Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing.

Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping control analysis for many years to uncover the misuse of endogenous steroids such as testosterone. Over the years, several challenges and limitations of this approach became apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the emergence of steroid preparations comprising identical CIRs as endogenous steroids. While the latter has been addressed recently by the implementation of hydrogen isotope ratios (HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented herein together with newly established reference values of those endogenous steroids being relevant for doping controls. From the fraction of glucuronidated steroids 5ß-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-Hydroxy-5ß-androstane-11,17-dione, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5ß-androstan-17-one (ETIO), 3ß-hydroxy-androst-5-en-17-one (DHEA), 5α- and 5ß-androstane-3α,17ß-diol (5aDIOL and 5bDIOL), 17ß-hydroxy-androst-4-en-3-one and 17α-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of ANDRO, ETIO, DHEA, 3ß-hydroxy-5α-androstan-17-one plus 17α- and androst-5-ene-3ß,17ß-diol were considered and analyzed after acidic solvolysis. The results obtained for the reference population encompassing n = 67 males and females confirmed earlier findings regarding factors influencing endogenous CIR. Variations in sample preparation influenced CIR measurements especially for 5aDIOL and 5bDIOL, the most valuable steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR of the same reference population enabled the evaluation of combined measurements of CIR and HIR and its usefulness regarding both steroid metabolism studies and doping control analysis. The combination of both stable isotopes would allow for lower reference limits providing the same statistical power and certainty to distinguish between the endo- or exogenous origin of a urinary steroid.

Development of a GC/C/IRMS method--confirmation of a novel steroid profiling approach in doping control.

In doping control, an athlete can only be convicted with the misuse with endogenous steroids like testosterone (T), if abnormal values of steroid metabolites and steroid ratios are observed and if the subsequent analysis with isotope ratios mass spectrometry (IRMS) confirms the presence of exogenously administered androgens. In this work, we compare the results of a novel steroid profiling approach with the performance an in-house developed IRMS method. The developed IRMS has the advantage over other methods to be relatively short in time and with target compounds androsterone, etiocholanolone, 5ß-androstane 3α,17ß-diol and 5α-androstane 3α,17ß-diol. Pregnanediol was used as an endogenous reference compound (ERC). Reference limits for the IRMS values were established and applied as decision limits for the evaluation of excretion urine from administration with oral T, T-gel, dihydrotestosterone (DHT) - gel and dehydroepiandrosterone (DHEA). Results indicated the importance of both androstanediols as important IRMS markers where relative values compared to an ERC (Δδ(13)C) yielded better detection accuracy than absolute δ(13)C-values. The detection times of all administered endogenous steroids were evaluated using the proposed thresholds. The results of traditional steroid profiling and a new approach based upon minor steroid metabolites monitoring introduced in a longitudinal framework were evaluated with IRMS. With traditional steroid profiling methods, 95% of the atypical samples could be confirmed whereas an additional 74% of IRMS confirmed was provided by a new biomarkers strategy. These results prove that the other steroid profiling strategies can improve the efficiency in detection of misuse with endogenous steroids.

Seized designer supplement named "1-Androsterone": identification as 3ß-hydroxy-5α-androst-1-en-17-one and its urinary elimination.

New analogues of androgens that had never been available as approved drugs are marketed as "dietary supplement" recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids. In the present study, we report the detection of a steroid in a product seized by the State Bureau of Criminal Investigation Schleswig-Holstein, Germany. The product "1-Androsterone" of the brand name "Advanced Muscle Science" was labeled to contain 100mg of "1-Androstene-3b-ol,17-one" per capsule. The product was analyzed underivatized and as bis-TMS derivative by GC-MS. The steroid was identified by comparison with chemically synthesized 3ß-hydroxy-5α-androst-1-en-17-one, prepared by reduction of 5α-androst-1-ene-3,17-dione with LS-Selectride (Lithium tris-isoamylborohydride), and by nuclear magnetic resonance. Semi-quantitation revealed an amount of 3ß-hydroxy-5α-androst-1-en-17-one in the capsules as labeled. Following oral administration to a male volunteer, the main urinary metabolites were monitored. 1-Testosterone (17ß-hydroxy-5α-androst-1-en-3-one), 1-androstenedione (5α-androst-1-ene-3,17-dione), 3α-hydroxy-5α-androst-1-en-17-one, 5α-androst-1-ene-3α,17ß-diol, and 5α-androst-1-ene-3ß,17ß-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common steroids of the urinary steroid profile were altered after the administration of "1-Androsterone". Especially the ratios of androsterone/etiocholanolone and 5α-/5ß-androstane-3α,17ß-diol and the concentration of 5α-dihydrotestosterone were influenced. 3α-Hydroxy-5α-androst-1-en-17-one appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to nine days after a single administration of one capsule.

Radical prostatectomy: influence on serum and urinary androgen levels.

BACKGROUND: The role of the prostate as an active endocrine organ and the hormonal changes after radical prostatectomy (RP) has not been well studied. The objective of this study was to investigate the serum and urine hormonal changes after RP. METHODS: Fifty-five healthy men with localized prostate cancer were enrolled in this cross-sectional study at a single academic center. We measured serum levels of testosterone, dihydrotestosterone (DHT), sex hormone binding globulin (SHBG), luteinizing hormone (LH), and follicle stimulating hormone (FSH) in all 55 patients preoperatively and in 53 patients 90 days postoperatively. Free testosterone was calculated in all patients. Inhibin B levels was analyzed in 44 patients pre- and postoperatively. Steroid urine profile including testosterone, DHT, 5alpha-androstane-3alpha,17beta-diol (3alphaAdiol), and androsterone (ADT) was also determined preoperatively and 90 days postoperatively in 18 patients. RESULTS: There were 53% increase in serum LH (P < 0.0001), 21% increase in serum FSH (P < 0.0001), and 13% decrease in DHT levels (P < 0.03). There were no significant changes in any other serum hormone investigated. Urinary levels of DHT glucuronides (DHT-G) decreased by 67% (P < 0.0003) while Androsterone-G and 3alphaAdiol-G increased by 37% (P = 0.019) and 44% (P = 0.023), respectively. There were no alterations in the urinary levels of the other steroids investigated. Inhibin B levels correlated inversely with both FSH (r = -0.67, P < 0.0001) and LH (r = -0.51, P = 0.0004). CONCLUSION: RP leads to significant increases in serum gonadotropins and significant DHT decrease in both serum and urine. These hormonal changes are independent of inhibin B.

[Detection of the metabolites of dehydroepiandrosterone in urine with gas chromatography-combustion-isotope ratio mass spectrometry].

AIM: To establish a method to determine the isotope ratios of 13C to 12C of dehydroepiandrosterone and its metabolites in urine, for detecting the source of dehydroepiandrosterone or its metabolites. METHODS: Preliminary separation of endogenous anabolic androgenic steroids could be achieved using solid phase extraction, enzymolysis and thin layer chromatography. The source of dehydroepiandrosterone and other endogenous anabolic androgenic steroids could be detected by their delta values with gas chromat ography-combustion-isotope ratio mass spectrometry. RESULTS: The 5 values of some metabolites of dehydroepiandrosterone reduced after the administration of dehydroepiandrosterone preparation. In these cases the data indicated that exogenous anabolic androgenic steroids were administrated. CONCLUSION: The source of dehydroepiandrosterone or its metabolites in urine could be detected by measuring their delta values with this method.

Urinary marker of oral pregnenolone administration.

Pregnenolone (PREG) can potentially be abused by athletes to maintain an equilibration of the steroidal environment after sex steroids administrations. Five men volunteers orally ingested 50 mg PREG to determine optimal urinary markers for detection of this steroid. Our findings show that ingestion of PREG has no significant effects on the testosterone/epitestosterone (T/E) and testosterone/luteinizing hormone (T/LH) ratios, whereas variable changes on the carbon isotopic values of three T metabolites: androsterone, etiocholanolone, 5beta-androstane-3alpha,17beta-diol (5beta-androstanediol) together with 16(5alpha)-androsten-3alpha-ol (androstenol) and 5beta-pregnane-3alpha,20alpha-diol (pregnanediol) have been observed. The difference between the carbon isotopic values (delta13C-values) of androstenol and pregnanediol is potentially the most reliable marker of exogenous PREG administration in males. For all subjects, the differences differ by 3.0 per thousand or more over a period of about 10 h and for both of them the detection window for positivity is extended over 40 h.

Urinary profile of androgen metabolites in a population of sportswomen during the menstrual cycle.

The aim of this investigation was to evaluate the urinary profile of androgen metabolites during menstrual cycle in both young-trained female athletes, and young sedentary women, not presenting any pathological signs. Urines were collected for 24 hours (08 : 00 a. m. the first day to 08 : 00 a. m. the second day) from all sportive and sedentary subjects. All steroids were measured by specific radioimmunological analysis, and the implications of these results in terms of concentrations and modifications by exercise will be discussed. During follicular phase, control values were respectively, testosterone glucuronide (TG): 1.67 +/- 0.70 nmol x mmol C -1; epitestosterone glucuronide (ETG): 2.51 +/- 0.88 nmol x mmol C -1; TG/ETG ratio: 0.72 +/- 0.26 and cortisol (FLU): 10.02 +/- 0.79 nmol x mmol C -1. No significant modifications were observed during luteal phase (respectively: TG: 1.48 +/- 0.50 nmol x mmol C -1; ETG: 2.65 +/- 0.93 nmol x mmol C -1; TG/ETG ratio: 0.67 +/- 0.31 and FLU: 9.29 +/- 3.37 nmol x mmol C -1. Similarly, no significant effect of physical training was observed on studied parameters between these two groups during either follicular phase (TG: 1.96 +/- 1.00 nmol x mmol C -1; ETG: 1.97 +/- 0.70 nmol x mmol C -1; TG/ETG ratio: 0.66 +/- 0.05 and FLU: 11.31 +/- 3.73 nmol x mmol C -1) or luteal phase (TG: 1.93 +/- 0.86 nmol x mmol C -1; ETG: 3.19 +/- 1.23 nmol x mmol C -1; TG/ETG ratio: 0.69 +/- 0.33 and FLU: 9.52 +/- 3.86 nmol x mmol C -1). It is concluded that although physical training could play a role in androgen metabolism, it has no significant incidence on urinary TG/ETG ratio. This study thus confirms that sportswomen can also be considered as normal subjects when they do not present any obvious endocrine disorder induced by physical activity.

A highly specific heterologous enzyme immunoassay for 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide (androstanediol-17G) and developmental patterns of urinary androstanediol-17G excretions.

We established a highly specific enzyme immunoassay (EIA) for 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide (androstanediol-17G). Rabbit antisera raised against 5 alpha-androstane-3 alpha, 11 alpha, 17 beta-triol 17-glucuronide 11-glutaryl bovine serum albumin and a heterologous tracer of androstanediol-17G conjugated with horseradish peroxidase at the glucuronic acid group were used. The EIA showed excellent specificity: there were no remarkable cross-reactivities with related androgens. The assay range for urine samples was 0.3-30 ng/ml. Recoveries of standards added to samples were 100-108%. Intra-assay and inter-assay coefficients of variation were 2.9-4.4% and 5.7-7.9%, respectively. The EIA was applied to urine samples of 407 males and 322 females to determine developmental patterns and normal ranges of androstanediol-17G excretions in 11 age groups (0 y, 1 y, 2-3 y, 4-5 y, 6-7 y, 8-9 y, 10-11 y, 12-13 y, 14-15 y, 16-17 y, and over 18 y). Urinary androstanediol-17G/creatinine (androstanediol-17G/Cre) ratios in both sexes were high in infancy, tended to decrease during childhood, and began to increase near adolescence. While androstanediol-17G/Cre ratio in girls increased at 8-9 y and reached a plateau during adolescence, that in boys increased at 10-11 y and continued to increase throughout adolescence. Androstanediol-17G/Cre ratios in girls were higher than those in boys at 6-7 y (P < 0.05) and at 8-9 y (P < 0.01). Androstanediol-17G/Cre ratios in boys were higher than those in girls at 12-13 y and at older ages (P < 0.01). These developmental patterns are parallel to age-related changes in androgenicity and serum androstanediol-17G, suggesting that urinary androstanediol-17G/Cre ratio could be a good marker for androgenicity in childhood.

Performance characteristics of a carbon isotope ratio method for detecting doping with testosterone based on urine diols: controls and athletes with elevated testosterone/epitestosterone ratios.

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.

Urinary 5 alpha-androstanediol and 5 beta-androstanediol measurement by gas chromatography after solid-phase extraction and high-performance liquid chromatography.

Urinary androstanediol measurement, often in association with other androgens, is commonly used to support the clinical diagnosis of idiopathic hirsutism. In addition, androgen excess has been shown to be the endocrine abnormality which characterizes patients with breast cancer. We recently developed a method for the measurement of urinary testosterone employing solid-phase extraction and HPLC purification before quantitative measurement by gas chromatography. In the present report we verify the feasibility of the method for the simultaneous measurement of 5 alpha-androstane-3 alpha,17 beta-diol and 5 beta-androstane-3 alpha,17 beta-diol in addition to testosterone in the same urine sample. The mean recovery for the whole procedure was 89.8% for 5 alpha-androstane-3 alpha,17 beta-diol and 87.8% for 5 beta-androstane-3 alpha, 17 beta-diol. The estimates of the coefficients of variation were 4.9% (95% confidence limits: 3.9-6.5%) and 3.9% (95% confidence limits: 3.1-5.2%), respectively. Accuracy was evaluated by standard addition and dilution assays and a linear relationship was found between expected and observed values (r2 = 0.997 for 5 alpha-androstane-3 alpha,17 beta-diol and r2 = 0.999 for 5 beta-androstane-3 alpha,17 beta-diol). The method is rapid, effective and suitable for the measurement of testosterone, 5 alpha-androstanediol and 5 beta-androstanediol in the same urine sample.

Screening urine for exogenous testosterone by isotope ratio mass spectrometric analysis of one pregnanediol and two androstanediols.

We propose a new screening method for testosterone (T) doping in sport. The current method for detecting T administration is based on finding a T to epitestosterone ratio (T/E) in urine that exceeds six. The difficulties with T/E are that T administration does not always result in a T/E>6 and that a rare individual will have T/E>6 in the absence of T administration. Our previous studies reveal that carbon isotope ratio helps to determine the origin of the urinary T because the values for T and its metabolites decrease after the administration of exogenous T. In this study, we present a rapid and efficient screening sample preparation method based on three successive liquid-solid extractions, deconjugation with E. coli beta-glucuronidase after the first extraction, acetylation after the second extraction, and a final extraction of the acetates. The 13C/12C of two T metabolites (5beta-androstane-3alpha,17beta-diol and 5alpha-androstane-3alpha,17beta-diol) and one pregnanediol as endogenous reference (5beta-pregnane-3alpha,20alpha-diol) was measured by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) on 10 ml of urine collected from 10 healthy men before and after T administration. Following T administration, the 13 C/12C of 5beta-androstane-3alpha,17beta-diol diacetate and 5alpha-androstane-3alpha,17beta-diol diacetate declined significantly from -26.2 per thousand to -30.8 per thousand and from -25.2 per thousand to -29.9 per thousand, respectively and the 13C/12C of 5beta-pregnane-3alpha,20alpha-diol diacetate was unchanged. In addition, the ratio of androstanediols to pregnanediol increased in the post-T urines.

Intramuscular administration of 5 alpha-dihydrotestosterone heptanoate: changes in urinary hormone profile.

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.

Androstanediol and 5-androstenediol profiling for detecting exogenously administered dihydrotestosterone, epitestosterone, and dehydroepiandrosterone: potential use in gas chromatography isotope ratio mass spectrometry.

The basis of a potential method for confirming intake of four natural androgens (testosterone, epitestosterone, dihydrotestosterone, and dehydroepiandrosterone is presented. The method relies on isolating from urine a steroid fraction containing androstenediol and androstanediol metabolites of these natural steroids and analyzing their 13C content by gas chromatography, combustion, isotope ratio mass spectrometry. The steroids were recovered from urine by conjugate hydrolysis with a Helix pomatia preparation (sulfatase and beta-glucuronidase), Girard T reagent separation to obtain a nonketonic fraction, and Sephadex LH-20 chromatography for purification. Metabolites appropriate for all of the natural steroids could be separated (as diacetates) by gas chromatography on a DB-17 capillary column viz.: 5 alpha (and beta)-androstane-3 alpha,17 alpha-diol (epitestosterone as precursor); 5 alpha (and beta)-androstane-3 alpha,17 beta-diol (testosterone as precursor); 5-androstene-3 beta,17 beta-diol (dehydroepiandrosterone precursor); and 5 alpha-androstane-3 alpha,17 beta- (and 17 alpha-) diol (dihydrotestosterone precursor). Measurement of the 13C content of the specific analytes after ingestion of the androgen precursors demonstrated a lowering of delta 13C/1000 value compared to normal values. Typically, in the male individual studied, delta 13C/1000 values for all components were -26 to -27 before drug administration and -29 to -30 at 6 h after, the latter values reflecting those obtaining for commercial synthetic steroid compared to in vivo synthesized steroid. While generally the metabolism of the steroids was as expected, this was not the case for 5 alpha-dihydrotestosterone. A major metabolite was 5 alpha-androstane-3 alpha,17 alpha-diol, which had presumably been formed by 17 beta/17 alpha isomerization, a process previously known for unnatural anabolics but not for natural hormones. The isolation, purification, and isotope ratio mass spectrometry techniques described may form the basis of a general method for confirming natural steroid misuse by sports participants.

Confirming testosterone administration by isotope ratio mass spectrometric analysis of urinary androstanediols.

A gas chromatographic combustion isotope ratio mass spectrometric (GC/C/IRMS) method was used for studying the incorporation of exogenous testosterone enanthate into excreted urinary 5 alpha- and 5 beta-androstane-3 alpha, 17 beta-diols. A multistep but straightforward work-up procedure produced a simple GC chromatogram of urinary steroid acetates composed principally of two androstanediols and pregnanediol. It is anticipated that such a method may form the basis of a doping control test for testosterone that could be used as a primary method during major sporting events or alternatively as a verification technique. Urine samples from five individuals were collected before and after administration of testosterone enanthate (250 mg). The delta 13C0/1000 value of andro-stanediols was around -26 to -28 during the baseline period and decreased to about -29 to -30 in the days following synthetic testosterone administration. One of the other major steroids in the chromatogram, pregnanediol, was utilized as the "internal standard," because its delta 13C0/1000 values did not markedly change following testosterone administration, remaining at -25 to -27. In all subjects studied, the delta 13C0/1000 values for androstanediols were reduced sufficiently over 8 days to confirm administration of synthetic testosterone. Although steroids isolated from urine of normal individuals from 12 different countries gave values between -24 and -28, this seemed not to be related to nationality or region. The most likely variable is the proportion of plants with low and high carbon 13 content in the diet. This variable is likely to be more affected by individual food preferences than broad ethnic food divisions. In this paper, we propose a ratio of delta 13C0/1000 for androstanediols to pregnanediol as a useful discriminant of testosterone misuse, a value above 1.1:1.0 being indicative of such misuse. The work-up procedure was designed for batch analysis and to use only simple techniques, rather than employ further instrumentation, such as high-performance liquid chromatography (HPLC), in purifying steroids for GC/C/IRMS.

The short-term effect of dietary pectin on plasma levels and renal excretion of dehydroepiandrosterone sulfate.

Studies specifically investigating the effects of single dietary components on plasma levels of dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) are rare. Especially no data is available with regard to specific dietary fibers. Therefore, the impact of pectin (a representative fiber that affects the enterohepatic recirculation of bile acids) was studied in a randomized crossover trial consisting of three diet periods characterized by the same food supply and daily doses of 0 g, 15 g or 30 g pectin. Blood and 24-h-urine samples were collected at the end of each 4-day diet period from 6 healthy male volunteers. Plasma levels of DHEA, cortisol and the major binding protein of DHEAS albumin remained unchanged with the varying pectin supplements. Also, no changes were observed for several urinary analytes including urinary DHEAS. However, effects of pectin intake (30, 15 versus 0 g/d) were seen for plasma DHEAS (9.3 +/- 2.8, 9.2 +/- 2.6, 8.0 +/- 3.1 mumol/L, p < 0.01) and total plasma cholesterol (4.4 +/- 0.7, 4.5 +/- 0.7, 4.7 +/- 0.8 mmol/L, p = 0.1). Obviously, the altered intake of fiber in the form of pectin affects plasma concentrations of DHEAS and cholesterol in an opposite direction. The reason for this is not known but a dietetically induced modulation of the binding properties of plasma albumin for DHEAS appears possible. Our findings suggest that the target tissue-available, not protein-bound fraction of circulating DHEAS (as reflected by the renal DHEAS output) is not necessarily altered when total plasma concentrations of DHEAS vary.

Proposed confirmatory procedure for detecting 5 alpha-dihydrotestosterone doping in male athletes.

Currently, there is no recommended confirmatory procedure for detecting doping with the anabolic steroid 5 alpha-dihydrotestosterone (DHT) in sportsmen. To develop a method, we determined ratios of hormone concentrations in urine from 120 healthy men and used these to set discrimination limits. These limits were then applied to results for urine specimens from 10 volunteers given DHT percutaneously (125 mg twice daily for 4 days). The ratio of DHT to epitestosterone (EpiT) was chosen as the primary marker of DHT administration, and ratios of 5 alpha-androstane-3 alpha, 17 beta-diol (5 alpha-ADIOL) to EpiT, 5 alpha-ADIOL to luteinizing hormone, and 5 alpha-ADIOL to 5 beta-androstane-3 alpha, 17 beta-diol were proposed as secondary markers. To evaluate method suitability, we analyzed 194 samples from sports competitors; results for 193 samples were negative, but the ratios in 1 sample greatly exceeded all the chosen limits. In conclusion, we propose that a test scheme based on our recommendations be considered for implementation in all Olympic drug-testing laboratories.

Urinary 3 alpha,17 beta-androstanediol glucuronide is a measure of androgenic status in Eld's deer stags (Cervus eldi thamin).

To determine the primary excretory by-products of testosterone (T), 85 microCi [3H]T was administered i.v. to two adult Eld's deer stags. Blood (10 ml) was collected by jugular venipuncture at 0, 5, 10, 15, 30, 45, 60, 90, 120, 150, 180, 240, and 480 min after isotope infusion, and all urine and feces were collected for 96 h after injection. Seventy percent of labeled circulating steroid was conjugated by 30 min postinfusion. The majority (80.4 +/- 3.2%) of T metabolites were excreted into urine, and 95.0 +/- 0.9% of these were conjugated, 95.8 +/- 0.2% being hydrolyzable with glucuronidase. Seven urinary androgen metabolites, including androstanediol (5 alpha-androstan-3 alpha-17 beta-diol and 5 beta-androstan-3 alpha-17 beta-diol), were identified in glucoronidase-hydrolyzed, ether-extracted Eld's deer urine pools after gas chromatography/mass spectrometry. A double-antibody 125I RIA for 5 alpha-androstanediol-3 alpha, 17 beta-diol,17-glucuronide (3 alpha-diol-G) was validated for unprocessed urine. Longitudinal assessments of urine samples collected from 13 stages for 3 yr revealed biological concordance between fluctuations in urinary 3 alpha-diol-G and serum T, as well as seasonal changes in secondary sexual characteristics. Overall correlation between "same-day" matched serum T and urinary 3 alpha-diol-G was 0.58, (n = 6; p < 0.001). Thus, monitoring urinary 3 alpha-diol-G provides a noninvasive, alternative method for characterizing male endocrine interrelationships in an endangered ungulate species.

Urinary profile of androgen metabolites at different stages of pubertal development in a population of sporting male subjects.

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)

[Unidentified steroid hormone of Cushing's syndrome and disease].

A previously unknown HPLC peak was recently observed in urine samples from patients with Cushing's syndrome and disease. We analysed dansylated derivatives of 17keto steroid glucuronides in urine samples from patients with Cushing's syndrome, Cushing disease and from healthy subjects using high-performance liquid chromatography (HPLC) on reversed-phase Cap Cell PakC8. All urine samples from patients with Cushing's syndrome caused by adrenal adenoma and Cushing's disease showed an unknown large peak at the point between [110HE-G] and [110HA-G] peaks and at a retention time of 25.4 min. The same unknown peak was also observed in urine samples from a patient with asymptomatic cortisol-producing adrenal adenoma and two patients with ectopic ACTH-producing tumor, though the peak height was low for the former and one of the latter but high for the second of the two patients. In contrast, healthy male and female urine only showed a very small peak at the same retention time. Urine samples from a Cushing disease treated with op'DDD and Cushing's syndrome bilaterally adrenalectomized and treating with cortisol showed no such peak. The retention time of this unknown peak is clearly different from that of seven 17keto steroid standard glucuronide conjugates. The structure of this substance may be closely related to [110HE-G] or [110HA-G].