Detection of 5α-androst-2-en-17-one and variants: Identification of main urinary metabolites in human urine samples by GC-MS and NMR.

Two steroids were identified in a supplement named D-2 following the detection of unknown compounds during the routine testing of an athlete's sample. The main glucuroconjugated metabolites were isolated from this urine by high performance liquid chromatography (HPLC) following enzymatic hydrolysis and identified by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) analyses as being 2α-hydroxy-5α-androst-3-en-17-one (M1) and 2ß,3α-dihydroxy-5α-androstan-17-one (M2). A third metabolite, 3α,4ß-dihydroxy-5α-androstan-17-one (M3) was also detected, however in lower amounts. The precursor steroids, 5α-androst-2-en-17-one (1) and 5α-androst-3-en-17-one (2) were present in the first D-2 products offered on the Internet. Later, the corresponding 17-hydroxyl compounds were offered as such or as esters (acetate, cypionate) in different relative ratios. Both M2 and M3 were synthesized from the trans-diaxial hydrolysis of the corresponding 2α,3α- and 3α,4α-epoxides (3). These were excreted in the hours following the controlled administration of the commercial product called D-2 R to a male volunteer and were also produced from the incubation of 1 and 2 with S9 liver fractions. Some preparations contain predominantly the alkene in C-2 and, therefore, an efficient detection method must include both primary metabolites M1 and M2. The latter was found equally in the fractions extracted following the enzymatic hydrolysis with ß-glucuronidase and the chemical solvolysis, which may ease its identification. Copyright © 2016 John Wiley & Sons, Ltd.

Targeted Metabolomics Approach To Detect the Misuse of Steroidal Aromatase Inhibitors in Equine Sports by Biomarker Profiling.

The use of anabolic androgenic steroids (AAS) is prohibited in both human and equine sports. The conventional approach in doping control testing for AAS (as well as other prohibited substances) is accomplished by the direct detection of target AAS or their characteristic metabolites in biological samples using hyphenated techniques such as gas chromatography or liquid chromatography coupled with mass spectrometry. Such an approach, however, falls short when dealing with unknown designer steroids where reference materials and their pharmacokinetics are not available. In addition, AASs with fast elimination times render the direct detection approach ineffective as the detection window is short. A targeted metabolomics approach is a plausible alternative to the conventional direct detection approach for controlling the misuse of AAS in sports. Because the administration of AAS of the same class may trigger similar physiological responses or effects in the body, it may be possible to detect such administrations by monitoring changes in the endogenous steroidal expression profile. This study attempts to evaluate the viability of using the targeted metabolomics approach to detect the administration of steroidal aromatase inhibitors, namely androst-4-ene-3,6,17-trione (6-OXO) and androsta-1,4,6-triene-3,17-dione (ATD), in horses. Total (free and conjugated) urinary concentrations of 31 endogenous steroids were determined by gas chromatography-tandem mass spectrometry for a group of 2 resting and 2 in-training thoroughbred geldings treated with either 6-OXO or ATD. Similar data were also obtained from a control (untreated) group of in-training thoroughbred geldings (n = 28). Statistical processing and chemometric procedures using principle component analysis and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) have highlighted 7 potential biomarkers that could be used to differentiate urine samples obtained from the control and the treated groups. On the basis of this targeted metabolomic approach, the administration of 6-OXO and ATD could be detected for much longer relative to that of the conventional direct detection approach.

Development of a sensitive GC-C-IRMS method for the analysis of androgens.

The administration of anabolic steroids is one of the most important issues in doping control and is detectable through a change in the carbon isotopic composition of testosterone and/or its metabolites. Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), however, remains a very laborious and expensive technique and substantial amounts of urine are needed to meet the sensitivity requirements of the IRMS. This can be problematic because only a limited amount of urine is available for anti-doping analysis on a broad spectrum of substances. In this work we introduce a new type of injection that increases the sensitivity of GC-C-IRMS by a factor of 13 and reduces the limit of detection, simply by using solvent vent injections instead of splitless injection. This drastically reduces the amount of urine required. On top of that, by only changing the injection technique, the detection parameters of the IRMS are not affected and there is no loss in linearity.

Carbon isotope ratio analysis of endogenous glucocorticoid urinary metabolites after cortisone acetate and adrenosterone administration for doping control.

Glucocorticoids are listed on the World Anti-Doping Agency (WADA) Prohibited List of substances. The detection of the administration of hydrocortisone and cortisone is complicated by the fact that the human body also produces these steroids naturally. Gas chromatography-combustion-isotope ratio mass spectrometry can be utilized to determine the use of endogenous glucocorticoids by measuring the carbon isotope ratio (CIR) of their resulting metabolites in human urine samples. A comprehensive sample preparation protocol for the analysis of endogenous glucocorticoid urinary metabolites was developed and validated, incorporating the use of high performance liquid chromatography (HPLC) for purification and chemical oxidation for derivatisation. Target compounds were tetrahydrocortisol and tetrahydrocortisone, and 11ß-hydroxyetiocholanolone, 11-oxoetiocholanolone and 11ß-hydroxyandrosterone, while pregnanediol functioned as the endogenous reference compound. Urine samples from a population of 50 volunteers were analyzed to determine CIR reference limits. Excretion studies of the endogenous glucocorticoid preparation cortisone acetate (25 mg oral) and the dietary supplement adrenosterone (75 mg oral) were conducted with six male individuals. Variable changes in steroid metabolite isotopic composition were found across subjects after administration. The study also revealed that CIR analysis of the major glucocorticoid metabolites tetrahydrocortisol and tetrahydrocortisone is necessary to unambiguously distinguish administration of cortisone and adrenosterone, the former officially restricted to out-of-competition use by athletes, the latter not being restricted at the current time. Moreover, this study reaffirms that CIR methods for the doping control of endogenous steroids should not rely upon a single ERC, as the administration of an appropriate precursor to that ERC could cause complications during analysis.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 3. Characterization, using GC-MS and GC-MS/MS, of androstanes and androstenes.

Urine from neonates with 21-hydroxylase deficiency contains a large range of androstane(ene)s, many of which have not been previously described. We present their characterization as the third part of a comprehensive study of urinary steroids, aiming to enhance the diagnosis of this disorder and to further elucidate steroid metabolism in neonates. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of hitherto undescribed androstane(ene)s. GC-MS/MS was pivotal for the structural characterization of 2-hydroxylated androstenediones but GC-MS was generally more informative for androstane(ene)s, in contrast to 17-hydroxylated pregnane(ene)s. Parallels were found between the GC-MS and GC-MS/MS characteristics of structurally similar androstenediones and progesterones without a substituent on the D-ring, but not with those of 17-hydroxylated progesterones. Assignment of 5α(ß) orientation, based on GC-MS characteristics, was possible for 11-oxo-androstanes. The major endogenous 3ß-hydroxy-5-enes in 21-hydroxylase deficiency did not differ from those in unaffected neonates. The key qualitative and quantitative differences encompassed 5α(ß)-androstanes and 3-oxo-androst-4-enes. Major positions of hydroxylation in these were C(2), C(6), C(11), C(16) and C(18). Additional oxo-groups were common at C(6), C(11) and C(16). We conclude that the presence of multiple further oxygenated metabolites of androstenedione in urine from neonates with 21-hydroxylase deficiency and their pattern indicate a predominance of the classical pathway of androgen synthesis and reflect an increased demand for clearance. The positions of oxygenation in androstane(ene)s are dependent on the configuration at C(3)-C(5).

Restricted-access nanoparticles for magnetic solid-phase extraction of steroid hormones from environmental and biological samples.

Restricted-access materials based on non-ionic surfactant-coated dodecyl-functionalized magnetic nanoparticles were prepared and applied to extract steroid hormones from environmental and biological samples. The magnetic nanoparticles were synthesized by co-precipitation, and were functionalized with dodecyltriethoxysilane, giving dodecyl-grafted magnetic nanoparticles (C12-Fe3O4). They were further modified with different non-ionic surfactants by self-assembly adsorption. Several types of non-ionic surfactants, Tween-20, 40, 60 and 85, and Span-40, 60 and 80, were investigated as the coatings. Tween surfactants coated C12-Fe3O4, named as TW-20 (40, 60, 85)-C12, exhibited good dispersibility in aqueous solution, which was a preferred character in extraction; besides, TW-20-C12 and TW-40-C12 showed good anti-interference ability and satisfactory reproducibility when they were used as magnetic solid-phase extraction (MSPE) sorbents. The factors that may influence the extraction, including the amount of magnetic nanoparticles, extraction and desorption time, the amount of salt addition, the type and volume of desorption solvent, the volume of methanol addition and pH of sample solution, were investigated in detail. High performance liquid chromatography-UV detection was employed for analysis of target analytes (steroid hormone compounds). The developed method was successfully used for the determination of the target analytes in environmental and urine samples. Both tested materials afforded good recovery, satisfactory reproducibility and low limits of detection for environmental samples, which indicates that the materials possessed anti-interference ability. However, compared to TW-40-C12, TW-20-C12 nanoparticles provided better recovery in relatively complex biological samples, which may indicate that the latter one is more appreciated in complex samples.

Gas chromatography coupled to mass spectrometry-based metabolomic to screen for anabolic practices in cattle: identification of 5α-androst-2-en-17-one as new biomarker of 4-androstenedione misuse.

The use of anabolic steroids as growth promoters for meat-producing animals is banned within the European Union. However, screening for the illegal use of natural steroid hormones still represents a difficult challenge because of the high interindividual and physiological variability of the endogenous concentration levels in animals. In this context, the development of untargeted profiling approaches for identifying new relevant biomarkers of exposure and/or effect has been emerging for a couple of years. The present study deals with an untargeted metabolomics approach on the basis of GC-MS aiming to reveal potential biomarkers signing a fraudulent administration of 4-androstenedione (AED), an anabolic androgenic steroid chosen as template. After a sample preparation based on microextraction by packed sorbent, urinary profiles of the free and deglucurono-conjugates urinary metabolites were acquired by GC-MS in the full-scan acquisition mode. Data processing and chemometric procedures highlighted 125 ions, allowing discrimination between samples collected before and after an administration of 4-AED. After a first evaluation of the signal robustness using additional and independent non-compliant samples, 17 steroid-like metabolites were pointed out as relevant candidate biomarkers. All these metabolites were then monitored using a targeted GC-MS/MS method for an additional assessment of their capacity to be used as biomarkers. Finally, two steroids, namely 5α-androstane-3ß,17α-diol and 5α-androst-2-en-17-one, were concluded to be compatible with such a definition and which could be finally usable for screening purpose of AED abuse in cattle.

In vitro and in vivo studies of androst-4-ene-3,6,17-trione in horses by gas chromatography-mass spectrometry.

This paper describes the application of gas chromatography-mass spectrometry (GC-MS) for in vitro and in vivo studies of 6-OXO in horses, with a special aim to identify the most appropriate target metabolite to be monitored for controlling the administration of 6-OXO in racehorses. In vitro studies of 6-OXO were performed using horse liver microsomes. The major biotransformation observed was reduction of one keto group at the C3 or C6 positions. Three in vitro metabolites, namely 6alpha-hydroxyandrost-4-ene-3,17-dione (M1), 3alpha-hydroxyandrost-4-ene-6,17-dione (M2a) and 3beta-hydroxyandrost-4-ene-6,17-dione (M2b) were identified. For the in vivo studies, two thoroughbred geldings were each administered orally with 500 mg of androst-4-ene-3,6,17-trione (5 capsules of 6-OXO((R))) by stomach tubing. The results revealed that 6-OXO was extensively metabolized. The three in vitro metabolites (M1, M2a and M2b) identified earlier were all detected in post-administration urine samples. In addition, seven other urinary metabolites, derived from a further reduction of either one of the remaining keto groups or one of the remaining keto groups and the olefin group, were identified. These metabolites included 6alpha,17beta-dihydroxyandrost-4-en-3-one (M3a), 6,17-dihydroxyandrost-4-en-3-one (M3b and M3c), 3beta,6beta-dihydroxyandrost-4-en-17-one (M4a), 3,6-dihydroxyandrost-4-en-17-one (M4b), 3,6-dihydroxyandrostan-17-one (M5) and 3,17-dihydroxyandrostan-6-one (M6). The longest detection time observed in urine was up to 46 h for the M6 metabolite. For blood samples, the peak 6-OXO plasma concentration was observed 1 h post administration. Plasma 6-OXO decreased rapidly and was not detectable 12 h post administration.

Development of criteria for the detection of adrenosterone administration by gas chromatography-mass spectrometry and gas chromatography-combustion-isotope ratio mass spectrometry for doping control.

Adrenosterone (androst-4-ene-3,11,17-trione, 11-oxoandrostenedione) is an endogenous steroid hormone that has been promoted as a dietary supplement capable of reducing body fat and increasing muscle mass. It is proposed that adrenosterone may function as an inhibitor of the 11beta-hydroxysteroid dehydrogenase type 1 enzyme (11beta-HSD1), which is primarily responsible for reactivation of cortisol from cortisone. The urinary metabolism of adrenosterone was investigated, after a single oral administration in two male subjects, by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Substantially increased excretion of 11beta-hydroxyandrosterone, 11beta-hydroxyetiocholanolone, 11-oxoandrosterone and 11-oxoetiocholanolone was observed. Minor metabolites such as 3alpha,17beta-dihydroxy-5beta-androstan-11-one, 3alpha-hydroxyandrost-4-ene-11,17-dione and 3alpha,11beta-dihydroxyandrost-4-en-17-one were also identified. The exogenous origin of the most abundant adrenosterone metabolites was confirmed by GC-C-IRMS according to World Anti-Doping Agency criteria. Through analysis of a reference population data set obtained from urine samples provided by elite athlete volunteers (n = 85), GC-MS doping control screening criteria are proposed: 11beta-hydroxyandrosterone concentration greater than 10 000 ng/mL (specific gravity adjusted to 1.020) or 11beta-hydroxyandrosterone/11beta-hydroxyetiocholanolone ratio greater than 20.Urine samples fulfilling these screening criteria may be subjected to GC-C-IRMS analysis for confirmation of adrenosterone administration.

Quantitative analysis of androst-4-ene-3,6,17-trione and metabolites in human urine after the administration of a food supplement by liquid chromatography/ion trap-mass spectrometry.

6-OXO, a new nutritional supplement commercially available on the internet, is sold as an aromatase-inhibitor and contains androst-4-ene-3,6,17-trione as active ingredient. This anabolic steroid is a prohibited substance in sports. Androst-4-ene-3,6,17-trione is metabolised to androst-4-ene-6alpha-ol-3,17-dione and androst-4-ene-6alpha,17beta-diol-3-one. A fast, sensitive and accurate LC/MS method was developed and validated for the quantification of androst-4-ene-3,6,17-trione and its metabolites in urine. The method is capable of determining the stereochemical position of the hydroxy-group at C-6 of the metabolites and consists of a liquid-liquid extraction step with diethylether after enzymatic hydrolysis, followed by separation on a reversed phase column. Ionisation of the analytes is carried out using atmospheric pressure chemical ionisation. The limit of quantification of the method was 5 ng/mL for all compounds. The accuracy ranged from 14.8 to 1.3% for androst-4-ene-3,6,17-trione, 9.4 to 1.6% for androst-4-ene-6alpha-ol-3,17-dione and 4.1 to 3.2% for androst-4-ene-6alpha,17beta-diol-3-one in the range of 5-1000 ng/mL. Using this method androst-4-ene-6alpha-ol-3,17-dione was identified as a major urinary metabolite, whereas androst-4-ene-6alpha,17beta-diol-3-one as a minor metabolite. While the parent compound is predominantly excreted in conjugated form, both metabolites are solely excreted as conjugates.

Detection of androst-4-ene-3,6,17-trione (6-OXO) and its metabolites in urine by gas chromatography-mass spectrometry in relation to doping analysis.

The metabolism and excretion of androst-4-ene-3,6,17-trione after administration of the 'nutritional' supplement 6-OXO was investigated by gas chromatography-mass spectrometry (GC-MS) in full-scan mode. The parent drug androst-4-ene-3,6,17-trione and androst-4-ene-6alpha,17beta-diol-3-one and androst-4-ene-6alpha-ol-3,17-dione were detected in the post-administration urine samples. Because androst-4-ene-3,6,17-trione is an anabolic steroid and an aromatase inhibitor, this substance is regarded as a doping agent. Hence, a selective and sensitive GC-MS method in selected ion monitoring mode for the detection of the TMS-enol-TMS-ether derivatives of these substances was developed and validated for doping control purposes. The limit of detection (LOD) of the investigated compounds ranged from 5 to 10 ng/mL. Using this method, the detection time for androst-4-ene-3,6,17-trione and androst-4-ene-6alpha,17beta-diol-3-one was 24 h, while androst-4-ene-6alpha-ol-3,17-dione could be detected up to 37 h after administration of the dose recommended by the manufacturer.

Newly identified steroid hormone in urine of patients with Cushing's syndrome: 3alpha,11 beta-dihydroxy-4-androsten-17-one.

We have previously reported that the urine of patients with Cushing's syndrome, including pituitary adenoma cases and adrenal adenoma cases, consistently show a conspicuous peak in the chromatographical analysis of 17-ketosteroid fraction but not in the urine of control subjects. The substance emerges just before 11beta-hydroxy-androsterone (11beta-OH-A) in capillary gas chromatography. In the present study, we have identified an "unknown peak substance" observed in the urine of Cushing's syndrome patients using gas chromatography-mass spectrometry (GC/MS). Trimethylsilylether (TMS)-derivative of the substance was found to have a molecular weight (MW) of 448, which is similar to that of 11-OH-A (MW: 450). From these findings, we hypothesized that the substance had the structure of a C-19 steroid with two hydroxyl groups at positions C-3 and C-11, one keto-group at C-17 and a double bond between C-4 and C-5 of the A ring. We hypothesized that the unknown peak substance was 3alpha,11beta-dihydroxy-4-androsten-17-one (3alpha,11beta-DH-A). To confirm this speculation we synthesized 3alpha,11beta-DH-A and compared the elution pattern of it with that of the "unknown peak substance" using GC and GC/MS. We found that both substances were indistinguishable by GC and GC/MS analysis. These results suggest that the unknown substance observed in the urine of patients with Cushing's syndrome is 3alpha,11beta-DH-A.

Demonstration of 2-unsaturated C19-steroids in the urine of female Asian elephants, Elephas maximus, and their dependence on ovarian activity.

Air-borne volatile substances have been demonstrated to signal oestrus, induce ovulation and synchronize ovarian activity in different mammals. An oestrous-related pheromone of the female Asian elephant (Elephas maximus) is known to induce behavioural responses in elephant bulls. Additional data revealed that timing of oestrus in females with close social relationships tends to be synchronized. Therefore, urine from female Asian elephants might be expected to contain luteal phase-dependent volatile substances, which may function as additional chemical signals in this species. The aim of the present study was to identify such compounds and to investigate their pattern of excretion throughout the ovarian cycle. Urine samples were collected three times a week during the follicular phase and one to three times a week during the luteal phase from five adult female Asian elephants from a total of 13 non-conception cycles and one conception cycle, including the first 72 weeks of pregnancy. A simple headspace solid-phase microextraction method has been developed for quantification of urinary volatile substances and analysis was performed by gas chromatography. The comparison of urine collected during the follicular and the luteal phase indicated the presence of two luteal phase-dependent substances. Mass spectrometry was used to identify one substance as 5alpha-androst-2-en-17-one and a second substance as the corresponding alcoholic compound 5alpha-androst-2-en-17beta-ol. The 5alpha-androst-2-en-17beta-ol and -17-one profiles reflected cyclic ovarian activity with clear (10-20-fold) luteal phase increases. Furthermore, measurements of both compounds were correlated positively with the concentration of urinary pregnanetriol and indicated cycle duration (15.1 +/- 1.2 weeks) similar to that obtained from pregnanetriol measurements (15.2 +/- 1.6 weeks). The results demonstrate the presence of two luteal phase-specific steroidal volatile compounds in elephant urine. One of the substances, 5alpha-androst-2-en-17-one, has been demonstrated in human axillary bacterial isolates. The measurement of both volatile substances in elephant urine can be used for rapid detection of the stage of the ovarian cycle, as the analysis can be completed within 2 h.

Disposition and pharmacokinetics of [14C]finasteride after oral administration in humans.

The disposition of [14C]finasteride, a competitive inhibitor of steroid 5 alpha-reductase, was investigated after oral administration of 38.1 mg (18.4 microCi) of drug in six healthy volunteers. Plasma, urine, and feces were collected for 7 days and assayed for total radioactivity. Concentrations of finasteride and its neutral metabolite, omega-hydroxyfinasteride (monohydroxylated on the t-butyl side chain), in plasma and urine were determined by HPLC assay. Mean excretion of radioactivity equivalents in urine and feces equaled 39.1 +/- 4.7% and 56.8 +/- 5.0% of the dose, respectively. The mean peak plasma concentrations reached for total radioactivity (ng equivalents), finasteride, and omega-hydroxyfinasteride were 596.5 +/- 88.3, 313.8 +/- 99.4, and 73.7 +/- 11.8 ng/ml, respectively, at approximately 2 hr; the mean terminal half-life for drug and metabolite was 5.9 +/- 1.3 and 8.4 +/- 1.7 hr, respectively. Of the 24-hr plasma radioactivity, 40.9% was finasteride, 11.8% was the neutral metabolite, and 26.7% was characterized as an acidic fraction of radioactivity. Binding of [14C]finasteride to plasma protein was extensive (91.3 to 89.8%), with a trend suggesting concentration dependency (range 0.02 to 2 micrograms/ml). Little of the dose was excreted in urine as parent (0.04%) or omega-hydroxyfinasteride (0.4%). Urinary excretion of radioactivity was largely in the form of acidic metabolite(s)--18.4 +/- 1.7% of the dose was eliminated as the omega-monocarboxylic acid metabolite. Finasteride was scarcely excreted unchanged in feces. In humans, finasteride is extensively metabolized through oxidative pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatographic determination of N-(2-methyl-2-propyl)-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta-carboxamide, a 4-azasteroid, in human plasma from a phase I study.

A sensitive and selective high-performance liquid chromatographic method has been developed for the quantitative determination of N-(2-methyl-2-propyl)-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta-carboxamide (I) in human plasma. I, a 5 alpha-reductase inhibitor and a potential therapeutic agent for benign prostatic hyperplasia, is a member of the family of compounds referred to as the 4-azasteroids. The 4-N-methyl analogue of the drug was used as the internal standard and calibration curves were developed at two levels of sensitivity to cover a large dynamic range of plasma concentrations. Drug was isolated from biological fluids with a solid-phase C18 extraction column; the analyte was further purified by adsorption and desorption from a second extraction column (CN cartridge). Evaluation of the isolation method revealed that it was reproducible and drug recoveries equalled ca. 90%. Chromatography was carried out on a C8 column (5 micron) with ultraviolet detection at 210 nm. The detection limit was ca. 10 ng/ml for I. Human plasma levels are reported for I following single-dose oral administration of 50,200 and 400 mg of drug; urinary excretion data are reported for a single volunteer given 400 mg of I.

Early diagnosis of 11 beta-hydroxylase deficiency in two siblings confirmed by analysis of a novel steroid metabolite in newborn urine.

Plasma and urinary steroid measurements are reported in 2 normotensive newborn female siblings with virilized external genitalia due to 11 beta-hydroxylase deficiency. Plasma 11-deoxycortisol concentrations were markedly elevated whereas 17OH-progesterone concentrations were not raised. Plasma renin activity was suppressed, but increased to levels characteristic of infancy within 4 weeks of treatment. The enzyme defect was confirmed by measurement of increased urinary excretion of tetrahydro-11-deoxycortisol. A more polar steroid metabolite, 6 alpha-hydroxytetrahydro-11-deoxycortisol was also determined by gas chromatographic and mass spectrometric analysis. Analysis of metabolites in urine is an additional specific marker to plasma 11-deoxycortisol measurement for the diagnosis of 11 beta-hydroxylase deficiency in early infancy.

Murine aggression induced by a boar chemosignal: a stimulus presentation dependency.

Male mice were used to assess for the presence of aggression-promoting cues in the boar chemosignal, 5 alpha-androst-16-ene-3-one. The findings of Experiment 1 indicated that this steroid has no aggression-promoting properties when mixed in water. When mixed in urine from castrated males, however, the steroid was shown to induce agonistic behavior in aggressors. Thus, the steroid was shown to possess aggression-promoting cues when associated with urine. Experiment 2 was designed to assess the chemosignal properties of the steroid when mixed in urine or juxtaposed either proximally or distally to urine. It was determined that the steroid needed to be either adjacent to or mixed in urine for chemosignal activity to be evidenced. It was concluded that (1) urine may function as an orienting stimulus for the appropriate detection of the steroid or (2) the conjoint presentation of the steroid and urine provided a qualitatively different stimulus complex than either stimulus presented alone. The findings of both experiments question the species specificity of the boar chemosignal and have methodological implications for studies attempting to isolate a chemocommunicative substance. Appropriate stimulus presentation procedures need to be considered for future research projects.

Immunization of pigs against the boar taint steroid androstenone.

Tissue concentrations of androstenone were measured in untreated control pigs and pigs immunized against 5 alpha-androst-16-en-3-one. Results confirmed that active immunization of male pigs against androstenone is unlikely to prevent the problem of "boar taint" in the carcass meat.

A note on the metabolism of 5 alpha-androst-16-en-3-one in the young boar in vivo.

The metabolism of plasma 5 alpha-androst-16-en-3-one (androstenone) was studied in two young boars weighing about 100 kg in which a single dose of tritiated androstenone was injected intravenously. The peripheral blood of one boar was continuously sampled for 6 h after injection; the total radioactivity per liter of plasma increased up to 14 min after the injection, and then declined rather slowly since plasma radioactivity was still measurable 7 days after injection. The metabolic clearance rate of androstenone was calculated to be about 80 000 liters per day. This quick disappearance of plasma androstenone was probably mainly due to storage in fatty tissue and, to a lesser extent, to catabolism into 5 alpha-androst-16-en-3 alpha-ol, 5 alpha-androst-16-en-3 beta-ol and particularly into unknown more polar compounds of which there were at least three. Radioactivity was mainly eliminated in the urine in the form of the same unknown polar compounds.

Studies of 16-androstenes in an infant with virilizing adrenal carcinoma.

An 18-month-old girl with virilization was found to have an encapsulated right adrenal carcinoma (2 x3 cm) with great variation in nuclear size, frequent mitoses, and possible blood vessel invasion. Preoperative urinary excretions of 17-ketosteroids, androsterone, etiocholanolone, dehydroepiandrosterone, testosterone, pregnanetriol, 3alpha-androstenol, and 3 beta-androstadienol were elevated; all showed a noticeable decrease postoperatively. Cortisol acetate, given preoperatively, produced a definite decrease in the urinary excretion of 17-ketosteroids and dehydroepiandrosterone; administration of corticotropin resulted in an increase in levels of urinary 17-ketosteroids, 17-hydroxycorticosteroids, and pregnanetriol. Urinary testosterone and 3beta-androstadienol may have diagnostic value since neither was suppressed by cortisol therapy. The behavior of both 3alpha-androstenol and 3beta-androstadienol in this study suggests that they are of adrenal origin.