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.

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.

Increased androgenic activity and breast cancer risk in premenopausal women.

Blood and urine specimens from 27 premenopausal breast cancer patients and 62 healthy controls have been compared with respect to concentration of testosterone and progesterone in blood and of testosterone and androstanediol in urine, measured in the luteal phase of the menstrual cycle. There was a strong positive association between the concentration of the two androgens, either in blood or urine, and breast cancer risk. A strong association was also observed with decreasing levels of progesterone. The association was statistically significant (p for trend less than 0.01) for each hormone; the rate ratios were 10.2 for serum testosterone (highest category), 5.6 for serum progesterone (lowest category), 8.4 for urinary testosterone (highest category), and 5.2 for androstanediol (highest category). The rate ratio for women presenting both high serum testosterone and low progesterone was 21.8 (4.1 to 116.1). Considering the exposure to at least one of three androgens at the highest level and low progesterone, the rate ratio was as high as 90.2 (8.2 to 989.7). This study provides evidence for the hypothesis that increased androgenic activity is an important risk indicator for breast cancer, particularly when associated with anovulation, as indicated by low serum progesterone level.

Anovulation and increased androgenic activity as breast cancer risk in women with fibrocystic disease of the breast.

The urine of 26 otherwise healthy women with fibrocystic disease of the breast was assayed by gas chromatography for testosterone and androstanediol (5alpha-androstane-3alpha, 17beta-diol), the major metabolite of dihydrotestosterone. The mean values for both androgens were significantly higher than in 18 normal women in the same age range. Sixteen of the 26 fibrocystic disease patients also had endometrial hyperplasia. Since the endometrial specimen was obtained in the premenstrual period, the presence of hyperplasia proved that the menstrual cycle in over two-thirds of the fibrocystic disease patients was nonovulatory.

PIP: Urine of 26 otherwise healthy women with fibrocystic breast disease (FCD) was assayed by gas chromatography for testosterone (Tes) and androstanediol (Ans), the major metabolite of dihydrotestosterone. The 26 patients under study had been subjected 3-5 months previously to biopsies of breast lumps, diagnosed as FCD. The histological features varied from simple cystic formations with moderate epithelial proliferation in 11 women (Group 1) to pronounced intraductal epithelial hyperplasia accompanied by epithelial cell atypia in 15 women (Group 2). The urinary Tes and Ans difference in FCD patients and controls (18 normal women) was significant at the level of p .01 for both androgens. In controls the mean excretion levels of Tes and Ans were 6.5 and 35 mcg/24 hours, respectively. In FCD patients, the mean Tes and Ans values were 17.4 and 68.5 mcg/24 hours, respectively. Group 2 presented a higher urinary Tes level than patients in Group 1, but the difference was not significant. The Ans level of Group 1 patients was significantly above normal (p .01) and near significantly higher (p .08) than that of the Group 2 patients; whereas the Ans level of Group 2 patients did not differ significantly from the normal value. Endometrial specimens showed that 16/26 FCD patients had endometrial hyperplasia. Since the endometrial specimen was obtained in the premenstrual period (Days 20-22), the presence of hyperplasia proved that the menstrual cycle in over two-thirds of the FCD patients was nonovulatory.

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.

[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 androstanediol and testosterone in adults.

Data are presented on the daily urinary excretion of androstanediol and testosterone in healthy adults using a sensitive radioligand assay. In nine men, the average urinary androstanediol (79 mug/day) was not significantly different from the urinary testosterone (84 mug/day). However, in women the average values of urinary androstanediol excretion (12 mug/day) were significantly higher than the urinary testosterone (4.2 mug/day). In each of the females, the urinary androstanediol was greater than the urinary testosterone. The data do not support the hypothesis that the daily urinary androstanediol excretion is a measure of the 5alpha-reduction of testosterone in androgen target tissues.

Urinary 5 alpha-androstane-3 alpha,17 beta-diol radioimmunoassay: a new clinical evaluation.

A rapid specific and reliable RIA for urinary 5 alpha-androstane-3 alpha,17 beta-diol (Adiol) is described using chromatographical purification and a specific antibody. Values are reported under some physiological and pathological conditions in 179 individuals. In 43 normal adult men, the mean (+/-SD) urinary Adiol excretion was 193 +/- 77 micrograms/24 h, and in 29 normal women it was 44 +/- 23 micrograms/24 h. These values are significantly different (P less than 0.01). In 49 hirsute women, urinary Adiol Excretion was elevated (137 +/- 51 micrograms/24 h) and significantly different from this value in normal women (P less than 0.01). The urinary Adiol excretion in 10 postmenopausal women was very low (less than 5 micrograms/24 h). In normal adult subjects, the theoretical contribution to urinary Adiol of the major secreted androgens was calculated. Whereas dehydroisoandrosterone and dehydroisoandrosterone sulfate yield the same amount of urinary Adiol in both sexes, testosterone is the main precursor of Adiol in men and androstenedione is the main precursor in normal premenopausal and hirsute women. However, the amount of Adiol recovered in the 24-h urine depends not only on the secretion rate of androstenedione and testosterone but is also related to the testosterone 5 alpha-reductase activity present in androgen target cells, especially in sexual skin.

Contribution of plasma androstenedione to 5 alpha-androstanediol glucuronide in women with idiopathic hirsutism.

To confirm that plasma delta 4 androstenedione (delta 4) is the main precursor for 5 alpha-androstane-3 alpha, 17 beta-diol glucuronide (Adiol G) in patients with idiopathic hirsutism (IH), delta 4 was cutaneously applied to five normal women and five women with IH. Several parameters of androgen metabolism were assayed basally and throughout the studies. Those included plasma delta 4, testosterone, and dihydrotestosterone as well as urinary Adiol G and testosterone glucuronide excretion. Under basal conditions plasma testosterone, delta 4, and dihydrotestosterone did not differ significantly between the two groups of subjects. Urinary Adiol G excretion was significantly higher (P less than 0.01) in IH patients [123 +/- 36 (SE) micrograms/24 h] than in the normal women group (45 +/- 20 micrograms/24 h). After percutaneous administration of delta 4, plasma delta 4 increased in both groups by nearly 600% and there was a 300% increase in Adiol G excretion in IH patients (336 +/- 57 micrograms/24 h), whereas only a 50% increase occurred in normal women (65 +/- 17 micrograms/24 h). We postulate that plasma delta 4 may be the main precursor accounting for the increased production of urinary Adiol G in women with IH, in whom hirsutism may be due to a high 5 alpha-reductase activity. Indeed, 5 alpha-reductase as measured in vitro in pubic skin was significantly higher in hirsute patients (224 +/- 66 fmol/mg skin X h) than in normal women (45 +/- 15 fmol/mg skin X h).

Elevated production and metabolic clearance rates of androgens in morbidly obese women.

Blood production rates of testosterone, dihydrotestosterone (DHT), and 3 alpha-androstanediol (3 alpha-diol) were found to be approximately 2-fold elevated in morbidly obese, nonhirsute, normally menstruating women. Values were intermediate between those found in normal women and those in a group of nonobese normally menstruating women with idiopathic hirsutism. Elevated androgen production rates in obese women were associated with 2- to 3-fold increases in MCRs, presumably due to decreased levels of sex hormone-binding globulin. Thus, increased production rates were offset by increased MCRs, resulting in plasma testosterone, DHT, and 3 alpha-diol concentrations that were similar in the obese and normal women. By contrast, women with hirsutism had increased production rates associated with elevated plasma androgens as well as increased MCRs. Urinary excretion of testosterone glucuronide and 3 alpha-diol glucuronide (3 alpha-diol G) were elevated in both obese and hirsute women, paralleling the increased androgen production rates. Despite increased production rates and excretion of androgens, obese women exhibited no menstrual abnormalities, hirsutism, or other signs of virilism. To explore the apparent ineffectiveness of increased androgen production to produce virilizing symptoms, we measured plasma 3 alpha-diol G levels as a measure of peripheral androgen action. The mean +/- SE plasma 3 alpha-diol G was 53 +/- 8 ng/dl in obese women and 36 +/- 6 in normal women; by contrast, women with idiopathic hirsutism had levels of 440 +/- 99, a 12-fold elevation. Plasma testosterone glucuronide in obese and hirsute women were only 2- to 3-fold elevated, while plasma DHT glucuronide was not increased in obese women and was only 2-fold elevated in hirsute women. Thus, obesity is a state of increased androgen production and accelerated clearance. 3 alpha-diol G levels in obese women were only minimally elevated, in contrast to values in the hirsute women, perhaps reflecting the apparent androgen ineffectiveness.

Comparative effects of cyproterone acetate or a long-acting gonadotropin-releasing hormone agonist in polycystic ovarian disease.

A randomized cross-over study was done to compare the therapeutic efficacy of cyproterone acetate (CPA) and a depot preparation of the LHRH superagonist (DTrp6-LHRH) in 10 patients with polycystic ovarian disease (PCO). All patients were treated with both agents (50 mg/day CPA, orally and (3 mg DTrp6-LHRH, im, approximately once a month) for 3 months, the 2 treatment periods being separated by 6 months. Both treatments resulted in marked clinical improvement, with diminished acne and seborrhoea and normalization of ovarian size by ultrasonographic criteria. In response to CPA treatment, basal plasma gonadotropin levels decreased, but the response to a LHRH test was not completely suppressed. Plasma estradiol, estrone, testosterone, and androstenedione levels significantly decreased, but urinary 3 alpha-androstanediol and plasma dehydroepiandrosterone sulfate levels did not change significantly. In contrast to CPA treatment, both basal and stimulated gonadotropin levels were completely suppressed after 3 weeks of treatment with DTrp6-LHRH. After a slight initial evaluation on day 2, plasma estrogen and androgen levels, with the exception of dehydroepiandrosterone sulfate fell into the castrate range urinary 3 alpha-androstanediol excretion decreased significantly. Thus, in patients with PCO, LHRH-A induced more complete gonadotropin inhibition than did CPA. After cessation of either therapy, the disease rapidly recurred.

Decreased urinary 5 alpha-androstane-3 alpha,17 beta-diol glucuronide excretion in patients with benign prostatic hyperplasia.

Urinary testosterone and 3 alpha-androstanediol (3 alpha diol G) glucuronides together with plasma testosterone, 5 alpha-dihydrotestosterone (DHT), and delta 4-androstenedione (delta 4) were measured in 43 normal young men (18-36 yr old), 23 elderly men without clinically evident prostatic pathology (54-89 yr old), 68 elderly men with benign prostatic hyperplasia (BPH group; 54-91 yr old), and 26 elderly men with well differentiated cancer of the prostate (K group; 63-97 yr old). Plasma testosterone decreased slightly with age in all 3 elderly groups (from 591 to 438, 479, and 444 ng/100 ml, respectively). Plasma DHT, on the contrary, was significantly (P less than 0.01) higher in the BPH group than in the other three groups (68 vs. 30, 37, and 32 ng/100 ml, respectively). Plasma delta 4 was significantly lower (P less than 0.01) in the elderly K group than in all other groups (59 vs. 109, 83, and 78 ng/100 ml, respectively). Urinary testosterone glucuronide decreased with age in all 3 elderly groups (from 109 to 55, 38, and 44 micrograms/24 h, respectively) as a result of decreased androgen production rates with age. All 3 elderly groups also had decreased urinary 3 alpha diol G, from 194 to 123, 55, and 118 micrograms/24 h, respectively. The group of elderly patients with BPH had the lowest mean urinary 3 alpha diol G excretion together with the highest mean plasma DHT. This low urinary 3 alpha diol G excretion, which reflects a decrease in both androgen production and DHT metabolism, suggests a decrease in 3 alpha-hydroxysteroid dehydrogenase activity, which, in turn, could explain the increased DHT availability and tissue retention in most target organs. Moreover, the extent of these modifications in androgen metabolism specific to the BPH condition raises the question of an overall alteration of androgen metabolism in patients with BPH which could be the cause of the disease.

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)

Possible indices for the detection of the administration of dihydrotestosterone to athletes.

Dihydrotestosterone (DHT) can be used by an athlete as an anabolic steroid to evade the current International Olympic Committee approved drug tests. To investigate the possibility of a method for its detection, the heptanoate ester of DHT was administered to two male subjects (150 mg i.m.). Urine samples, collected before and after the injection, were subjected to enzymatic hydrolysis and the excretion rates of DHT, 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and testosterone (T) were determined by radioimmunoassay. Relative changes in the excretion of DHT, 3 alpha-diol, 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol), 5 beta-androstane-3 alpha, 17 beta-diol (5 beta-diol), T and epitestosterone (17 alpha hydroxyandrost-4-en-3-one; Epi-T) were determined by gas chromatography-mass spectrometry (GC-MS). Following administration of DHT, the urinary excretion rates of DHT, 3 alpha-diol and 3 beta-diol increased when compared to those of T, Epi-T, 5 beta-diol and luteinizing hormone (LH). Concentrations of DHT in the plasma increased whereas those of T, LH and follicle stimulating hormone decreased. The changes following such modest doses of DHT suggest that these ratios of urinary hormones may be used for the detection of doping with DHT.

Decreased 3 alpha-androstanediol glucuronide levels in plasma and random urines in male pseudohermaphroditism caused by 5 alpha-reductase deficiency.

Concentrations of 3 alpha-diol glucuronide (3 alpha-diol G) in plasma and/or random urine samples were determined in seven subjects with familial male pseudohermaphroditism (FMP) due to 5 alpha-reductase deficiency (5 alpha-RD). All subjects were natives of an isolated Turkish village with a high incidence of consanguineous marriage. A specific and sensitive antibody to 3 alpha-diol was used for radioimmunoassay of 3 alpha-diol G after hydrolysis and chromatographic purification. The mean plasma 3 alpha-diol G in three subjects (31 ng/dL) was much lower than the normal male concentration (516 +/- 50) (+/- SE) and was even lower than normal female values (119 +/- 10.9 ng/dL). In five subjects, mean urinary 3 alpha-diol G in random urine samples was 7.6 (range 2.1 to 12.7) ng/mg creatinine. This was considerably decreased compared with the mean adult male concentration of 65.4 +/- 9.4 and even lower than normal age-matched nonhirsute female values (19.6 +/- 2.1 ng/mg Cr). To validate the use of 3 alpha-diol G/creatinine ratios in random urine samples, correlations of three consecutive eight-hour samples with 24-hour values were determined in 8 male and 3 female age-matched controls. There was an excellent correlation (r = .95) and the linear regression line (y = 0.51x + 2.58) indicates that the 24-hour excretion of 3 alpha-diol G in microgram/24 h is approximately twice the random urinary concentration in ng/mg Cr.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurements of 3 alpha,17 beta-androstanediol glucuronide in serum and urine and the correlation with skin 5 alpha-reductase activity.

Serum and urinary measurements of 3 alpha,17 beta-androstanediol glucuronide (3 alpha-diol G) reflect peripheral androgen action and have been useful clinically. This study was designed to compare these levels in hirsute women, normal premenopausal and postmenopausal women, and in men and to correlate each measurement with skin 5 alpha-reductase activity (5 alpha-RA), an excellent correlate of androgenicity. Although serum 3 alpha-diol G values were similar in premenopausal and postmenopausal women, values were higher in hirsute women and in men. This pattern was similar for urinary 3 alpha-diol G but with greater overlap in values between hirsute and nonhirsute women and men. Serum 3 alpha-diol G showed a highly significant correlation with levels of genital 5 alpha-RA (r = 0.839, P less than 0.001), whereas urinary 3 alpha-diol G did not correlate. Serum and urinary 3 alpha-diol G also did not correlate with one another (r = 0.03). These data suggest that while both serum and urinary 3 alpha-diol G may be useful clinically, serum 3 alpha-diol G appears to correlate better with androgenicity and 5 alpha-RA. It is suggested further that the sources of serum and urinary 3 alpha-diol G may be somewhat different.

Formation of androstanediol from 13C-labeled testosterone in humans.

The determination of urinary 5 alpha-androstane-3 alpha,17 beta-diol (3a-Diol) by gas chromatography/mass spectometry during and after the infusion of stable-labeled testosterone (T) represents an alternative to the use of radioactive label for turnover studies in vivo. Using this methodology to assess the urinary excretion rates of T and 3a-Diol in healthy men (n = 6) and women (n = 5) during and after the intravenous infusion (t = 4 hours) of 20 mg (men) or 5 mg (women) [13C]testosterone, the cumulative renal excretion of 13C-labeled T was found to be 15.6 +/- 9.6 micrograms/24 hours (men) and 1.1 +/- 1.6 micrograms/24 hours (women), equivalent to 0.08% +/- 0.05% and 0.02% +/- 0.03% of the infused amount of 13C-T, respectively. The cumulative excretion of 13C-3a-Diol was 67.7 +/- 19.9 micrograms/24 hours (men) and 10.0 +/- 6.0 micrograms/24 hours (women), equivalent to 0.3 +/- 0.1% and 0.2 +/- 0.1% of the infused dose of 13C-labeled testosterone, respectively.

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.

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.

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.