their biochemical and pharmacological relevance, these outcomes could be leveraged to refine the analytical strategy currently followed in the antidoping field for the analysis of the urinary steroidal markers, with potential implications also in other forensic and/or clinical investigations.

Codeine influences the serum and urinary profile of endogenous androgens but does not interact with the excretion rate of administered testosterone.

Today's doping tests involve longitudinal monitoring of urinary steroids including the testosterone glucuronide and epitestosterone glucuronide ratio (T/E) in an Athlete Biological Passport (ABP). The aim of this study was to investigate the possible influence of short-term use of codeine on the urinary excretion of androgen metabolites included in the steroidal module of the passport prior to and after the co-administration with testosterone. The study was designed as an open study with the subjects being their own control. Fifteen healthy male volunteers received therapeutic doses of codeine (Kodein Meda) for 6 days. On Day 3, 500 mg or 125mg of testosterone enanthate (Testoviron®-Depot) was administered. Spot urine samples were collected for 17 days, and blood samples were collected at baseline, 3, 6, and 14days after codeine intake. The circulatory concentration of total testosterone decreased significantly by 20% after 3days' use of codeine (p=0.0002) and an atypical ABP result was noted in one of the subjects. On the other hand, the concomitant use of codeine and testosterone did not affect the elevated urinary T/E ratio. In 75% of the individuals, the concentration of urinary morphine (a metabolite of codeine) was above the decision limit for morphine. One of the participants displayed a morphine/codeine ratio of 1.7 after codeine treatment, indicative of morphine abuse. In conclusion, our study shows that codeine interferes with the endogenous testosterone concentration. As a result, the urinary steroid profile may lead to atypical findings in the doping test.

Dose-dependent testosterone sensitivity of the steroidal passport and GC-C-IRMS analysis in relation to the UGT2B17 deletion polymorphism.

The newly implemented Steroid Module of the Athlete Biological Passport has improved doping tests for steroids. A biomarker included in this passport is the urinary testosterone glucuronide to epitestosterone glucuronide (T/E) ratio, a ratio greatly affected by a deletion polymorphism in UGT2B17. Suspect urine doping tests are further analyzed with gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to determine the origin of the androgen. In this study, we investigated the sensitivity of the steroidal module and the IRMS analysis, in subjects administered with three doses of testosterone enanthate (500, 250, and 125 mg), in relation to the UGT2B17 polymorphism. All subjects carrying the UGT2B17 enzyme reached the traditionally used threshold, a T/E ratio of 4, after all three administered doses, whereas none of the subjects devoid of this enzyme reached a T/E of 4. On the other hand, using the athlete biological passport and IRMS analysis, all three doses could be detected to a high degree of sensitivity. The concentrations of all steroids included in the steroidal module were dose dependently increased, except for epitestosterone which decreased independent of dose. The decrease in epitestosterone was significantly associated with circulatory levels of testosterone post dose (rs =0.60 and p=0.007). In conclusion, these results demonstrate that administration of a single dose of 125-500 mg testosterone enanthate could be detected using the athlete biological passport, together with IRMS. Since IRMS is sensitive to testosterone doping independent of UGT2B17 genotype, also very small changes in the steroidal passport should be investigated with IRMS.

Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

The screening of testosterone (T) misuse for doping control is based on the urinary steroid profile, including T, its precursors and metabolites. Modifications of individual levels and ratio between those metabolites are indicators of T misuse. In the context of screening analysis, the most discriminant criterion known to date is based on the T glucuronide (TG) to epitestosterone glucuronide (EG) ratio (TG/EG). Following the World Anti-Doping Agency (WADA) recommendations, there is suspicion of T misuse when the ratio reaches 4 or beyond. While this marker remains very sensitive and specific, it suffers from large inter-individual variability, with important influence of enzyme polymorphisms. Moreover, use of low dose or topical administration forms makes the screening of endogenous steroids difficult while the detection window no longer suits the doping habit. As reference limits are estimated on the basis of population studies, which encompass inter-individual and inter-ethnic variability, new strategies including individual threshold monitoring and alternative biomarkers were proposed to detect T misuse.The purpose of this study was to evaluate the potential of ultra-high pressure liquid chromatography (UHPLC) coupled with a new generation high resolution quadrupole time-of-flight mass spectrometer (QTOF-MS) to investigate the steroid metabolism after transdermal and oral T administration. An approach was developed to quantify 12 targeted urinary steroids as direct glucuro- and sulfo-conjugated metabolites, allowing the conservation of the phase II metabolism information, reflecting genetic and environmental influences. The UHPLC–QTOF-MSE platform was applied to clinical study samples from 19 healthy male volunteers, having different genotypes for the UGT2B17 enzyme responsible for the glucuroconjugation of T. Based on reference population ranges, none of the traditional markers of T misuse could detect doping after topical administration of T, while the detection window was short after oral TU ingestion. The detection ability of the 12 targeted steroids was thus evaluated by using individual thresholds following both transdermal and oral administration. Other relevant biomarkers and minor metabolites were studied for complementary information to the steroid profile, including sulfoconjugated analytes and hydroxy forms of glucuroconjugated metabolites. While sulfoconjugated steroids may provide helpful screening information for individuals with homozygotous UGT2B17 deletion, hydroxy-glucuroconjugated analytes could enhance the detection window of oral T undecanoate (TU) doping.

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.

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.

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.

Non-steroidal anti-inflammatory drugs interact with testosterone glucuronidation

Testosterone and epitestosterone are secreted mainly as glucuronide metabolites and the urinary ratio of testosterone glucuronide to epitestosterone glucuronide, often called T/E, serves as a marker for possible anabolic steroids abuse by athletes. UDP-glucuronosyltransferase (UGT) 2B17 is the most important catalyst of testosterone glucuronidation. The T/E might be affected by drugs that interact with UGT2B17, or other enzymes that contribute to testosterone glucuronidation. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used by sportsmen and we have examined the effect of two NSAIDs, diclofenac and ibuprofen, on testosterone and epitestosterone glucuronidation in human liver microsomes. In parallel, we have studied the inhibitory effect of these NSAIDs on recombinant UGT2B17 and UGT2B15, as well as other human hepatic UGTs that revealed low but detectable testosterone glucuronidation activity, namely UGT1A3, UGT1A4, UGT1A9 and UGT2B7. Both diclofenac and ibuprofen inhibited testosterone glucuronidation in microsomes, as well as UGT2B15 and UGT2B17. Interestingly, UGT2B15 was more sensitive than UGT2B17 to the two drugs, particularly to ibuprofen. Human liver microsomes lacking functional UGT2B17 exhibited significantly higher sensitivity to ibuprofen, suggesting that UGT2B15 plays a major role in the residual testosterone glucuronidation activity in UGT2B17-deficient individuals. Nonetheless, a minor contribution of other UGTs, particularly UGT1A9, to testosterone glucuronidation in such individuals cannot be ruled out at this stage. The epitestosterone glucuronidation activity of human liver microsomes was largely insensitive to ibuprofen and diclofenac. Taken together, the results highlight potential interactions between NSAIDs and androgen glucuronidation with possible implications for the validity of doping tests.

Determination of sulfates and glucuronides of endogenic steroids in biofluids by high-performance liquid chromatography/orbitrap mass spectrometry

the possibility of selective determination of testosterone and epitestosterone glucuronides in urine by high-performance liquid chromatography/high-resolution mass spectrometry using solid phase microextraction on a meps cartridge was studied. the effect of the biological matrix on the spectra of conjugated steroids can be taken into account by using the spectra of conjugates recorded for urine samples after hydrolysis as reference spectra. the conditions of fragmentation in the ion source were optimized for separate analytes. this method was used for analyzing real samples with different testosterone/epitestosterone ratios. variations in conjugate contents and qualitative changes in the steroid profile of endogenic compounds were observed.

Evaluation of equine urine reactivity towards phase II metabolites of 17‐hydroxy steroids by liquid chromatography/tandem mass spectrometry

Proper storage conditions of biological samples are fundamental to avoid microbiological contamination that can cause chemical modifications of the target analytes. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method through direct injection of diluted samples, without prior extraction, was used to evaluate the stability of phase II metabolites of boldenone and testosterone (glucuronides and sulphates) in intentionally poorly stored equine urine samples. We also considered the stability of some deuterated conjugated steroids generally used as internal standards, such as deuterated testosterone and epitestosterone glucuronides, and deuterated boldenone and testosterone sulphates. The urines were kept for 1 day at room temperature, to mimic poor storage conditions, then spiked with the above steroids and kept at different temperatures (-18 degrees C, 4 degrees C, room temperature). It has been possible to confirm the instability of glucuronide compounds when added to poorly stored equine urine samples. In particular, both 17beta- and 17alpha-glucuronide steroids were exposed to hydrolysis leading to non-conjugated steroids. Only 17beta-hydroxy steroids were exposed to oxidation to their keto derivatives whereas the 17alpha-hydroxy steroids were highly stable. The sulphate compounds were completely stable. The deuterated compounds underwent the same behaviour as the unlabelled compounds. The transformations were observed in urine samples kept at room temperature and at a temperature of 4 degrees C (at a slower rate). No modifications were observed in frozen urine samples. In the light of the latter results, the immediate freezing at -18 degrees C of the collected samples and their instant analysis after thawing is the proposed procedure for preventing the transformations that occur in urine, usually due to microbiological contamination.

Genetic Variations in UDP-Glucuronosyl Transferase 2B17: Implications for Testosterone Excretion Profiling and Doping Control Programs

The use of drugs and ergogenic substances to augment athletic performance, commonly referred to as doping, has evolved along with sporting events. Ancient Olympic athletes consumed mushrooms, plants, and herbs in an attempt to gain a competitive edge. The modern Olympic Games made their debut in 1896, and mixtures of cocaine, ephedrine, and strychnine were used to enhance performance. Anabolic androgenic steroids (substances similar to the hormone testosterone) were used after World War II by Soviet athletes to increase muscle mass and power in weightlifting and bodybuilding events. Anabolic androgenic steroids rapidly spread to athletes in other sporting events and are still a problem in today’s sports world. To deal with the problem of doping in sports, the International Olympic Committee established a Medical Commission. The first list of prohibited substances was created in 1967; drug testing was implemented at Olympic Games the following year. In 1999, an independent international organization, the World Anti-Doping Agency (WADA), was created to combat doping in sports and provide unified standards for doping control. Anabolic androgenic steroids are the most abused class of prohibited substances, with testosterone accounting for many positive cases. Testosterone abuse is problematic because synthetic testosterone is indistinguishable from endogenous testosterone by routine screening methods such as gas chromatography–mass spectrometry. In the 1980s, it was discovered that testosterone use alters the ratio of testosterone glucuronide to epitestosterone glucuronide (T/E ratio)1 in urine. Epitestosterone is a naturally occurring biologically inactive epimer of testosterone that remains relatively constant in urine. A population-based T/E ratio cutoff of 6.0 was initially used to indicate synthetic testosterone use; the T/E ratio cutoff was lowered to 4.0 in 2005. Based on data from several laboratories, the average T/E ratio ranges from 0.9 to 1.6 for healthy male adolescents and men. At the UCLA Olympic Analytical Laboratory, …

The detection of androstenedione abuse in sport: a mass spectrometry strategy to identify the 4‐hydroxyandrostenedione metabolite

Studies have shown that the administration of androstenedione (ADIONE) significantly increases the urinary ratio of testosterone glucuronide to epitestosterone glucuronide (T/E) - measured by gas chromatography/mass spectrometry (GC/MS) - in subjects with a normal ( approximately 1) or naturally high (>1) initial values. However, the urinary T/E ratio has been shown not to increase in subjects with naturally low (<1) initial values. Such cases then rely on the detection of C(6)-hydroxylated metabolites shown to be indicative of ADIONE administration. While these markers may be measured in the routine GC/MS steroid profile, their relatively low urinary excretion limits the use of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to specifically confirm ADIONE administration based on depleted (13)C content. A mass spectrometry strategy was used in this study to identify metabolites of ADIONE with the potential to provide compound-specific detection. C(4)-hydroxylation was subsequently shown to be a major metabolic pathway following ADIONE administration, thereby resulting in urinary excretion of 4-hydroxyandrostenedione (4OH-ADIONE). Complementary analysis of 4OH-ADIONE by GC/MS and GC/C/IRMS was used to confirm ADIONE administration.

UDP-Glucuronosyltransferases (UGTs) 2B7 and UGT2B17 Display Converse Specificity in Testosterone and Epitestosterone Glucuronidation, whereas UGT2A1 Conjugates Both Androgens Similarly

Testosterone and epitestosterone are endogenous steroids that differ in the configuration of the hydroxyl-bearing carbon at C-17. Testosterone is the predominant male sex hormone, whereas the role of epitestosterone is largely unclear. In humans, both androgens are excreted mainly as glucuronide conjugates and the urinary ratio of testosterone to epitestosterone (T/E), used to expose illicit testosterone abuse by male athletes, indicates the relative concentrations of the respective glucuronides. Some male athletes have T/E values greater than the accepted threshold value (4.0), even without testosterone abuse. We have analyzed athletes' urine samples and found that the main reason for such false-positive results in doping tests was a low epitestosterone glucuronide concentration not a high level of testosterone glucuronide. Sulfate conjugates of both testosterone and epitestosterone were also detected in the different urine samples. Glucuronidation assays with the 19 human UDP-glucuronosyltransferases (UGTs) of subfamilies UGT1A, UGT2A, and UGT2B revealed that UGT2B17 is the most active enzyme in testosterone glucuronidation. UGT2B17 does not glucuronidate epitestosterone, but inhibition studies revealed that it binds epitestosterone with affinity similar to that of testosterone. Epitestosterone glucuronidation is catalyzed mainly by UGT2B7, and the K(m) of this reaction is significantly lower than the K(m) of UGT2B17 for testosterone. Although UGT2B7 and UGT2B17 exhibited high, although converse, stereoselectivity in testosterone and epitestosterone glucuronidation, UGT2A1, an extrahepatic enzyme that is expressed mainly in the nasal epithelium, catalyzed the glucuronidation of both steroids at considerable rates and similar kinetics. The results shed new light on the substrate specificity and stereoselectivity of human UGTs.

Genetic aspects of epitestosterone formation and androgen disposition: influence of polymorphisms in CYP17 and UGT2B enzymes

Testosterone is a commonly abused androgen in sports and in the gym culture of the society. Its abuse is conventionally disclosed by urinary assay of the testosterone/epitestosterone (T/E) glucuronide ratio, which should not exceed 4. A noteworthy number of athletes, however, have higher natural ratios than 4, most likely because of decreased excretion of epitestosterone glucuronide. Falsely positive doping test results are of great concern for the legal rights of the sportsman. Our objective was to study the genetic aspects of epitestosterone formation, and to elucidate the impact of genetic variation in androgen-metabolizing enzymes. Urine from different study populations was analysed for androgen glucuronides by gas chromatography-mass spectrometry. All men were genotyped for the uridine diphospho-glucuronosyltransferase (UGT) 2B17 deletion polymorphism and single nucleotide polymorphisms in the cytochrome P-450c17alpha (CYP17), UGT2B15 and UGT2B7 genes. Expression of UGT2B15 mRNA in human liver samples was analysed using real-time PCR. A T>C (A1>A2) promoter polymorphism in the CYP17 gene was associated with the urinary glucuronide levels of epitestosterone and its putative precursor androstene-3beta, 17alpha-diol, resulting in 64% higher T/E ratios in A1/A1 homozygotes. Individuals devoid of UGT2B17 had significantly higher UGT2B15 mRNA levels in liver than individuals carrying two functional UGT2B17 alleles. The CYP17 promoter polymorphism may partly explain high natural (>4) T/E ratios. Our data indicate that 5-androstene-3beta, 17alpha-diol is an important precursor of epitestosterone and that CYP17 is involved in its production. In addition, we found that lack of the UGT2B17 enzyme may be compensated for by increase in UGT2B15 transcription.

Ion trap MS/MS of intact testosterone and epitestosterone conjugates—Adducts, fragile ions and the advantages of derivatisation

In ion trap mass spectrometry, fragile ions may fragment under the application of resonance ejection during precursor mass isolation, reducing MS/MS spectral intensity. In this study the steroidal epimers testosterone glucuronide (TG) and epitestosterone glucuronide (EG) have been chosen as a model for exploring whether compound structure is linked to ion trap fragility. Both compounds form multiple adducts by ESI-MS, namely protonation, ammonium and sodium, however, the mass spectrum of EG displays a more intense ammonium adduct peak than TG. [TG + NH 4] +, [EG + NH 4] + and [EG + H] + were found to be fragile ions. To explain the differences in adduct formation and fragility, molecular modelling was employed. Ammonium adduction was localised to the glucuronide ring oxygens and while EG has eight possible adduction sites, only seven were located for TG explaining the increased ammonium adduct abundance with EG. In EG the bond between the steroid and the glucuronide was slightly longer and the oxygen in this bond was more basic than TG. This shows that the EG bond is weaker which may contribute to the fact that [EG + H] + but not [TG + H] + is fragile. To investigate whether stability could be restored by chemical means, EG was derivatised with tris(trimethoxyphenyl)phosphonium chloride or methylated on the carboxylic acid and Girard P or methoxylamine on the 3-keto group. Derivatisation of the steroid rather than the glucuronide eliminated fragility and using a charged derivative eliminated adduct formation. This work demonstrates the importance of carefully considering the nature of the derivative and the site of derivatisation.

Short-term stability of testosterone and epitestosterone conjugates in urine samples: Quantification by liquid chromatography–linear ion trap mass spectrometry

A simple method using liquid chromatography–linear ion trap mass spectrometry for simultaneous determination of testosterone glucuronide (TG), testosterone sulfate (TS), epitestosterone glucuronide (EG) and epitestosterone sulfate (ES) in urine samples was developed. For validation purposes, a urine containing no detectable amount of TG, TS and EG was selected and fortified with steroid conjugate standards. Quantification was performed using deuterated testosterone conjugates to correct for ion suppression/enhancement during ESI. Assay validation was performed in terms of lower limit of detection (1–3 ng/mL), recovery (89–101%), intraday precision (2.0–6.8%), interday precision (3.4–9.6%) and accuracy (101–103%). Application of the method to short-term stability testing of urine samples at temperature ranging from 4 to 37 °C during a time-storage of a week lead to the conclusion that addition of sodium azide (10 mg/mL) is required for preservation of the analytes.

Stability studies of testosterone and epitestosterone glucuronides in urine

The stability of testosterone glucuronide (TG), epitestosterone glucuronide (EG) and the T/E ratio in urine has been studied. Samples were analyzed by gas chromatography coupled to mass spectrometry (GC/MS). Urine samples were submitted to a solid-liquid cleanup followed by extraction of unconjugated testosterone (T) and epitestosterone (E) with tert-butyl methyl ether (free fraction). The remaining aqueous phase was hydrolyzed with beta-glucuronidase and extracted at alkaline pH with n-pentane. Analytes were analyzed by GC/MS as their enol-trimethylsilyl (TMS) derivatives. The urine for stability testing was obtained from an excretion study after the administration of T to healthy volunteers. The homogeneity of the sample was verified before starting the stability study. The stability of TG and EG was evaluated at different storage conditions. For long-term stability testing, analyte concentration in urine stored at 4 degrees C and -20 degrees C was determined at different time intervals for 22 months. For short-term stability testing, analyte concentration was evaluated in urine stored at 37 degrees C for 3 and 7 days. The effect of repeated freezing (at -20 degrees C) and thawing (at room temperature) was studied for up to three cycles. Data obtained in this work demonstrated the stability of TG, EG and the T/E ratio in sterilized urine samples stored at 4 and -20 degrees C for 22 months and after going through repeated freeze/thaw cycles. Decreases in concentration were observed after 7 days of storage at 37 degrees C due to the partial cleavage of the glucuronide conjugates; however, the T/E ratio was not affected. These results show the feasibility of preparing reference materials containing TG and EG to be used for quality control purposes.