The incubation of [4-14C]testosterone with adult male hamster liver cytosol at pH 6.7 yielded 5 beta-androstane-3 alpha, 17 beta-diol and small quantities of 5 beta-androstane-3 beta, 17 beta-diol, 17 beta-hydroxy-5 beta-androstan-3-one, 3 alpha-hydroxy-5 beta-androstan-17-one and androstenedione. The use of [4-14C]androstenedione as substrate yielded the same 5 beta-metabolites and also testosterone and a trace of epitestosterone. 5 beta-Androstane-3 alpha, 17 beta-diol was the major metabolite at "low" concentrations of substrate but testosterone and 3 alpha-hydroxy-5 beta-androstan-17-one became the major metabolites as the concentration of the substrate was increased. Small quantities of 5 beta-androstane-3,17-dione and 3 beta-hydroxy-5 beta-androstan-17-one were detected at "high" while 5 beta-androstane-3 alpha, 17 alpha-diol was detected at "low" concentrations of androstenedione. NADPH was more effective than NADH except in the formation of the 3 beta-steroids. Furthermore, the 3 beta-steroids were formed in maximum quantities at a lower pH than the other metabolites. The relative production of the metabolites was consistent with their respective spectrophotometrically determined degree of hydroxyl dehydrogenation.

Urinary excretion of testosterone, epitestosterone, androstendion, and 7-keto-dehydroepistendion was studied in 34 healthy men, aged from 20 to 72 years. The maximal excretion of these steroids and observed in men, aged between 20 and 30 years; their reduction was noted with the advance of age.

Testosterone has been identified by mass spectrometry in blood and follicular fluid aspirated from mature Graafian follicles of mares. Quantitative measurements made by gas chromatography-mass spectrometry have validated the determination of plasma testosterone made by radioimmunoassay. However, because of high levels of epitestosterone (17 alpha-hydroxyandrost-4-en-3-one) in the follicular fluid, radioimmunoassay overestimates the true concentrations of testosterone. The occurrence of testosterone in mare follicular fluid at a concentration which is two orders of magnitude higher than that in peripheral plasma suggests that the follicle may contribute to the production of circulating testosterone. A biosynthetic pathway for oestradiol-17 beta which involves testosterone is therefore likely to occur in the mare ovary as in many other mammalian species.

Voltammetric investigation of two corticoid isomers--testosterone and epitestosterone has been carried out at bare and single-wall carbon nanotubes (SWNT)-modified edge plane pyrolytic graphite electrode (EPPGE). Square wave voltammetry (OSWV) has been used for the simultaneous determination of isomeric steroids. The reduction of the two isomers occurred in a pH dependent, 2e, 2H+ process and well-defined voltammetric peaks were observed. Under the optimum experimental conditions, linear calibration curves are obtained within the concentration range 5-1000 nM for both the steroids with the limit of detection 2.8 x 10(-9) and 4.1 x 10(-9) M for testosterone and epitestosterone respectively. The developed protocol is successfully implemented for the analysis of both the compounds in the urine samples of normal subjects as well as in patients undergoing treatment with testosterone. The results obtained from the proposed voltammetric method were also compared with HPLC analysis and found to be similar.

For an effective detection of doping with pseudo-endogenous anabolic steroids, the urinary steroid profile is of high value. In this work, the aim was to investigate steroid metabolism disruption after exogenous intramuscular administration of different testosterone esters. The investigation focused on both sulfo - and glucoro conjugated androgens. A single intramuscular injection of either 1000 mg testosterone undecanoate (Nebido®) or a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Sustanone®), was given to six healthy volunteers. Urine was collected throughout a testing period of 60 days. A LC-MS method was developed and validated for the analysis of eight conjugated steroids in their intact form. The results show that urinary changes in both sulfo - and glucuro conjugated steroid levels are prominent after the injection of testosterone esters. A promising potential marker for the intake of exogenous testosterone is the combined ratio of epitestosterone sulfate/epitestosterone glucuronide to testosterone sulfate/testosterone glucuronide ((ES/EG)/(TS/TG)) as a complementary biomarker for testosterone abuse. This represents a new piece of evidence to detect testosterone doping, representing a new approach and being independent from the metabolic connections of the markers in the steroid passport.

A novel method for determining the testosterone/epitestosterone concentration ratio in human urine was established by capillary electrophoresis with diode-array detector. The urine samples were firstly purified by the solid extraction. The optimal experimental conditions were: running buffer pH = 4.74, 15.0 mmol L^-1 HAc-NaAc, separation voltage 25 kV, temperature 25 °C, sample injection pressure 3.43 × 10³ Pa, and duration 10 s. The testosterone and epitestosterone linear range were determined as 8.0-960.0 ng mL^-1, respectively. The testosterone and epitestosterone detection limits were determined as 4.6 and 4.5 ng mL^-1, respectively. The relative standard deviation was less than 0.36%.

Keywords: Capillary electrophoresis; Determination; Epitestosterone; Testosterone; Urine.

The use of anabolic substances is prohibited in food-producing animals throughout the European Union. No method is available to reliably detect the misuse of natural hormones in cattle. A method was developed to detect the abuse of testosterone in cattle fattening. Synthesized testosterone is rather depleted in the (13)C/(12)C ratio. Hence, the method is based on gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis of urine. To select testosterone metabolites and endogenous reference compounds (ERC), the concentration of urinary steroids of cattle was investigated. Dehydroepiandrosterone and androst-5ene-3beta,17alpha-diol were chosen as ERCs to show endogenous (13)C/(12)C ratios. Etiocholanolone and 5alpha-androstane-3beta,17alpha-diol were chosen as the most important testosterone metabolites. Other metabolites known from literature like epitestosterone were less promising. In principle, GC/C/IRMS is a nonspecific method because finally carbon dioxide is analyzed. Therefore, a dedicated cleanup procedure for the selected steroids was developed. By means of proposed confidence intervals in the isotopic composition of ERCs and metabolites, the administration of testosterone to cattle could be detected reliably. Differences of up to 11 per thousand on the delta-scale between ERC and testosterone metabolites were found after testosterone administration, whereas endogenous differences did not exceed 2 per thousand.

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 other important ones 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 K_m values, between 0.4 and 4 µM, while the K_m values for glucuronides of testosterone and dehydroepiandrosterone were 14 and 51 µM, respectively. MRP2 transported the glucuronides at lower affinity, as indicated by K_m 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.

The laboratory profile of intranasal testosterone gel has not been previously reported from an anti-doping perspective. Because intranasal testosterone gel is newly available as a commercial product, we sought to examine the laboratory parameters following administration of this formulation, with particular attention to anti-doping guidelines. Five healthy and active male subjects were administered testosterone intranasal gel three times daily for four weeks, using a pattern of five consecutive days on, two days off. Urine was collected after each five-day round of drug administration and analyzed using a full steroid screen and isotope ratio mass spectrometry (IRMS). Windows of detection for elevated testosterone/epitestosterone (T/E) and other steroid ratios, World Anti-Doping Agency (WADA) athlete biological passport (ABP) findings, and IRMS results were analyzed in this study. In the 0-24 h window post-administration, 70% of samples were flagged with a suspicious steroid profile and 85% were flagged as atypical passport findings according to the WADA ABP steroid module. In the 24-48 h window, 0% of samples displayed suspicious steroid profiles while 40% resulted in atypical passport findings. IRMS testing confirmed the presence of exogenous testosterone in 90% and 40% of samples in the 0-24 h and 24-48 h windows post-administration, respectively. Additionally, IRMS data were analyzed to determine commonalities in the population changes in δ13 C values of testosterone, androsterone, etiocholanolone, 5αAdiol, and 5βAdiol. Though no discernible metabolic trend of the route of administration was identified, we discovered that intranasal gel testosterone is detectable using conventional anti-doping tests. Copyright © 2016 John Wiley & Sons, Ltd.

A pharmacological dose of a long-acting testosterone ester, testosterone hexahydrobenzoate, was administered intramuscularly to two mares. The time course for some characteristic metabolites in blood and urine was then studied using an analytical method based on gas chromatography-mass spectrometry associated with stable isotope dilution. Among the plasma analytes, testosterone glucuronide was found to be the most adequate indicator for the monitoring of exogenous testosterone up to 2 weeks postadministration if a threshold value of 200 ng/L was applied. In urine, the simultaneous measurement of the concentrations of testosterone sulfate (TS) and epitestosterone sulfate (ES) allowed the calculation of the concentration ratio, TS/ES, which was independent of urine flow and which offered the possibility of detecting testosterone misuse 20 to 30 days after dosing if a tentative threshold value of 8 was adopted. In addition to this ratio, particularly when the TS/ES ratio was close to the cutoff point, it seemed advisable to take into account the concentrations of 5 alpha-androstane-3 beta, 17 alpha-diol (glucuronide) and its 17 beta-isomer (sulfate), which should not exceed 50 micrograms/L.

Glucuronidation is one of the most important metabolic processes which eliminates androgens from the human organism. Three enzymes (UDP-glucuronosyltransferases) are responsible for transfering the glucuronic group from glucuronic acid to androgens: UGT2B7, UGT2B15 and UGT2B17. Glucuronide products are more polar, water soluble, less toxic and easily extracted form the body. Testosterone is a common androgen abused in sport. The norm for urinary testosterone/epitestosterone ratio is below 4.0. Large testosterone excretion is associated with a deletion polymorphism of the UGT2B17 gene. This polymorphism decreases T/E ratio level.

Detection of testosterone and/or its pro-drugs in the gelding is currently regulated by the application of an international threshold for urinary testosterone of 20 ng/mL. The use of steroid ratios may provide a useful supplementary approach to aid in differentiating between the administration of these steroids and unusual physiological conditions that may result in atypically high testosterone concentrations. In the current study, an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify testosterone (T) and epitestosterone (E). The method was used to analyze 200 post-race urine samples from geldings in order to generate the ratios for the reference population. Following statistical analysis of the data, an upper limit of 5 for T:E ratio in geldings is proposed. Samples collected from 15 geldings with atypical urinary testosterone concentrations (>15 ng/mL) but otherwise normal steroid profile, had T:E ratios within those observed for the reference population. The applicability of an upper T:E ratio to detect an administration was demonstrated by the analysis of a selection of incurred samples from testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi-Bolic®) administrations. These produced testosterone concentrations above the threshold of 20 ng/mL, but also T:E ratios above the proposed limit of 5. In conclusion, consideration of the T:E ratio appears to be a valuable complementary aid to evaluate whether an atypical testosterone concentration could be caused by a natural biological outlier as opposed to the administration of these steroids. Copyright © 2016 John Wiley & Sons, Ltd.

Plasma (solvolyzed and unsolvolyzed) from the male lizard Tiliqua rugosa was analyzed using gas chromatography, high-performance liquid chromatography (HPLC), and mass spectroscopy. HPLC with electrochemical detection was also used to characterize ketosteroid conjugates. Testosterone was identified in the solvolyzed plasma extract, and a compound corresponding to testosterone sulfate was detected in unsolvolyzed extracts. Concentrations were approximately 500 nmol/liter in intact male plasma, less than 100 nmol/liter in castrates, and undetectable in female plasma. Steroid glucuronides appeared to be absent from plasma. Epitestosterone conjugates were not detected, although the free steroid is found in high concentrations in T. rugosa. The presence of testosterone sulfate in the blood of T. rugosa may indicate a possible role for sulfoconjugates in controlling the availability of biologically active androgens.

The presence of exogenous testosterone has been monitored mainly in the urine and blood. However, other biological matrices such as hair, nail, and saliva samples can be used successfully for in vivo measurement. Chromatographic analysis requires pretreatment to obtain free testosterone and its metabolites. Among the pretreatment procedures, digestion, hydrolysis and solvolysis steps are conducted to reach the analytical purpose. Digestion assay is indicated for hair and nail samples. First, it is recommended to perform the decontamination step. After that, alkaline solution (NaOH), organic solvents and other reagents can be added to the samples and incubated under determined conditions for the digestion step. Hydrolysis assay is recommended to urine and blood samples. Acid hydrolysis cleaves conjugated testosterone and its metabolites using HCl or H2SO4 solution at appropriate time and temperature. However, there is formation of interferent compounds, degradation of dehydroepiandrosterone and decrease of peak resolution for epitestosterone. Enzymatic hydrolysis is an alternative technique able to promote free testosterone and its metabolites with low degradation. It is important to establish the best conditions according to the biological fluid and the amount of the sample. Sulfatase enzyme is recommended together with β-glucuronidase to cleave sulfoconjugate steroids. Solvolysis assay is similar to acid hydrolysis, but organic solvents are responsible to promote steroid deconjugation. Other approaches such as combination of different pretreatments, surface response or ultrasonic energy have been used to obtain the total of free steroids. So, the biological matrix defines the best procedure for pretreatment to achieve the analytical purpose, knowing its advantages and limitations.

Explants of canine prostate were cultured in a defined medium for periods of up to 5 days with and without added steroids. Testosterone and 5 alpha-dihydrotestosterone failed to maintain their histological integrity and induced a greatly increased formation of stromal elements. Epitestosterone and 5 alpha-dihydroepitestosterone were partially successful in maintaining epithelial height although secretory activity was not preserved. The only steroid that sustained epithelial height and secretory activity whilst keeping stromal growth at a minimum was 5 alpha-androstane-3 alpha, 17 alpha-diol. The three other epimeric androstanediols were ineffective.

The specificity of the neonatal, andreogen-induced, irreversible programming of hepatic steroid metabolism in the rat was investigated. 5-alpha-Dihydrotestosterone propionate and estradiol benzoate were as efficient as testosterone propionate in inducing a male type of liver metabolism in the adult animal, whereas epitestosterone propionate, etiocholanolone propionate, and o,p'-DDT were practically inactive in this respect. These findings indicate that different mechanisms are involved in neonatal imprinting of hepatic steroid metabolism and in the well-known neonatal androgenic and estrogenic induction of persistent estrus and acyclic gonadotropin secreqion.

An accurate method for the measurement of carbon isotope ratios of steroids in human urine has been developed at the National Measurement Institute, Australia (NMIA) for the certification of a freeze-dried human urine reference material (CRM NMIA MX005). The method measures δ(13)C values by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis following hydrolysis, solvent extraction and high performance liquid chromatography (HPLC) purification. Reference δ(13)C values for testosterone metabolites etiocholanolone, androsterone, and endogenous reference compounds (ERCs) 11β-hydroxyandrosterone and pregnanediol were determined, as well as information δ(13)C values for testosterone, epitestosterone, 11-oxoetiocholanolone, and a range of differences (Δ(13)C) between testosterone metabolites and ERCs. The measurement uncertainty was rigorously evaluated with expanded uncertainties for the reference δ(13)C values between 1.1 and 1.6 ‰ at the 95% coverage level.

Blood and testicular extracts of the scincid lizard Tiliqua (Trachydosaurus) rugosa were analyzed using thin-layer, column, and high-performance liquid chromatography. The major androgens isolated were epitestosterone, testosterone, and androstenedione. Epitestosterone was characterized by chromatography in several systems, and by derivative formation. Epitestosterone was further identified in testicular extracts by gas chromatographymass spectrometry. Immunoreactive material was detected in tissue extracts using an antiserum specific to epitestosterone. The maximum concentration of epitestosterone in blood, measured by radioimmunoassay, was four times that of testosterone (approximately 900 and 200 nmol/l, respectively). Epitestosterone could not be detected in the blood of intact females and the concentration of this steroid was low in castrate males. The maximum testicular concentrations (pmol/testis) were 390 (nonincubated) and 2050 (incubated) for epitestosterone, and 2025 (nonincubated) and 1040 (incubated) for testosterone. Both plasma and testicular concentrations showed considerable seasonal variation. The identification of endogenous epitestosterone confirms the results of earlier investigations using radioactive substrates. The occurrence of this steroid as a major product of the testis in T. rugosa is discussed in relation to androgens in reptiles and other vertebrates.

Generally, compound-specific isotope analysis of steroids is carried out by gas chromatography combined with isotope ratio mass spectrometry. Thus, a derivatization of the steroids prior to the measurement is compulsory, and a correction of the isotopic data is often necessary. To overcome this limitation, we present a new approach of high-temperature liquid chromatography coupled with photodiode array detection and isotope ratio mass spectrometry (HT-LC/PDA/IRMS) for the carbon isotope ratio analysis of unconjugated steroids. A steroid mixture containing 19-norandrosterone, testosterone, epitestosterone, androsterone, and 5β-pregnane-3α,17α,20α-triol was fully separated on a C4 column under high-temperature elution with water as the sole eluent. The accuracy for isotope analysis (±0.5 ‰) was around 20 μg g(-1) for testosterone, epitestosterone (79 ng steroid absolute on column), and 30 μg g(-1) for 19-norandrosterone, androsterone, and 5β-pregnane-3α,17α,20α-triol (119 ng steroid absolute on column). The applicability of the method was tested by measuring a pharmaceutical gel containing testosterone. With this work, the scope of LC/IRMS applications has been extended to nonpolar compounds.

BACKGROUND

Aging men suffer from a decrease of androgen-anabolic steroids known as "Partial Androgen Decline in the Aging Male" (PADAM) that is sometimes compensated by the use of androgen replacement therapy.

RESULTS

To decide whether androgen therapy should be considered, hormonal serum levels are commonly tested. In addition, the St. Louis questionnaire is used as and indication. Accordingly, serum levels of testosterone, androstendione, dihydrotestosterone, dehydroepiandrosterone, its sulphate, epimers of 7-hydroxy-dehydroepiandrosterone, epitestosterone, lutropin, follitropin, prolactin and sex hormone binding globulin were measured in 216 men over 50 years of age. Further, the Sr. Louis questionnaire was applied and the extent of the relations among the data was evaluated. RIA and IRMA kits from Immunotech and Orion-Diagnostica were used for measurement of the hormones except epitestosterone and 7-hydroxy-dehydroepiandrosterone epimers, which were determined using specific radioimmunoassays.

CONCLUSIONS

The decline in the index of free testosterone, given by the increase of SHBG levels, as well as the decrease in the levels of DHEA and its derivates were the most prominent. No correlation was found between the levels of hormones and the individual items of the questionnaire or with the total score calculated from the questionnaire data. In conclusion, the University of Saint Louis questionnaire did not give a reliable indication of an androgen deficit. However, the found values and their changes can still be helpful during the decision process concerning indication and initiation of hormone replacement.