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Context: Little is known about how exogenous testosterone (T) affects the steroid profile in women. More knowledge would give the antidoping community keys as to how to interpret tests and detect doping., Objective: This work aimed to investigate the steroid profile in serum and urine in young healthy women after T administration., Methods: In a randomized, double-blind, placebo-controlled study, 48 healthy young women were assigned to daily treatment with T cream (10 mg) or placebo (1:1) for 10 weeks. Urine and blood were collected before and at the end of treatment. Serum steroids were analyzed with liquid chromatography-tandem mass spectrometry, and urine levels of T, epitestosterone (E), and metabolites included in the Athlete Biological Passport (ABP) were analyzed with gas chromatography-tandem mass spectrometry., Results: In serum, T and dihydrotestosterone levels increased, whereas sex hormone-binding globulin and 17-hydroxyprogesterone decreased after T treatment as compared to placebo. In urine, T and 5α-androstanediol increased in the T group. The median T increase in serum was 5.0-fold (range, 1.2-18.2) and correlated to a 2.2-fold (range, 0.4-14.4) median increase in T/E in urine (rs = 0.76). Only 2 of the 24 women receiving T reached the T/E cutoff ratio of 4, whereas when the results were added to the ABP, 6 of 15 participants showed atypically high T/E (40%). In comparison, 22/24 women in the T group increased serum T more than 99.9% of the upper confidence interval of nontreated values., Conclusion: It seems that the T/E ratio is not sufficient to detect exogenous T in women. Serum total T concentrations could serve as a complementary marker of doping., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In Europe, chemical castration has been adopted as a treatment for paraphilia since the 1930s. Among the various chemical castration agents, luteinizing hormone-releasing hormone (LHRH) agonists are now used widely because of their effectiveness and safety. In South Korea, a legislation of chemical castration to control the sexual impulses of sexual offenders was enforced in July 2011. Most of these subjects are treated with leuprorelin acetate, an LHRH agonist, for chemical castration. Despite this, there are few studies that address the long-term influence of LHRH agonists on testosterone (T) and epitestosterone (E) levels in chemical castration subjects. In order to analyze the urinary levels of T in chemical castration subjects, whose T levels are extremely low, we developed and validated an analytical method for the detection of both T and E in human urine using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The urine samples were hydrolyzed, extracted, and analyzed by LC-MS/MS with electrospray ionization in the positive-ion mode. The limits of detection were 0.02 ng/mL and the limits of quantitation were 0.05 ng/mL, which provided great sensitivity. The established method was applied to urine samples from chemical castration subjects and healthy male volunteers. The chemical castration subjects showed significantly lower urinary T levels than the control subjects. In addition, the urinary E levels were also lower in the chemical castration subjects; however, the T/E ratios were constant and did not show a notable decrease because of the simultaneous decrease in both urinary T and E. The urinary T levels and T/E ratio did not exceed the doping control criteria for exogenous T ingestion for any subject. This study shows the trend of urinary T and E levels in long-term treated chemical castration subjects by establishing a highly sensitive LC-MS/MS method, that provides useful information for monitoring chemical castration., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Context: Obesity is known to impact reproductive function in adults, but little is known about its effects on reproductive hormones during puberty., Objective: To assess sex differences in effects of obesity on reproductive hormones and their relation to insulin sensitivity and secretion., Design: Cross-sectional study including anthropometrics, serum and urine reproductive hormone concentrations, and intravenous glucose tolerance testing (IVGTT) to assess acute insulin response to glucose (AIRg), and insulin sensitivity (Si)., Setting: Outpatient academic clinical research center., Patients: Girls (52%) and boys (48%) who were normal weight (NW; n = 51, BMI-Z score = -0.11 ± 0.77, age = 11.5 ± 1.7 years) and obese (n = 53, BMI-Z score = 2.22 ± 0.33, age = 10.9 ± 1.5 years), Tanner stage 2 to 3., Results: Boys with obesity had lower total testosterone (P < 0.0001) and higher concentrations of the urinary estradiol metabolite, E1c, (P = 0.046) than boys with NW. Girls with obesity had higher free androgen index (FAI; P = 0.03) than NW girls. Both boys and girls with obesity had lower sex hormone-binding globulin (SHBG; P < 0.0001) than NW. AIRg was inversely related to SHBG in boys (R = 0.6, P < 0.0001) and girls (R = 0.53, P = 0.0001). Si correlated with higher SHBG in boys (R2 = 0.67, P < 0.0001) and girls (R = 0.5, P = 0.0003), higher total testosterone for boys (R = 0.39, P = 0.01), and lower FAI for girls (R = -0.2, P = 0.04)., Conclusion: Youth with obesity have lower SHBG than youth with NW, but obesity has differential effects on reproductive hormones in girls versus boys, which are apparent early in puberty. Ongoing longitudinal studies will evaluate the impact of obesity on reproductive hormones in girls and boys as puberty progresses., (Copyright © 2019 Endocrine Society.)

Background: The impact of testosterone (T) treatment on antidoping detection tests in female-to-male (F2M) transgender men is unknown. We investigated urine and serum sex steroid and luteinizing hormone (LH) profiles in T-treated F2M men to determine whether and, if so, how they differed from hypogonadal and healthy control men., Method: Healthy transgender (n = 23) and hypogonadal (n = 24) men aged 18 to 50 years treated with 1000 mg injectable T undecanoate provided trough urine and blood samples and an additional earlier postinjection sample (n = 21). Healthy control men (n = 20) provided a single blood and urine sample. Steroids were measured by mass spectrometry-based methods in urine and serum, LH by immunoassay, and uridine 5'-diphospho-glucuronosyltransferase 2B17 genotype by polymerase chain reaction., Results: Urine LH, human chorionic gonadotropin, T, epitestosterone (EpiT), androsterone (A), etiocholanolone (Etio), A/Etio ratio, dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), and 5α,3α- and 5β,3α-androstanediols did not differ between groups or by time since last T injection. Urine T/EpiT ratio was <4 in all controls and 12/68 (18%) samples from T-treated men, but there was no difference between T-treated groups. Serum estradiol, estrone, and DHEA were higher in transgender men, and serum T and DHT were higher in earlier compared with trough blood samples, but serum LH, follicle-stimulating hormone, and 3α- and 3β,5α-diols did not differ between groups., Conclusion: Urine antidoping detection tests in T-treated transgender men can be interpreted like those of T-treated hypogonadal men and are unaffected by time since last T dose. Serum steroids are more sensitive to detect exogenous T administration early but not later after the last T dose.

Context: Urinary cadmium (Cd) excretion is associated with cancer and cardiovascular morbidity. A potential mechanism could be disturbance of steroidogenesis in gonads and adrenal glands., Objective: We tested whether urinary excretion of Cd is correlated with that of cortico- and sex steroid metabolites in the general adult population., Setting: The Swiss Kidney Project on Genes in Hypertension is a multicentric, family-based population study., Measures: Urinary excretions of steroid hormone metabolites and Cd were measured with separate day and night collections. Associations were analyzed by mixed linear models., Results: Urinary Cd and testosterone excretions in men were significantly correlated (respective day and night β values [standard error (SE)], 1.378 [0.242], P < 0.0005; and 1.440 [0.333], P < 0.0005), but not in women [0.333(0.257), P = 0.2; and 0.674 (0.361), P = 0.06]. Urinary Cd and cortisol excretions were positively associated in both sexes [day: β = 0.475 (SE, 0.157), P = 0.0025, and 0.877 (SE, 0.194), P < 0.0005, respectively; night: β = 0.875 (SE, 0.253), P < 0.0005 and 1.183 (SE, 0.277), P = 0.00002, respectively]. Cd excretion was correlated with mineralocorticoid metabolites excretion, except tetrahydroaldosterone, in both sexes (P < 0.01). There was an independent effect of Cd on sex hormone and corticosteroid synthesis and an interdependent effect on gluco- and mineralcorticoid production., Conclusion: Our findings provide evidence for a global stimulating effect on steroid synthesis already at low-dose Cd exposure. These findings might explain the association of Cd with diseases such as steroid-sensitive cancers or metabolic disorders., (Copyright © 2017 Endocrine Society)

Context: Steroid sulfatase (STS) cleaves the sulfate moiety off steroid sulfates, including dehydroepiandrosterone (DHEA) sulfate (DHEAS), the inactive sulfate ester of the adrenal androgen precursor DHEA. Deficient DHEA sulfation, the opposite enzymatic reaction to that catalyzed by STS, results in androgen excess by increased conversion of DHEA to active androgens. STS deficiency (STSD) due to deletions or inactivating mutations in the X-linked STS gene manifests with ichthyosis, but androgen synthesis and metabolism in STSD have not been studied in detail yet., Patients and Methods: We carried out a cross-sectional study in 30 males with STSD (age 6-27 y; 13 prepubertal, 5 peripubertal, and 12 postpubertal) and 38 age-, sex-, and Tanner stage-matched healthy controls. Serum and 24-hour urine steroid metabolome analysis was performed by mass spectrometry and genetic analysis of the STS gene by multiplex ligation-dependent probe amplification and Sanger sequencing., Results: Genetic analysis showed STS mutations in all patients, comprising 27 complete gene deletions, 1 intragenic deletion and 2 missense mutations. STSD patients had apparently normal pubertal development. Serum and 24-hour urinary DHEAS were increased in STSD, whereas serum DHEA and testosterone were decreased. However, total 24-hour urinary androgen excretion was similar to controls, with evidence of increased 5α-reductase activity in STSD. Prepubertal healthy controls showed a marked increase in the serum DHEA to DHEAS ratio that was absent in postpubertal controls and in STSD patients of any pubertal stage., Conclusions: In STSD patients, an increased 5α-reductase activity appears to compensate for a reduced rate of androgen generation by enhancing peripheral androgen activation in affected patients. In healthy controls, we discovered a prepubertal surge in the serum DHEA to DHEAS ratio that was absent in STSD, indicative of physiologically up-regulated STS activity before puberty. This may represent a fine tuning mechanism for tissue-specific androgen activation preparing for the major changes in androgen production during puberty.

Context: Sebaceous gland hypertrophy (SGH) and acne-like skin eruptions are frequent during the first months of life, yet the etiology and prevalence of these conditions in infants are not clear., Objective: The objective of the study was to evaluate the association of postnatal androgens with SGH and acne in infants., Design: This was a longitudinal, monthly follow-up from 1 wk (D7) to 6 months of age (M1-M6)., Patients: Patients included 54 full-term (FT; 26 boys) and 48 preterm (PT; gestational age at birth 27.7-36.6 wk, 22 boys) infants., Main Outcome Measures: The occurrence of SGH (present/absent) and acne (5-10, 10-50, and >50 papules) was registered and compared with urinary levels of dehydroepiandrosterone and its sulphate and testosterone measured by liquid chromatography-tandem mass spectrometry., Results: SGH was observed in 89% of FT and 96% of PT infants (P = 0.28). Acne (more than five papules) was observed in 91% of FT infants and in 75% of PT infants (P = 0.06). Both SGH and acne were associated with developmental rather than calendar age: SGH was limited to postmenstrual age less than 46 wk and acne was not observed less than 37 wk of postmenstrual age. Urinary androgen levels showed severalfold differences in magnitude between sexes and between the FT and PT groups. After grouping according to sex and maturity, the occurrence of SGH and the severity of acne were associated with higher urinary dehydroepiandrosterone sulphate and testosterone levels in each group., Conclusions: SGH and acne are common during the first months of life and associated with endogenous, physiologically elevated levels of androgens originating from the adrenals and gonads. These data suggest a novel role for postnatal androgen secretion in infancy.

Context: Limited data suggest that testosterone is decreased during space flight, which could contribute to bone and muscle loss., Objective: The main objective was to assess testosterone and hormone status in long- and short-duration space flight and bed rest environments and to determine relationships with other physiological systems, including bone and muscle., Design: Blood and urine samples were collected before, during, and after long-duration space flight. Samples were also collected before and after 12- to 14-d missions and from participants in 30- to 90-d bed rest studies., Setting: Space flight studies were conducted on the International Space Station and before and after Space Shuttle missions. Bed rest studies were conducted in a clinical research center setting. Data from Skylab missions are also presented., Participants: All of the participants were male, and they included 15 long-duration and nine short-duration mission crew members and 30 bed rest subjects., Main Outcome Measures: Serum total, free, and bioavailable testosterone were measured along with serum and urinary cortisol, serum dehydroepiandrosterone, dehydroepiandrosterone sulfate, and SHBG., Results: Total, free, and bioavailable testosterone was not changed during long-duration space flight but were decreased (P < 0.01) on landing day after these flights and after short-duration space flight. There were no changes in other hormones measured. Testosterone concentrations dropped before and soon after bed rest, but bed rest itself had no effect on testosterone., Conclusions: There was no evidence for decrements in testosterone during long-duration space flight or bed rest.

Context: 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., Objective: 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., Participants: 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: Intervention included a single im dose of 500 mg testosterone enanthate., Main Outcome Measures: Urinary sulfo- and glucuronide-conjugated steroids were measured., Results: 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., Conclusion: We propose that the androsterone glucuronide to epitestosterone glucuronide ratio may serve as a complementary biomarker to reveal testosterone abuse.

The authors investigated whether soy intake is associated with sex steroid levels in Japanese children. This cross-sectional study was conducted in autumn 2006. Subjects were substantially healthy preschoolers, 230 boys and 198 girls, aged 3-6 years. Dietary data, including soy intake, were assessed using 3-day dietary records. Each child's dietary intake was controlled for total energy intake using the Willett method (Nutritional Epidemiology. Oxford, United Kingdom: Oxford University Press; 1990:245-271). Urinary estrone, estradiol, testosterone, and 5-androstene-3β,17α diol levels measured using liquid chromatography-electrospray ionization tandem mass spectrometry, and urinary dehydroepiandrosterone level measured with a radioimmunoassay, were adjusted for urinary creatinine levels. In the analysis of covariance for sex steroids after adjustments for age and body mass index, soy intake was significantly negatively related to estrone and estradiol in boys and positively related to testosterone and 5-androstene-3β,17α diol in girls. Isoflavone had a significant tendency to be negatively associated with estradiol in boys and to be positively associated with testosterone in girls. Total energy intake was not associated with any sex steroids in boys or girls. These results suggest that soy intake might affect the secretion or metabolism of sex steroids in childhood and that the effects might differ by sex.

This work describes the development, validation, and application of a novel methodology for the determination of testosterone and methenolone in urine matrices by stir bar sorptive extraction using polyurethane foams [SBSE(PU)] followed by liquid desorption and high-performance liquid chromatography with diode array detection. The methodology was optimized in terms of extraction time, agitation speed, pH, ionic strength and organic modifier, as well as back-extraction solvent and desorption time. Under optimized experimental conditions, convenient accuracy were achieved with average recoveries of 49.7 8.6% for testosterone and 54.2 ± 4.7% for methenolone. Additionally, the methodology showed good precision (<9%), excellent linear dynamic ranges (>0.9963) and convenient detection limits (0.2-0.3 μg/L). When comparing the efficiency obtained by SBSE(PU) and with the conventional polydimethylsiloxane phase [SBSE(PDMS)], yields up to four-fold higher are attained for the former, under the same experimental conditions. The application of the proposed methodology for the analysis of testosterone and methenolone in urine matrices showed negligible matrix effects and good analytical performance.

Previous studies of the round goby (Neogobius melanostomus Pallas, 1814), an invasive fish species in the Laurentian Great Lakes of North America, have shown that this species has the ability to both synthesize and smell steroids that have a 5 beta-reduced and 3 alpha-hydroxyl (5 beta,3 alpha) configuration. An enzyme-linked immunoassay (EIA) for 3 alpha-hydroxy-5 beta-androstane-11,17-dione (11-O-ETIO) has been used to show a substantial rise in the rate of release of immunoreactive compounds into the water when males are injected with salmon gonadotropin releasing hormone analogue. Similar increases were noted for 11-ketotestosterone and 17,20 beta-dihydroxypregn-4-en-3-one. Partitioning of the extracts between diethyl ether and water showed the presence of both free and conjugated immunoreactive 11-O-ETIO. Only conjugated immunoreactivity was found in urine (implying that free steroid is released via the gills). The identity of the conjugates was probed by using HPLC, EIA, and mass spectrometry and removal of sulfate and glucosiduronate groups. Immunoreactivity in the conjugated fraction was found to be due mainly to 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-sulfate. However, the evidence was also strong for the presence in water extracts of substantial amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-glucosiduronate (which could be detected only by EIA after removal of the glucosiduronate group with beta-glucuronidase). There were also small amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-sulfate and 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-glucosiduronate. These studies give some idea of the types, amounts, and ratios of 11-O-ETIO derivatives that are released by reproductive N. melanostomus and will aid further research into the putative pheromonal roles of 5 beta,3 alpha-reduced androgens in this species.

Background: Congenital adrenal hyperplasia is a group of disorders caused by defects in the adrenal steroidogenic pathways. In its most common form, 21-hydroxylase deficiency, patients develop varying degrees of glucocorticoid and mineralocorticoid deficiency as well as androgen excess. Therapy is guided by monitoring clinical parameters as well as adrenal hormone and metabolite concentrations., Content: We review the evidence for clinical and biochemical parameters used in monitoring therapy for congenital adrenal hyperplasia. We discuss the utility of 24-h urine collections for pregnanetriol and 17-ketosteroids as well as serum measurements of 17-hydroxyprogesterone, androstenedione, and testosterone. In addition, we examine the added value of daily hormonal profiles obtained from salivary or blood-spot samples and discuss the limitations of the various assays., Summary: Clinical parameters such as growth velocity and bone age remain the gold standard for monitoring the adequacy of therapy in congenital adrenal hyperplasia. The use of 24-h urine collections for pregnanetriol and 17-ketosteroid may offer an integrated view of adrenal hormone production but target concentrations must be better defined. Random serum hormone measurements are of little value and fluctuate with time of day and timing relative to glucocorticoid administration. Assays of daily hormonal profiles from saliva or blood spots offer a more detailed assessment of therapeutic control, although salivary assays have variable quality.

Background: Testosterone (T) is excreted in urine as water-soluble glucuronidated and sulfated conjugates. The ability to glucuronidate T and other steroids depends on a number of different glucuronidases (UGT) of which UGT2B17 is essential. The aim of the study was to evaluate the influence of UGT2B17 genotypes on urinary excretion of androgen metabolites in pubertal boys., Study Design: A clinical study of 116 healthy boys aged 8-19 yr. UGT2B17 genotyping was performed using quantitative PCR. Serum FSH, LH, T, estradiol (E2), and SHBG were analyzed by immunoassays, and urinary levels of androgen metabolites were quantitated by gas chromatography/mass spectrometry in all subjects., Results: Ten of 116 subjects (9%) presented with a homozygote deletion of the UGT2B17 gene (del/del), whereas 52 and 54 boys were hetero- and homozygous carriers of the UGT2B17 gene (del/ins and ins/ins), respectively. None of the reproductive hormones were affected by UGT2B17 genotype. In all subjects, mean urinary T/epitestosterone ratio was 1.56 [1.14 (SD); 0.1-6.9 (range)] and unaffected by age or pubertal stage. Subjects with homozygous deletions of UGT2B17 had significantly lower urinary levels of T and 5alpha- and 5beta-androstanediol. Mean urinary T/epitestosterone was significantly reduced in del/del subjects [0.29 (0.30); 0.1-1.0 (range), P < 0.0001]., Conclusion: In pubertal boys, a common homozygous deletion in the UGT2B17 gene strongly affected urinary excretion pattern of androgen metabolites but did not influence circulating androgen levels.

Context: Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone (T/E) ratio, levels above 4.0 being considered suspicious. The large variation in testosterone glucuronide (TG) excretion and its strong association with a deletion polymorphism in the uridine diphospho-glucuronosyl transferase (UGT) 2B17 gene challenge the accuracy of the T/E ratio test., Objective: Our objective was to investigate whether genotype-based cutoff values will improve the sensitivity and specificity of the test., Design: This was an open three-armed comparative study., Participants: A total of 55 healthy male volunteers with either two, one, or no allele [insertion/insertion, insertion/deletion, or deletion/deletion (del/del)] of the UGT2B17 gene was included in the study., Intervention: A single im dose of 500 mg testosterone enanthate was administered., Main Outcome Measures: Urinary excretion of TG after dose and the T/E ratio during 15 d were calculated., Results: The degree and rate of increase in the TG excretion rate were highly dependent on the UGT2B17 genotype with a 20-fold higher average maximum increase in the insertion/insertion group compared with the del/del group. Of the del/del subjects, 40% never reached the T/E ratio of 4.0 on any of the 15 d after the dose. When differentiated cutoff levels for the del/del (1.0) and the other genotypes (6.0) were applied, the sensitivity increased substantially for the del/del group, and false positives in the other genotypes were eliminated., Conclusions: Consideration of the genetic variation in disposition of androgens will improve the sensitivity and specificity of the testosterone doping test. This is of interest not only for combating androgen doping in sports, but also for detecting and preventing androgen abuse in society.

We developed a test that compares sequential measurements of a biomarker against previous readings performed on the same individual. A probability mass function expresses prior information on interindividual variations of intraindividual parameters. Then, the model progressively integrates new readings to more accurately quantify the characteristics of the individual. This Bayesian framework generalizes the two main approaches currently used in forensic toxicology for the detection of abnormal values of a biomarker. The specificity is independent of the number n of previous test results, with a model that gradually evolves from population-derived limits when n = 0 to individual-based cutoff thresholds when n is large. We applied this model to detect abnormal values in an athlete's steroid profile characterized by the testosterone over epitestosterone (T/E) marker. A cross-validation procedure was used for the estimation of prior densities as well as model validation. The heightened sensitivity/specificity relation obtained on a large data set shows that longitudinal monitoring of an athlete's steroid profile may be used efficiently to detect the abuse of testosterone and its precursors in sports. Mild assumptions make the model interesting for other areas of forensic toxicology.

Context: The reproductive endocrinology in Asians and Caucasians is of great interest in view of large differences in prostate cancer rate and sensitivity to pharmacological male contraception. In addition, interpretation of certain antidoping tests is confounded by interethnic variation in androgen disposition. Uridine diphosphoglucuronosyl transferases have a key role in the homeostasis and metabolism of androgens. Recently a deletion polymorphism was detected in the UGT2B17 gene., Objective: The objective of the study was to evaluate the contribution of the UGT2B17 deletion polymorphism to the interindividual and interethnic variation of androgen metabolism and excretion., Methods and Results: Urine from 122 Swedish and 74 Korean healthy men was analyzed for several androgen glucuronides including testosterone. The distribution of the natural logarithms of urinary testosterone concentrations showed a distinct bimodal pattern in both groups, suggesting a monogenic inheritance. When the UGT2B17 genotypes were compared with urinary testosterone levels, all of the individuals of the UGT2B17 homozygous deletion/deletion genotype had no or negligible amounts of urinary testosterone. The deletion/deletion genotype was seven times more common in the Korean (66.7%) than the Swedish population (9.3%). In addition, the Swedes had significantly higher levels of serum testosterone, compared with the Koreans., Conclusions: Our results show that the UGT2B17 polymorphism is strongly associated with the bimodal distribution of the testosterone excretion and also with the large differences in testosterone excretion between Koreans and Swedes.

Urinary steroid excretion after androstenedione intake has been examined after a single dose of 50 mg and single doses of 100 or 300 mg/d for 7 d. We evaluated the effects of 28 d of 100 mg three times a day (t.i.d.) androstenedione intake on urinary steroid excretion. Twenty healthy men, ages 30-39 yr (33.5 +/- 0.6), consumed 100 mg androstenedione t.i.d. or placebo for 28 d. Urine samples were analyzed for testosterone, epitestosterone, androsterone, and etiocholanolone via HPLC/tandem mass spectrometry on d 0 and 28. Androstenedione intake increased (P < 0.05) urinary testosterone 35.1 +/- 10.5 ng/ml vs. 251.6 +/- 87.5 ng/ml, epitestosterone 35.3 +/- 8.8 ng/ml vs. 99.7 +/- 28.7 ng/ml, androsterone 2,102 +/- 383 ng/ml vs. 15,767 +/- 3,358 ng/ml, and etiocholanolone 1,698 +/- 409 ng/ml vs. 11,329 +/- 2,656 ng/ml (means +/- se). Although the testosterone to epitestosterone ratio (T/E) tended to increase with androstenedione intake (1.2 +/- 0.3 vs. 4.0 +/- 1.6; P = 0.12), only one subject had a urinary T/E greater than the current Olympic criteria (>6.0) for a positive drug test. Chronic intake of 100 mg androstenedione t.i.d. increases the urinary excretion of steroid metabolites. Due to inconsistent increases in the T/E ratio, the T/E ratio may not effectively detect androstenedione use.

The most frequently used method to demonstrate testosterone abuse is the determination of the testosterone and epitestosterone concentration ratio (T/E ratio) in urine. Nevertheless, it is known that factors other than testosterone administration may increase the T/E ratio. In the last years, the determination of the carbon isotope ratio has proven to be the most promising method to help discriminate between naturally elevated T/E ratios and those reflecting T use. In this paper, an excretion study following oral administration of 40 mg testosterone undecanoate initially and 13 h later is presented. Four testosterone metabolites (androsterone, etiocholanolone, 5 alpha-androstanediol, and 5 beta-androstanediol) together with an endogenous reference (5 beta-pregnanediol) were extracted from the urines and the delta(13)C/(12)C ratio of each compound was analyzed by gas chromatography-combustion-isotope ratio mass spectrometry. The results show similar maximum delta(13)C-value variations (parts per thousand difference of delta(13)C/(12)C ratio from the isotope ratio standard) for the T metabolites and concomitant changes of the T/E ratios after administration of the first and the second dose of T. Whereas the T/E ratios as well as the androsterone, etiocholanolone and 5 alpha-androstanediol delta(13)C-values returned to the baseline 15 h after the second T administration, a decrease of the 5 beta-androstanediol delta-values could be detected for over 40 h. This suggests that measurements of 5 beta-androstanediol delta-values allow the detection of a testosterone ingestion over a longer post-administration period than other T metabolites delta(13)C-values or than the usual T/E ratio approach.

An extraction procedure for clostebol metabolites in urine is developed including enzymatic hydrolysis of conjugated metabolites with Helix pomatia juice (SHP) and solid-phase extraction (SPE) with further cleanup of sample extracts. For the enzymatic deconjugation step, variables such as buffer pH, amount of enzyme, incubation time, and temperature are examined. For the SPE step, different wash solutions and combinations with subsequent liquid-liquid extractions are examined. Incurred bovine urine samples, obtained through oral and intramuscular administration of clostebol acetate to animals, are used to test the performance of the developed method. In addition to the optimization of the sample pretreatment procedure, an interlaboratory study for the analysis of the incurred urine samples with ELISA and GC-MS is performed and good agreements are observed.

An isocratic high-performance liquid chromatographic method for the determination of testosterone (T) and epitestosterone (ET) in human urine using liquid-liquid or solid-phase extraction (SPE) is developed and validated. The optimum separation is achieved using a Hypersil C(18) column, water-acetonitrile (57:43, v/v) as the mobile phase and UV-absorbance detection at 245 nm. The recoveries obtained for T and ET in liquid-liquid and SPE demonstrate that these procedures are interchangeable. Quantitation limits for T and ET are 8.6 and 5.4 ng/mL using solvent extraction and 7.3 and 5.7 ng/mL using SPE, respectively. The proposed method is used to evaluate the urinary T, ET, and the T/ET ratio for a healthy male population using liquid-liquid extraction, and the T and ET excretion profile for nine healthy men using SPE.

Clostebol acetate (4-chloro-testosterone acetate) is an anabolic steroid used for fattening purposes in cattle breeding. To safeguard public health, its use has been prohibited by the European Commission since 1986. Screening for its urinary metabolites is therefore an important tool for the control of possible violations. Because those metabolites appear conjugated to glucuronic acid or sulfate, deconjugation prior to analysis is necessary. This work describes the variability in results seen with the use of various commercial preparations of Helix pomatia (SHP) for enzymatic hydrolysis of the conjugates. A simultaneous oxidative side reaction was observed, converting metabolites with a 3-OH-4-ene structure into a 3-oxo-4-ene structure. This was not observed when samples were incubated without enzyme or in the presence of heat-inactivated SHP. GC-MS analysis revealed oxidation of some metabolites of clostebol acetate, 4-chloro-4-androsten-3alpha-ol-17-one and 4-chloro-4-androsten-3alpha,17beta-diol, changing them into other metabolites, 4-chloro-4-androsten-3,1 7-dione and clostebol (4-chloro-testosterone), respectively. Based on the difference in cross-reactivities of the antibodies for these metabolites, comparative analysis in enzyme immunoassay, following enzymatic hydrolysis, confirmed this transformation. This oxidative conversion phenomenon could be of great importance when considering the choice or target analytes for screening bovine urine.

Androstenedione is a steroid hormone and the major precursor to testosterone. It is available without prescription and taken with the expectation that it will be converted to testosterone endogenously and increase strength and athletic performance. The metabolism of orally administered testosterone has not been well studied. We randomly assigned 37 healthy men to receive 0, 100, or 300 mg oral androstenedione in a single daily dose for 7 d. Single 8-h urine collections were performed on the day before the start of the androstenedione administration and on d 1 and 7 to assess excretion rates of free and glucuronide- conjugated testosterone, androsterone, etiocholanolone, and dihydrotestosterone. Serum testosterone glucuronide concentrations were measured by frequent blood sampling over 8 h on d 1 in 16 subjects (5 each in the 0 and 100 mg group and 6 in the 300 mg group). In the control group, mean (+/-SE) d 1 and 7 excretion rates for testosterone, androsterone, etiocholanolone, and dihydrotestosterone were 3 +/- 1, 215 +/- 26, 175 +/- 26, and 0.4 +/- 0.1 microg/h, respectively. In the 100 mg group, mean d 1 and 7 excretion rates for testosterone, androsterone, etiocholanolone, and dihydrotestosterone were 47 +/- 11, 3,836 +/- 458, 4,306 +/- 458, and 1.6 +/- 0.2 microg/h, respectively. In the 300 mg group, mean d 1 and 7 excretion rates for testosterone, androsterone, etiocholanolone, and dihydrotestosterone were 115 +/- 39, 8,142 +/- 1,362, 10,070 +/- 1,999, and 7.7 +/- 1.5 microg/h, respectively. Urinary excretion rates of all metabolites were greater in both the 100 and 300 mg groups than in controls (P < 0.0001). Urinary excretion rates of testosterone (P = 0.007), androsterone (P = 0.009), etiocholanolone (P = 0.0005), and dihydrotestosterone (P < 0.0001) were greater in the subjects who received 300 mg androstenedione than in those who received 100 mg. In the treated groups, excretion of free testosterone accounted for less than 0.1% of the total excreted testosterone measured. Serum testosterone glucuronide levels increased significantly during frequent blood sampling in both the 100 and 300 mg groups compared with controls (P = 0.0005 for the 100 mg group; P < 0.0001 for the 300 mg group). The net mean changes in area under the curve for serum testosterone glucuronide were -18 +/- 25%, 579 +/- 572%, and 1267 +/- 1675% in the groups receiving 0, 100, and 300 mg/d androstenedione, respectively. We conclude that the administration of both 100 and 300 mg androstenedione increases the excretion rates of conjugated testosterone, androsterone, etiocholanolone, and dihydrotestosterone and the serum levels of testosterone glucuronide in men. The magnitude of these increases is much greater than the changes observed in serum total testosterone concentrations. These findings demonstrate that orally administered androstenedione is largely metabolized to testosterone glucuronide and other androgen metabolites before release into the general circulation.

Background: Carbon isotope ratio methods are used in doping control to determine whether urinary steroids are endogenous or pharmaceutical., Methods: Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was used to determine the delta(13)C values for 5 beta-androstane-3 alpha,17 beta-diyl diacetate (5 beta A), 5 alpha-androstane-3 alpha,17 beta-diyl diacetate (5 alpha A), and 5 beta-pregnane-3 alpha,20 alpha-diyl diacetate (5 beta P) in a control group of 73 healthy males and 6 athletes with testosterone/epitestosterone ratios (T/E) >6., Results: The within-assay precision SDs for 5 beta A, 5 alpha A, and 5 beta P were +/- 0.27 per thousand, +/- 0.38 per thousand, and +/- 0.28 per thousand, respectively. The between-assay precision SDs ranged from +/- 0.40 per thousand to +/- 0.52 per thousand. The system suitability and batch acceptance scheme is based on SDs. For the control group, the mean delta(13)C (SD) values were -25.69 per thousand (+/- 0.92 per thousand), -26.35 per thousand (+/- 0.68 per thousand), and -24.26 per thousand (+/- 0.70 per thousand), for 5 beta A, 5 alpha A, and 5 beta P, respectively. 5 beta P was greater than 5 beta A and 5 alpha A (P <0.01), and 5 beta A was greater than 5 alpha A (P <0.01). The means - 3 SD were -28.46 per thousand, -28.39 per thousand, and -26.37 per thousand for 5 beta A, 5 alpha A, and 5 beta P, respectively. The maximum difference between 5 beta P and 5 beta A was 3.2 per thousand, and the maximum 5 beta A/5 beta P was 1.13. Three athletes with chronically elevated T/Es had delta(13)C values consistent with testosterone administration and three did not., Conclusions: This GC-C-IRMS assay of urine diols has low within- and between-assay SDs; therefore, analysis of one urine sample suffices for doping control. The means, SDs, +/-3 SDs, and ranges of delta(13)C values in a control group are established. In comparison, testosterone users have low 5 beta A and 5 alpha A, large differences between 5 beta A or 5 alpha A and 5 beta P, and high 5 beta A/5 beta P and 5 alpha A/5 beta P ratios.

The ratio of the concentration of testosterone glucuronide to the concentration of epitestosterone glucuronide (T/E ratio) as determined in urine is the most frequently used method to prove testosterone abuse by athletes. A T/E ratio higher than 6 has been considered as proof of abuse in the past; however, cases of naturally occurring higher T/E ratios have been described. Since the introduction of the T/E ratio in doping analysis, the parameters that may or may not influence the T/E ratio, possibly leading to false-positive results, have been debated. To achieve more insight on the influencing circumstances, an overview is given to obtain an objective view on the merits of the urinary T/E ratio. Relevant analytical aspects of the T/E ratio, potential parameters of endogenous and exogenous origins, as well as some alternative methods to determine testosterone abuse, such as the urinary testosterone/luteinizing hormone ratio, gas chromatography-combustion-isotope-ratio mass spectrometry, hair analysis, and high-performance liquid chromatography-mass spectrometry, are discussed.

The development of the reproductive axis is thought to be a gradual process, but our understanding of the complex endocrine changes that accompany the transition from premenarche to reproductive life in women has been hampered by the paucity of longitudinal studies. We studied 112 premenarchal Caucasian females at 6-month intervals over 4 yr and obtained a detailed reproductive and dietary history. We quantified reproductive hormones in 24-h urine collections as a measure of daily output and measured body composition biometrically and with the use of dual energy x-ray absortiometry scans. The percent body fat did not change appreciably in the study period (range, 21-24%) and was unrelated to menarche. Sex steroid and gonadotropin levels changed exponentially in the year approaching menarche. FSH levels peaked at menarche and then progressively declined thereafter. Estradiol output increased rapidly in the year approaching menarche and then plateaued thereafter. The frequency of menstrual bleeding increased rapidly and plateaued at 1 yr postmenarche. At 1 yr, 65% of these adolescent women had established a pattern of 10 or more menstrual episodes/yr, and by 3 yr postmenarche this figure exceeded 90%. There were no significant changes in dietary intake of protein, carbohydrate, or fat in the same period. Menarche occurs as a result of rapid maturation of the reproductive axis and heralds the reestablishment of a negative sex steroid feedback loop that parallels the adult threshold. These events appear to develop independent of changes in body composition and diet, but may reflect the improved nutrition and socioeconomic status of the late 20th century.

Dehydroepiandrosterone (DHEA) is an endogenous androgenic steroid produced by the ovaries and adrenal glands. Research suggests that DHEA can be converted to testosterone in peripheral tissues. Classified as a nutritional supplement, this compound may be purchased without a prescription. The military and international sports organizations prohibit the use of exogenous androgenic/anabolic steroids. Steroid-screening results are considered "positive" when the urinary ratio of testosterone to epitestosterone (T/E), an inactive synthetic byproduct, exceeds 6:1. Human volunteers ingested the recommended daily dose of 50 mg each morning for 30 days to determine if DHEA causes an adverse effect on this ratio. Urinary samples were collected before ingestion and 2-3 h after ingestion. Urine samples were extracted using solid-phase columns and analyzed using a previously developed gas chromatography-mass spectrometry method. T/E results were compared to an average baseline generated from three urine samples obtained before the study. Mean baseline T/E ratios averaged 0.67 for the seven subjects (range 0.1-1.2). The mean T/E ratio after DHEA ingestion ranged from 0.03 to 2.11. Individual postdose T/E ratios ranged from 0.01 to 3.7. The results from these individuals indicate that the administration of DHEA at this dose, for this period of time, has a minimal effect on urine T/E ratios and would not be expected to result in a positive screen for testosterone abuse. One subject agreed to take a single dose of 250 mg. This acute, high dose caused his T/E ratio to increase by 40% relative to the predose value.

The use of 4-chlorotestosterone acetate by farmers for cattle fattening was recently demonstrated although the use of this anabolic steroid is strictly forbidden in the European Union. We investigated the metabolism of 4-chlorotestosterone acetate in the bovine species after intramuscular and oral administration. Nineteen metabolites were detected in urine after intramuscular injection, and eight metabolites were identified. For this purpose, preparative HPLC, mass spectrometry with different ionization modes (electronic impact and chemical ionization), and different acquisition techniques were used (high resolution, selected ion monitoring, and scan measurement). Metabolite stereoisomerism was determined on the basis of retention time and organic synthesis. 4-Chloroepitestosterone (M2), 4-chloroandrost-4-en-3alpha-ol-17-one (M3), and 4-chloroandrost-4-ene-3,17-dione (M4) were identified as the main urinary markers of intramuscular administration. On the other hand, 4-chloroandrost-4-ene-3alpha,17beta-diol (M7), 4-chloroandrostan-3beta-ol-17-one (M5), and M2 were the primary indicators of an oral administration. In addition, we have shown that 95% of the metabolites were sulfo-conjugated, except for M3, which was partially conjugated to glucuronic acid. Finally, the main metabolites (M2, M3, and M4) were easily identified for 1.5 months after intramuscular administration.

A recommended confirmatory procedure for detecting 5 alpha-dihydrotestosterone (DHT) doping in male athletes proposed the use of the urinary concentration ratio of DHT to epitestosterone (EpiT) as the primary marker and those of 5 alpha-androstane-3 alpha,17 beta-diol (5 alpha-Adiol) to EpiT, luteinizing hormone (LH), and 5 beta-androstane-3 alpha,17 beta-diol (5 beta-Adiol) as secondary markers. Here we investigate the effects on these markers of intramuscular administration of DHT heptanoate (250 mg) to six healthy men. Within 24 h of administration all four markers greatly exceeded the published discrimination limits, remaining above these limits for 10-14 days. All ratios returned to basal values by day 28. In contrast to results after percutaneous administration, 5 beta-Adiol excretion decreased, probably as a consequence of greater suppression of testicular steroidogenesis. Results were largely in agreement with those obtained after percutaneous administration, although probably augmented by the larger dose and the different route of delivery.

Over the last decade the number of laboratories accredited by the International Olympic Committee (IOC) has grown to 25. Nearly half of the approximately 90,000 samples tested annually are collected on short notice-the most effective means to deter the use of anabolic androgenic steroids (AAS). The major urinary metabolites of AAS have been characterized and are identified by their chromatographic retention times and full or partial mass spectra. The process of determining if an athlete has used testosterone (T) begins with finding a T to epitestosterone (E) ratio > 6 and continues with a review of the T/E-time profile. For the user who discontinues taking T, the T/E reverts to baseline (typically approximately 1.0). For the extremely rare athlete with a naturally increased T/E ratio, the T/E remains chronically increased. Short-acting formulations of T transiently increase T/E, and E administration lowers it. Among ancillary tests to help discriminate between naturally increased T/E values and those reflecting T use, the most promising is determination of the carbon isotope ratio.

Our goal in this study was to determine whether the urinary ratio of testosterone to luteinizing hormone (T/LH) as an indicator of exogenous anabolic steroid (AS) use is superior to the urinary ratio of testosterone to epitestosterone (T/E). After 2 weekly placebo injections, 19 subjects were given testosterone cypionate (TC) injections of 250 or 500 mg/week for 14 weeks followed by 14 weekly placebo injections. Patients were considered to have ceased taking TC if they tested negative 9 weeks after their last injection. For detection of illicit or supraphysiological TC (AS) use, the urinary T/E ratio of > or = 6 yielded a false-negative rate of 46% and a false-positive rate of 4%. However, a urinary T/LH ratio of > or = 30 produced a false-negative rate of only 24% and a false-positive rate of 13%. We conclude that the urinary T/LH ratio of > or = 30 is a more sensitive marker of AS use than the urinary T/E ratio of > or = 6 and remains sensitive for twice as long as urinary T/E.

We examined the response of different biochemical markers of bone resorption to bisphosphonate therapy (400 mg of etidronate daily for 6 months) in mild Paget disease (n = 14). Urinary markers included hydroxyproline (OHP), total (T) and free (F) pyridinolines (Pyds) determined by HPLC, immunoreactive FPyds, immunoreactive TPyds, and the N- and C-terminal telopeptides of type I collage (NTx, CL). Serum measurements included tartrate-resistant acid phosphatase (TRAcP) and the C-terminal telopeptide of type I collagen (ICTP). ICTP and TRAcP showed a minimal response to therapy (% change at 6 months, -13.1 +/- 6.8 and -6.7 +/- 3.4, respectively). The response was greatest for urinary telopeptides (NTx and CL; % change -75.7 +/- 7.5 and -73.4 +/- 8.9, respectively). The response was somewhat greater for TPyds than for FPyds. We conclude that: (a) ICTP and TRAcP are unreliable indicators of changes in bone turnover; (b) oligopeptide-bound Pyds and telopeptide fragments of type I collagen in urine show a somewhat greater response to therapy than do FPyds and may be more sensitive indicators of bone resorption; and (c) as yet no evidence suggests that these markers are substantially better predictors of the clinical response to therapy than serum total alkaline phosphatase or urinary OHP. There are several problems with the interpretation of these measurements in Paget disease, and the clinical utility of these measurements remains uncertain.

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.

The ratio of urinary testosterone (u-T) to epitestosterone (T/EpiT) is used to disclose testosterone (T) administration in the doping control of sports, and a ratio greater than 6 constitutes an offence. Nevertheless, the possibility of biological outliers must not be discounted, and the use of ketoconazole has been suggested for a dynamic test to distinguish between such athletes and those using T. In this investigation, ketoconazole was administrated to three groups of T-pretreated and two groups of untreated healthy male subjects. The subjects in one of the pretreated groups were patients with mild hypogonadism. One untreated group consisted of athletes that had been tested three times with high urinary T/EpiT levels. The effects of ketoconazole administration on serum T (s-T) level and urinary T/EpiT ratio were monitored every 2 h for an 8-h period and clearly separated T-pretreated and untreated subjects into two clusters (P < 0.0001). The T/EpiT ratio increased and the s-T level remained unchanged in pretreated individuals during the ketoconazole test, whereas T/EpiT decreased by 60% and s-T by almost 90% in untreated subjects. The statistical power of the test increased by using several time points and combining the urinary T/EpiT with the s-T data. In conclusion, the ketoconazole test is suitable as a supportive dynamic test for the urinary T/EpiT ratio measurements in the doping control of athletes.

Combined self-administration of testosterone (T) and epitestosterone (ET) by athletes counteracts the efficiency of the corresponding urinary glucuronides (G) ratio, (T/ET)G, as an indicator of T abuse. I therefore propose 5-androstene-3 beta, 17 alpha-diol (5A3 beta 17 alpha), the immediate metabolic precursor of ET, as a new reference compound for the expression of relative excretions of T and ET. Thus (T/5A3 beta 17 alpha)G and (ET/5A3 beta 17 alpha)G become potential criteria to indicate joint administration of T and ET, since their respective threshold values (2.5 and 1.5) are exceeded even when (T/ET)G remains below the critical value of 6.

In this cross-sectional study on 140 subjects, several testosterone and epitestosterone metabolites have been analyzed by gas chromatography-mass spectrometry associated with stable isotope dilution, a technique requested for doping analysis in general, and detection of exogenous testosterone supply in particular. Urinary excretions of luteinizing hormone, testosterone and epitestosterone glucuronides and sulfates, as well as glucuronides of 5-androstene-3 beta, 17 alpha-diol, 5 alpha- and 5 beta-androstane-3 alpha, 17 alpha-diol and the corresponding 17 beta-isomers, present similar patterns of increase throughout pubertal development, from stage 1 up to stage 5. Excretion levels are significantly different in general between stages 1, 2, 3 and 4, the highest percentage increase being observed between stages 3 and 4. None of the ratios of testosterone to epitestosterone glucuronides are beyond the threshold value of 6, where testosterone abuse by athletes is suspected. No particular pubertal stage exceeded this critical value with a probability higher than p = 0.006, a value that was determined on the whole population. This is consistent with the non-significant differences between correlation slopes of regression curves, relating either testosterone or epitestosterone glucuronide to chronological age. The ratio of testosterone glucuronide to luteinizing hormone increases significantly throughout puberty and this might be a limitation to the widespread use of this ratio for the detection of testosterone misuse.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the ratio of testosterone to epitestosterone (T/E) in urine is used to detect testosterone administration in athletes, with a ratio > 6 considered as evidence of an offense. We show that administration of ketoconazole, which inhibits testosterone biosynthesis, may be useful for differentiating between an athlete who is using testosterone and one who naturally gives a ratio > 6. In a control subject pretreated with testosterone, ketoconazole caused the ratio to increase; conversely, it caused a decrease in the ratio in an athlete under investigation. Repeated administration of ketoconazole to two normal men caused a decrease in the ratio due to a large decrease in the urinary excretion rate of testosterone relative to epitestosterone. Stimulation with human chorionic gonadotropin exacerbated the differences in excretion rates. A single administration of ketoconazole to six normal men caused the T/E ratios to decrease significantly within 8 h, a suitable time scale for use in a dynamic test.

An increased ratio between urinary testosterone (T) and epitestosterone (epiT) has been accepted by the International Olympic Committee as a marker for T doping. However, in a few subjects, we and others have observed constantly above-normal urinary T/epiT ratios that are unlikely to be related to exogenous T administration. To find a better test for T doping, we studied several serum and urinary androgens and androgen precursors, estrogens, and luteinizing hormone (LH) in seven healthy volunteers for 35 days after an intramuscular injection of 250 mg of testosterone enanthate. Among urinary analyses, only the T/epiT ratio was a suitable marker of T doping; of the serum assays, 17 alpha-hydroxyprogesterone (17OHP), T/17OHP ratio, LH, and T/LH ratio were fair to good markers of T doping. The serum T/17OHP ratio was the best marker of those tested, with all seven subjects having above-normal values for this in the first 3 days of the observation period. No other marker showed abnormal values in all subjects at any time. Moreover, the T/17OHP ratio was affected by neither diurnal variation nor physical stress. The value of this marker for T doping was further supported by the finding of normal T/17OHP ratios in a subject with increased urinary T/epiT ratios caused by an abnormally low testicular epiT production, probably related to genetic factors.

The determination of oral turinabol (4-chloro-17 alpha-methyl-17 beta-hydroxy-1,4-androstadien-3-one) [1] in the 'free' fraction of human urine samples by gas chromatography and capillary column gas chromatography/mass spectrometry was studied. After administration to man, three major metabolites are formed whose structures were identified as 6 beta-hydroxy-turinabol [2], 6 beta, 12-dihydroxy-turinabol [4], and 6 beta, 16-dihydroxy-turinabol [5], respectively. In much smaller quantities at least another three metabolites are excreted, one of which could be identified as 17 epi-turinabol [6]. No measurable amounts of 1 itself were detected in any of the urine samples investigated. The rate of metabolism and urinary excretion is reasonably fast. The total amount of recovered material, in the form of the three main metabolites, is on the order of 15%. Clean-up procedures and chromatographic conditions are presented in detail.

14C ring-labelled hydrocortisone, testosterone and benzoic acid dissolved in acetone were applied to the backs of guinea-pigs (4 microgram/cm2). Percutaneous absorption was quantified by following the excretion of tracer in urine and faeces for 5 days. Absorption of hydrocortisone and benzoic acid was 2.4% (s.d. = 0.5; n = 3) and 31.4% (s.d. = 9.1; n = 3) of the applied dose respectively, similar to published human absorption data. Testosterone was absorbed to a greater extent in guinea-pigs (34.9% +/- 5.4; n = 5) than man. A thioglycollate based depilatory cream significantly increased the skin absorption of testosterone, while the absorption velocity was unaltered. Two analytical methods were compared, direct counting versus wet ashing; results were in the same range for the three compounds. Two methods of quantifying skin absorption were compared; urine recovery corrected for incomplete urinary excretion after parenteral administration versus the addition of urine and faecal recovery. For benzoic acid, excreted mainly in the urine (88.1%), the two methods gave similar results; for testosterone, excreted in the urine to a lesser extent (46.8%), the method of addition of urine and faecal recovery appeared to be more correct.

The development of radioimmunoassay of steroid hormones in peripheral venous blood has led to a more direct index of gonadal production and concurrent monitoring of glandular function. While serial analyses of total urinary oestrogens can be correlated with luteinizing hormone (LH) excretion in treating amenorrhoea, radioimmunoassay of plasma oestrogens measures acute changes in ovarian responsiveness. For studies of androgens radioimmunoassay of plasma testosterone is the method of choice, but fractionation of urinary 17-oxosteroids may be useful and can be extended to include the pregnane series and corticoids. Urinary pregnanediol, however, may not reflect the endogenous secretion of progesterone and should be reserved for assessing induction of ovulation. Luteal function can be evaluated rapidly and specifically by radioimmunoassay of plasma progesterone. Although radioimmunoassay offers a new approach to ovarian physiology, its systematic application to disease is still in the future.

The urinary testosterone levels of 40 premenopausal and postmenopausal patients with advanced breast carcinoma were assayed before and after ovariectomies. Histologic examination of the ovaries of 25 patients whose hormone levels were above normal revealed interstitial cell hyperplasia. Two months after the operations, the amounts of testosterone excreted by the womaen decreased significantly. We considered this as indirect evidence that the ovaries contributed to androgen synthesis. However, 15 patients had normal hormone excretion values that did not change 2 months postoperatively. Testosterone levels for 6 of the women 5 months after surgery were higher than at the previous testing. We surmised that the increased gonadotropic activity that followed ovariectomy migh have stimulated adrenal androgen secretion. To elucidate this point, we gave injections of 15,000 IU human chorionic gonadotropin (HCG) to breast cancer patients who were free of any recurrence and, 3 or 24 months previously, had undergone prophylactic ovariectomies (5 and 4 patients, respectively). In 5 of these women, the amount of testosterone excreted increased significantly after HGG was administered. We inhibited adrenal androgen secretion in 11 patients, whose presurgical urinary testosterone levels were above normal. After their ovaries were removed, these women received 1.5 mg dexamethaxone (DXM) daily for 30 days at a time, after which the amount of testosterone excreted was evaluated and, if the level was above 5.0 mug/24 hours, therapy was repeated for another 30 days. Of the patients who showed objective remission after ovarietomies, there were 4 (26.3%) of 15 who had normal androgen excretion levels; 8 (57.1%) of 14 had increased amounts and were treated by surgery alone; and 10 (90.9%) of 11 received a combination of ovariectomies and DXM therapy. The duration of regression was the highest in the latter group.