Investigations into the concentration and metabolite profiles of stanozolol and LGD-4033 in blood plasma and seminal fluid using liquid chromatography high-resolution mass spectrometry.

Potential scenarios as to the origin of minute amounts of banned substances detected in doping control samples have been a much-discussed problem in anti-doping analysis in recent years. One such debated scenario has been the contamination of female athletes' urine with ejaculate containing doping agents and/or their metabolites. The aim of this work was to obtain complementary information on whether relevant concentration ranges of doping substances are excreted into the ejaculate and which metabolites can be detected in the seminal fluid (sf) and corresponding blood plasma (bp) samples. A method was established to study the concentration and metabolite profiles of stanozolol and LGD-4033-substances listed under anabolic substances (S1) on the World Anti-Doping Agency's Prohibited List-in bp and sf using liquid chromatography high-resolution mass spectrometry (LC-HRMS). For sf and bp, methods for detecting minute amounts of these substances were developed and tested for specificity, recovery, linearity, precision, and reliability. Subsequently, sf and bp samples from an animal administration study, where a boar orally received stanozolol at 0.33 mg/kg and LGD-4033 at 0.11 mg/kg, were measured. The developed assays proved appropriate for the detection of the target substances in both matrices with detection limits between 10 and 40 pg/mL for the unmetabolized drugs in sf and bp, allowing to estimate the concentration of stanozolol in bp (0.02-0.40 ng/mL) and in sf (0.01-0.25 ng/mL) as well as of LGD-4033 in bp (0.21-2.00 ng/mL) and in sf (0.03-0.68 ng/mL) post-administration. In addition, metabolites resulting from different metabolic pathways were identified in sf and bp, with sf resembling a composite of the metabolic profile of bp and urine.

Organ-on-a-chip: Determine feasibility of a human liver microphysiological model to assess long-term steroid metabolites in sports drug testing.

A fundamental challenge in preventive doping research is the study of metabolic pathways of substances banned in sport. However, the pharmacological predictions obtained by conventional in vitro or in vivo animal studies are occasionally of limited transferability to humans according to an inability of in vitro models to mimic higher order system physiology or due to various species-specific differences using animal models. A more recently established technology for simulating human physiology is the "organ-on-a-chip" principle. In a multichannel microfluidic cell culture chip, 3-dimensional tissue spheroids, which can constitute artificial and interconnected microscale organs, imitate principles of the human physiology. The objective of this study was to determine if the technology is suitable to adequately predict metabolic profiles of prohibited substances in sport. As model compounds, the frequently misused anabolic steroids, stanozolol and dehydrochloromethyltestosterone (DHCMT) were subjected to human liver spheroids in microfluidic cell culture chips. The metabolite patterns produced and circulating in the chip media were then assessed by LC-HRMS/(MS) at different time points of up to 14 days of incubation at 37°C. The overall profile of observed glucurono-conjugated stanozolol metabolites excellently matched the commonly found urinary pattern of metabolites, including 3'OH-stanozolol-glucuronide and stanozolol-N-glucuronides. Similarly, but to a lower extent, the DHCMT metabolic profile was in agreement with phase-I and phase-II biotransformation products regularly seen in postadministration urine specimens. In conclusion, this pilot study indicates that the "organ-on-a-chip" technology provides a high degree of conformity with traditional human oral administration studies, providing a promising approach for metabolic profiling in sports drug testing.

Stanozolol-N-glucuronide metabolites in human urine samples as suitable targets in terms of routine anti-doping analysis.

The exogenous anabolic-androgenic steroid (AAS) stanozolol stays one of the most detected substances in professional sports. Its detection is a fundamental part of doping analysis, and the analysis of this steroid has been intensively investigated for a long time. This contribution to the detection of stanozolol doping describes for the first time the unambiguous proof for the existence of 17-epistanozolol-1'N-glucuronide and 17-epistanozolol-2'N-glucuronide in stanozolol-positive human urine samples due to the access to high-quality reference standards. Examination of excretion study samples shows large detection windows for the phase-II metabolites stanozolol-1'N-glucuronide and 17-epistanozolol-1'N-glucuronide up to 12 days and respectively up to almost 28 days. In addition, we present appropriate validation parameters for the analysis of these metabolites using a fully automatic method online solid-phase extraction (SPE) method already published before. Limits of identification (LOIs) as low as 100 pg/ml and other validation parameters like accuracy, precision, sensitivity, robustness, and linearity are given.

A novel HPLC-MRM strategy to discover unknown and long-term metabolites of stanozolol for expanding analytical possibilities in doping-control.

Stanozolol is one of the most commonly abused anabolic androgenic steroids (AAS) by athletes and usually detected by its parent drug and major metabolites. However, its metabolic pathway is complex, varied and individually different, it is important to characterize its overall metabolic profiles and discover new and long-term metabolites for the aims of expanding detection windows. High performance liquid chromatography coupled with triple quadrupole mass spectrometer (HPLC-MS/MS) was used to analyze the human urine after oral administration of stanozolol. Multiple reaction monitoring (MRM), one of the scan modes of triple quadrupole mass spectrometer showing extremely high sensitivity was well used to develop a strategy for metabolic profiles characterization and long-term metabolites detection based on typical precursor to product ion transitions of parent drug and its major metabolites. Utilizing the characteristic fragment ions of stanozolol and its major metabolites as the product ions, and speculating unknown precursor ions based on the possible phase I and phase II metabolic reactions in human body, the metabolite profiles of stanozolol could be comprehensively discovered, especially for those unknown and low concentration metabolites in human urine. Then these metabolites were further well structure identified by targeted high resolution MS/MS scan of quadrupole-time of flight mass spectrometry (Q-TOF). Applying this strategy, 27 phase I and 21 phase II metabolites of stanozolol were identified, in which 13 phase I and 14 phase II metabolites have not been reported previously. The 9 out of 48 metabolites could be detected over 15days post drug administration. This strategy could be employed effectively to characterize AAS metabolic profiles and discover unknown and long-term metabolites in sports drug testing.

Detection of stanozolol O- and N-sulfate metabolites and their evaluation as additional markers in doping control.

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC-MS/MS to establish potential long-term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M-I to M-XI) were detected and characterized by LC-MS/MS. This paper provides valuable data on the ionization and fragmentation of O-sulfates and N-sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long-term metabolite (epistanozolol-N-glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.

Clinical pattern and acute and long-term management of hereditary angioedema due to C1-esterase inhibitor deficiency

Background: Hereditary angioedema due to C1-esterase inhibitor deficiency (HAE-C1-INH) is a life-threatening disease. Objectives: To describe the clinical characteristics and management of patients with HAE-C1-INH during routine clinical practice. Methods: An observational, retrospective study was performed in patients with HAE-C1-INH. Demographic, clinical, and analytical data were collected from 2 periods: period A (October 2009-September 2010) and period B (October 2007-September 2009). Results: We studied 112 patients with HAE-C1-INH (57.1% females). Age at onset of symptoms was 14.4 years (lower in patients who had experienced attacks in the previous year). In period B (n=87), 62.1% of patients presented at least 1 edema attack (median, 3.5 attacks/patient/2 years), and 19.1% of attacks were treated. In period A (n=77), 58.4% of patients were on maintenance therapy. Stanozolol was the most widely used drug (48.9%), with a mean weekly dose of 6.7 mg. At least 1 attack was recorded in 72.7% of patients (median, 3.0 attacks/patient/year), and 31.5% of the attacks were treated. Treatment of acute attacks increased by 12.4%. Conclusion: Age at onset of symptoms is associated with clinical expression of disease. The higher age at onset of symptoms, the fewer number of attacks per patient and year, and the lower dose of attenuated androgens necessary to control the disease than in other series lead us to hypothesize that HAE-C1-INH could have a less severe expression in Spain. Acute attacks seem to be treated increasingly often (AU)

Antecedentes: El angioedema hereditario por déficit del inhibidor de la C1 esterasa (AEH-C1-INH) es potencialmente mortal. Objetivos: Describir las características clínicas y el manejo de pacientes con AEH-C1-INH durante la práctica clínica habitual. Métodos: Estudio retrospectivo observacional de pacientes con AEH-C1-INH. Se recogieron datos demográficos, clínicos y analíticos en los periodos A (Octubre 2009-Septiembre 2010) y B (Octubre 2007-Septiembre 2009). Resultados: Se estudiaron 112 pacientes con AEH-C1-INH (57,1% mujeres) con edad de inicio de los síntomas de 14,4 años (inferior en aquellos pacientes con ataques en el último año). En el periodo B (n=87) 62,1% tuvo al menos un ataque (mediana: 3,5 ataques/paciente /2 años) y el 19,9% de los ataques se trataron. En el periodo A (n=77) 58,4% recibieron tratamiento de mantenimiento, siendo el estanozolol el fármaco más utilizado (48,9%) (dosis media semanal 6,7mg). El 72,7% de los pacientes tuvo al menos un ataque (mediana: 3,0 ataques / paciente / año), el 31,5% se trataron. Hubo un incremento del 12,4% de tratamientos de ataques agudos. Conclusiones: La edad de inicio de los síntomas está relacionada con la expresión clínica de la enfermedad. La edad superior del inicio de los síntomas, el menor número de ataques por paciente/año, y una dosis inferior de andrógenos atenuados para controlar la enfermedad, comparado con otros países, permite hipotetizar que el AEH-C1-INH en España tendría una expresión clínica menos grave. Existe una tendencia al alza en la frecuencia de tratamiento de ataques agudos (AU)

Drug-drug interaction and doping, part 2: an in vitro study on the effect of non-prohibited drugs on the phase I metabolic profile of stanozolol.

The present study was designed to provide preliminary information on the potential impact of metabolic drug-drug interaction on the effectiveness of doping control strategies currently followed by the anti-doping laboratories to detect the intake of prohibited agents. In vitro assays based on the use of human liver microsomes and recombinant cytochrome P450 isoforms were developed and applied to characterize the phase I metabolic profile of the prohibited agent stanozolol, both in the absence and in the presence of substances (ketoconazole, itraconazole, miconazole, cimetidine, ranitidine, and nefazodone) not included in the World Anti-Doping Agency (WADA) list of prohibited substances and methods and frequently administered to athletes. The results show that the in vitro model utilized in this study is adequate to simulate the in vivo metabolism of stanozolol. Furthermore, our data showed that ketoconazole, itraconazole, miconazole, and nefazodone caused a marked modification in the production of the metabolic products (3'-hydroxy-stanozolol, 4ß-hydroxy-stanozolol and 16ß-hydroxy-stanozolol) normally selected by the anti-doping laboratories as target analytes to detect stanozolol intake. On the contrary, moderate variations were registered in the presence of cimetidine and no significant modifications were measured in the presence of ranitidine. This evidence confirms that the potential effect of drug-drug interactions is duly taken into account also in anti-doping analysis.

Use of in vitro technologies to study phase II conjugation in equine sports drug surveillance.

BACKGROUND: Within equine drug surveillance, there is significant interest in analyzing intact phase II conjugates of drugs in urine, but progress has been limited by a lack of reference material. METHOD: In this study, in vitro techniques using equine liver fractions were employed to produce glucuronide and sulfate conjugates of stanozolol, 16ß-hydroxystanozolol and nandrolone, the glucuronide conjugate of morphine and the glutathione metabolite of chlordinitrobenzene for the first time in equine sports drug surveillance. RESULTS: The glucuronide conjugate of the synthetic progestagen altrenogest was also produced in vitro, removing the requirement for sample hydrolysis during routine urinalyses. CONCLUSION: These results highlight the potential of in vitro studies for the production of phase II reference material, allowing the development of assays based on intact conjugates.

Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry.

The applicability of LC-MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4xi,16xi-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3'-hydroxy-stanozolol, 16beta-hydroxy-stanozolol and 4beta-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC-QTOF MS.

Simultaneous immunochemical detection of stanozolol and the main human metabolite, 3'-hydroxy-stanozolol, in urine and serum samples.

Two enzyme-linked immunosorbent assays (ELISAs) have been established for the analysis of stanozolol (St) and 3'-hydroxy-stanozolol (3'OH-St), the main metabolite found in humans. The immunizing hapten N2'-(5-valeric acid)-androst-2-eno[3,2-c]-pyrazol-17a-methyl-17b-ol (hapten 8) has been designed with the aid of molecular modeling and theoretical tools to allow immunochemical detection of both compounds. Using an ELISA based on a homologous antisera/coating antigen combination, St can be selectively quantified without significant interference of the St metabolites or other steroids potentially present in the biological samples. On the other hand, St immunoreactivity equivalents due to the additional presence of 3'OH-St can also be quantified using an ELISA based on a heterologous antisera/coating antigen combination, in which the metabolite can be detected with 51% cross-reactivity. Thus, As147/5BSA detects 3'OH-St and St in buffer with IC(50) values of 1.46 and 0.68 microg L(-1), respectively. In contrast, As147/8BSA is highly specific for St with an IC(50) of 0.16 microg L(-1) and a limit of dection of just 0.022 microg L(-1). Performance of both assays in urine and serum samples has been evaluated and demonstrate that inappropriate use of stanozolol by athletes or young people can be detected in these matrices after simple cleanup methods, with IC(50) values below the minimum performance required levels established by the World Antidoping Agency.

Mass spectrometry of stanozolol and its analogues using electrospray ionization and collision-induced dissociation with quadrupole-linear ion trap and linear ion trap-orbitrap hybrid mass analyzers.

Mass spectrometric identification and characterization of growth-promoting anabolic-androgenic steroids in biological matrices has been a major task for doping control as well as food safety laboratories. The fragmentation behavior of stanozolol, its metabolites 17-epistanozolol, 3'-OH-stanozolol, 4alpha-OH-stanozolol, 4beta-OH-stanozolol, 17-epi-16alpha-OH-stanozolol, 16alpha-OH-stanozolol, 16beta-OH-stanozolol, as well as the synthetic analogues 4-dehydrostanozolol, 17-ketostanozolol, and N-methyl-3'-OH-stanozolol, was investigated after positive electrospray ionization and subsequent collision-induced dissociation utilizing a quadrupole-linear ion trap and a novel linear ion trap-orbitrap hybrid mass spectrometer. Stable isotope labeling, H/D-exchange experiments, MS3 analyses and high-resolution/high mass accuracy measurements of fragment ions were employed to allow proposals for charge-driven as well as charge-remote fragmentation pathways generating characteristic product ions of stanozolol at m/z 81, 91, 95, 105, 119, 135 and 297 and 4-hydroxylated stanozolol at m/z 145. Fragment ions were generated by dissociation of the steroidal A- and B-ring retaining the introduced charge within the pyrazole function of stanozolol and by elimination of A- and B-ring fractions including the pyrazole residue. In addition, a charge-remote fragmentation causing the neutral loss of methanol was observed, which was suggested to be composed by the methyl residue at C-18 and the hydroxyl function located at C-17.

Photoaffinity labeling identification of thyroid hormone-regulated glucocorticoid-binding peptides in rat liver endoplasmic reticulum: an oligomeric protein with high affinity for 16beta-hydroxylated stanozolol.

Steroid-binding proteins unrelated to the classical nuclear receptors have been proposed to play a role in non-genomic actions of the17alpha-alkylated testosterone derivative (17alpha-AA) stanozolol (ST). We have previously reported that male rat liver endoplasmic reticulum contains two steroid-binding sites associated with high molecular mass oligomeric proteins: (1) the ST-binding protein (STBP); and (2) the low-affinity glucocorticoid-binding protein (LAGS). To further explore the role of LAGS on the mechanism of action of ST, we have now studied: (1) the interaction of ST and its hydroxylated metabolites with solubilized LAGS and the cytosolic glucocorticoid receptor (GR); and (2) the effects of hormones on the capability of STBP to bind ST. We found that, unlike 17alpha-methyltestosterone, neither ST nor its hydroxylated metabolites bind to GR. However, the 16beta-hydroxylation of ST significantly increases the capability of LAGS to bind ST. Interestingly, 3'-hydroxylation of ST abrogates the capability of LAGS to bind ST. ST (k(i)=30 nM) and 16beta-hydroxystanozolol (k(i)=13 nM) bind with high affinity to LAGS, and are capable of accelerating the rate of dissociation of previously bound dexamethasone from the LAGS. STBP and LAGS are strongly induced by ethinylestradiol. However, unlike STBP, LAGS is regulated by thyroid hormones and growth hormone, which proves that these steroid-binding activities are associated with different binding sites. These findings seem to suggest a novel mechanism for ST whereby membrane-associated glucocorticoid-binding activity is targeted by the 16beta-hydroxylated metabolite of ST. ST and its 16beta-hydroxylated metabolite modulate glucocorticoid activity in the liver through negative allosteric modulation of LAGS, with the result of this interaction an effective increase in classical GR-signaling by increasing glucocorticoid availability to the cytosolic GR.

Hepatic lipase activity influences high density lipoprotein subclass distribution in normotriglyceridemic men. Genetic and pharmacological evidence.

Several studies have reported an inverse relationship between hepatic lipase activity and plasma high density lipoprotein (HDL) cholesterol concentrations. The purpose of the present study was to determine whether genetic and pharmacological variation in hepatic lipase activity alters the distribution of HDL subclasses. Two independent analytical methods (nuclear magnetic resonance and gradient gel electrophoresis) were used to compare HDL subclass distributions in 11 homozygotes for the -514C allele of hepatic lipase and in 6 homozygotes for the -514T allele. Mean hepatic lipase activity was 45 +/- 15 mmol. l(-1). hr(-1) in -514C homozygotes and 20 +/- 7 mmol. l(-1). hr(-1) in -514T homozygotes. Both analytical methods indicated that HDL(2b) was significantly higher and HDL(3a) was significantly lower in -514T homozygotes than in -514C homozygotes. No differences were noted in the other HDL fractions (HDL(2a), HDL(3b), and HDL(3c)). To determine the effects of increased hepatic lipase activity, 20 men were given the synthetic anabolic steroid, stanozolol. Stanozolol treatment increased hepatic lipase activity more than two-fold (38 +/- 18 to 85 +/- 25 mmol. l(-1). hr(-1) ), and markedly reduced the plasma concentrations of the larger HDL subclasses (HDL(2b) and HDL(2a)). The plasma concentrations of the smallest HDL subclasses (HDL(3b) and HDL(3c)) were unchanged by stanozolol treatment. Taken together, these genetic and pharmacological data indicate that variation in hepatic lipase activity has highly specific effects on the distribution of HDL subclasses in the circulation.-Grundy, S. M., G. L. Vega, J. D. Otvos, D. L. Rainwater, and J. C. Cohen. Hepatic lipase activity influences high density lipoprotein subclass distribution in normotriglyceridemic men: genetic and pharmacological evidence.

Multi-laboratory study of the analysis and kinetics of stanozolol and its metabolites in treated calves.

The European Union banned the use of anabolic steroids for cattle fattening in 1988. Analytical techniques able to detect trace amounts of the parent drugs and their metabolites are mandatory for the control of abuse. Stanozolol (Stan) is an anabolic steroid that is often found in injection sites and cocktails. However, it has never been detected in tissues (kidney fat, meat) or excreta (urine, faeces) taken during regulatory inspection. The difference between the structure of Stan and the other steroids (a pyrazole ring fused to the androstane ring system) is probably the cause of this phenomenon. In the multi-laboratory study described here, veal calves were treated with intramuscular doses of Stan. In the excreta of these calves the presence, absence and/or concentration of Stan and of its major metabolites 16 beta-hydroxystanozolol and 3'-hydroxystanozolol were determined. For the determination of these analytes the different laboratories used different extraction and clean-up procedures and also evaluated different analytical techniques such as GC-MS (negative chemical ionization) and LC-MS-MS. The aim of this investigation was to explore which analyte should be validated for veterinary inspection purposes.

Identification of a specific binding site for the anabolic steroid stanozolol in male rat liver microsomes.

Male rat liver microsomes contain a [3H]dexamethasone binding site, capable of binding glucocorticoids and progesterone. We have shown previously that the 17 alpha-alkylated androgen, stanozolol, can inhibit the [3H]dexamethasone binding to microsomes through a negative allosteric mechanism, which gives rise to the possibility of its interaction with a different binding site. In this study, the existence of a single-saturating binding site, capable of binding the radioactive steroid with a maximum number of the specific binding site of 49 +/- 2 pmol/mg of protein and a Kd of 37 +/- 1.3 nM was demonstrated by using [3H]stanozolol. In competition experiments, only stanozolol and danazol were able to compete with [3H]stanozolol for its binding to microsomes, among more than 60 steroids and other compounds tested. The binding of [3H]stanozolol was depressed after protease treatment of the microsomes, or after the administration of cycloheximide to adult male rats for 24 hr, which suggest its proteic nature. The [3H]stanozolol binding site was detected in many tissues of the rat, with the highest concentrations being found in the liver. It was detected from birth, increasing afterward in concentration and reaching a peak at 2 to 3 months of age. This is the first experimental verification of the existence in liver microsomes of a specific binding site for some 17 alpha-alkylate androgens, such as stanozolol and danazol, different from the androgen receptor or the [3H]dexamethasone binding site.

Direct interactions of androgenic/anabolic steroids with the peripheral benzodiazepine receptor in rat brain: implications for the psychological and physiological manifestations of androgenic/anabolic steroid abuse.

The peripheral benzodiazepine receptor (PBR) is a mitochondrial protein involved in regulating steroid synthesis and transport. We report here the effects of androgenic/anabolic steroids (AAS) on the binding of the PBR-specific ligand [3H] PK11195 to male rat brain cortical synaptoneurosomes. Two synthetic AAS, stanozolol and 17beta-testosterone cypionate (17beta-cyp), significantly inhibited 1 nM [3H] PK11195 binding at concentrations greater than 5 and 25 microM, respectively. Stanozolol was the most effective inhibitor, reducing [3H] PK11195 binding by up to 75%, compared to only 40% inhibition by 17beta-cyp, at 50 microM AAS concentration. Two other AAS, 17alpha-methyltestosterone and nortestosterone decanoate, were incapable of inhibiting [3H] PK11195 binding at concentrations up to 50 microM. On the basis of Scatchard/Rosenthal analysis, [3H] PK11195 binds to two classes of binding sites, and the inhibition of [3H] PK11195 binding by stanozolol appears to be allosteric, primarily reducing binding to the higher affinity [3H] PK11195 binding site. These results, in combination with earlier studies indicating the direct effects of AAS on the function of additional central nervous system receptor complexes, suggest that the behavioral and psychological effects of AAS result from the interactions of AAS with multiple regulatory systems in the brain.

Studies on anabolic steroids--12. Epimerization and degradation of anabolic 17 beta-sulfate-17 alpha-methyl steroids in human: qualitative and quantitative GC/MS analysis.

The epimerization and dehydration reactions of the 17 beta-hydroxy group of anabolic 17 beta-hydroxy-17 alpha-methyl steroids have been investigated using the pyridinium salts of 17 beta-sulfate derivatives of methandienone 1, methyltestosterone 4, oxandrolone 7, mestanolone 10 and stanozolol 11 as model compounds. Rearrangement of the sulfate conjugates in buffered urine (pH 5.2) afforded the corresponding 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes in a ratio of 0.8:1. These data indicated that both epimerization and dehydration of the 17 beta-sulfate derivatives were not dependent upon the respective chemical features of the steroids studied, but were instead inherent to the chemistry of the tertiary 17 beta-hydroxy group of these steroids. Interestingly, in vivo studies carried out with human male volunteers showed that only methandienone 1, methyltestosterone 4 and oxandrolone 7 yielded the corresponding 17-epimers 2, 5 and 8 and the 18-nor-17,17-dimethyl-13(14)-enes 3, 6 and 9 in ratios of 0.5:1, 2:1 and 2.7:1, respectively. No trace of the corresponding 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes derivatives of mestanolone 10 and stanozolol 11 was detected in urine samples collected after administration of these steroids. These data suggested that the in vivo formation of the 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes derivatives of 17 beta-hydroxy-17 alpha-methyl steroids is also dependent upon phase I and phase II metabolic reactions other than sulfation of the tertiary 17 beta-hydroxyl group, which are probably modulated by the respective chemical features of the steroidal substrates. The data reported in this study demonstrate that the 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes are not artifacts resulting from the acidic or microbial degradation of the parent steroids in the gut as previously suggested by other authors, but arise from the rearrangement of their 17 beta-sulfate derivatives. Unchanged oxandrolone 7 was solely detected in the unconjugated steroid fraction whereas unchanged steroids 1, 4 and 11 were recovered from the glucuronide fraction. These data are indirect evidences suggesting that the glucuronide conjugates of compounds 1 and 4 are probably enol glucuronides and that of compound 11 is excreted in urine as a N-glucuronide involving its pyrazole moiety. The urinary excretion profiles of the epimeric and 18-nor-17,17-dimethyl-13(14)-ene steroids are presented and discussed on the basis of their structural features.

Metabolism of stanozolol: identification and synthesis of urinary metabolites.

Urinary metabolites of stanozolol (17 alpha-methyl-17 beta-hydroxy-5 alpha-androst-2-eno(3,2-c)-pyrazole) following oral administration were isolated by chromatography on XAD-2 and by preparative high-performance liquid chromatography (HPLC) and identified by gas chromatography-mass spectrometry (GC/MS) with electron impact (EI)-ionisation. Stanozolol is excreted as a conjugate but is metabolized to a large extent. All identified metabolites are hydroxylated, namely at C-3' of the pyrazole ring and at C-4 beta, C-16 alpha and C-16 beta of the steroid. Less than 5% of the metabolites are found in the unconjugated urine fraction: 3'-hydroxy-stanozolol (II) and 3'-hydroxy-17-epistanozolol (III). Conjugated excreted metabolites are 3'-hydroxystanozolol (II), stanozolol (I), 4 beta-hydroxy-stanozolol (IV), 16 beta-hydroxystanozolol (V), 16 alpha-hydroxystanozolol (VI), two isomers of 3',16-dihydroxystanozolol (VII, VIII), two isomers of 4 beta, 16-dihydroxystanozolol (IX, X) and a 3',?-dihydroxystanozolol (XI). 3'-Hydroxystanozolol, 4 alpha-hydroxystanozolol, 4 beta-hydroxystanozolol, 16 alpha-hydroxy-, 16 alpha-hydroxy-17-epi- and 16 beta-hydroxystanozolol were synthesised to confirm the structural assignment of the main metabolites.