Automated sample preparation and gas chromatographic–mass spectrometric analysis of urinary androgenic anabolic steroids

This paper presents an automated method for extracting anabolic agents from urine samples for their GC–MS analysis by selected-ion monitoring. The sample preparation was carried out in a Hewlett-Packard 7686 SPE PrepStation system. Each 0.6-ml aliquot was hydrolyzed, extracted, dried and trimethylsilyl (TMS) derivatized in a 2-ml vial without any hands-on labor. When sample preparation was finished 2 μl of the extract was injected into the gas chromatograph by split (1:10) mode. Due to the small amount of free space in the 2-ml vials for handling the sample, parameters like time of hydrolysis, type of shaking, number of extractions and some TMS derivatization parameters had to be adjusted to achieve the best recovery for all of the compounds in the screening. Manual and automated sample preparation schemes were compared in terms of linearity, precision, accuracy, limit of detection and recovery data. When large concentrations were analyzed using the automated method no carry-over effect was observed.     

Alterations in Glial Fibrillary Acidic Protein (GFAP) mRNA Levels in the Hamster Facial Motor Nucleus: Effects of Axotomy and Testosterone

Testosterone propionate (TP) administered at the time of facial nerve injury in the hamster accelerates the rate of regeneration. In this study, we tested the hypothesis that the mechanism by which TP augments peripheral nerve regeneration involves regulation of glial fibrillary acidic protein (GFAP) mRNA in the facial motor nucleus. Castrated male hamsters were subjected to right facial nerve transection, with half the animals implanted subcutaneously with Silastic capsules containing exogenous TP and the remainder sham implanted. Postoperative survival times were 0.25, 1, 2, 4, 7, and 14 d. Qualitative/quantitative analyses of both film and emulsion autoradiograms were accomplished. Axotomy, with or without TP, resulted in a dramatic increase in GFAP mRNA levels by 1 d postoperative on the axotomized side, relative to controls. GFAP mRNA levels remained elevated throughout all postoperative times in both the nonhormone- and TP-treated animals. Qualitative examination of the film autoradiograms indicated a generalized decrease in the amount of GFAP mRNA in the control and axotomized nuclei of TP-treated animals when compared to the control and axotomized nuclei, respectively, of nonhormone-treated animals. Statistical comparison of the values obtained for both the film and emulsion autoradiograms confirmed this impression. Thus, while the injury-induced increases in GFAP mRNA expression were not blocked by TP, the overall extent of the increase was significantly tempered by steroid treatment. These data suggest that hormonal modulation of the astrocytic response to peripheral nerve injury may be a contributing factor in the ability of steroids to enhance the regenerative capacities of injured motor neurons.     

Reproductive cycles in a reared strain of the mummichog, a daily spawner

Annual, lunar, and diel samplings were taken from a strain of mummichog (Arasaki strain) reared in outdoor tanks under natural conditions, to examine gonadal maturity. Gonads of yearling fish were quite immature in September. During late autumn and winter, a gradual increase in the GSI of both sexes was observed, and the growth of cortical alveolus phase oocytes in females and basal spermatogenesis in males progressed. In late February, a rapid increase in the GSI of both sexes, vitellogenesis in females, and active spermatogenesis in males, occurred. The spawning period of the yearling fish was from late March to August judging from the presence of milt-producing males and ovulated females. The spawning period of the underyearling fish started in the same month as the yearlings, but terminated 1 month earlier. Plasma levels of oestradiol-17 β (E₂) in females and testosterone in males were high during the spawning period in the yearlings. In the underyearlings, however, E₂ levels peaked in early spring, and declined in the latter part of the spawning period. Neither a lunar nor semilunar cycle was evident in the reproductive activity of this fish, which proved to be a typical daily spawner. Females showed an apparent daily reproductive cycle; oocyte maturation commenced at about 1200 hours, germinal vesicle breakdown (GVBD) occurred at about 2400 hours, and ovulation was completed by 2400 hours, 24 h after GVBD. Such clear annual and daily reproductive cycles make this strain of mummichog a suitable model for the study of environmental and endocrine regulation of reproductive cycles in marine and estuarine teleosts.     

Plasma and ovarian thyroxine levels in relation to sexual maturation and gestation in female Sebastes inermis

Plasma T₄ (L-thyroxine) concentrations in the viviparous rockfish Sebastes inermis showed a peak (119·3±27·8 ng ml⁻¹) during the development of embryos. Ovarian T₄ concentrations increased during vitellogenesis and final maturation and decreased during embryogenesis.However, total T₄ content within the ovary increased continuously up to the larval stages. Plasma oestradiol-17β, (E₂) concentrations peaked at the final maturation stage and then recovered to the level seen during the non-reproductive periods. Plasma testosterone (T) concentrations showed a peak (5·33±1·08 ng ml⁻¹) at the embryo stage and high levels were maintained throughout the gestation period. Plasma T₄ concentrations during the embryo stage and ovarian T₄ content during vitellogenesis were much higher than the levels reported in oviparous fishes. These data suggest that in viviparous rockfish, T₄ may be required for sustaining gestation or embryo development within the maternal body. In addition, the high content of total T₄ in the ovary implies that there probably does exist a maternal-embryo relationship during gestation as well as during vitellogenesis.     

Prolonged-release gonadotrophin-releasing hormone analogue implants enhance oocyte final maturation and ovulation, and increase plasma concentrations of sulphated C21 steroids in North Sea plaice

Reproductively mature female plaice were implanted with or without 50 μg of gonadotrophin-releasing hormone analogue (GnRHa), suspended in either coconut oil or methacrylate resin. The weight of the GnRHa-treated fish increased significantly (due to hydration of the oocytes) and reached a peak between 10 and 14 days. The fish produced several batches of eggs, which were consistently bigger than those produced by control fish. Plasma concentrations of free 17β-oestradiol and glucuronidated testosterone rose briefly (4 days) in response to the GnRHa, but then fell continuously till the end of the experiment (20 days). Plasma concentrations of sulphated 5β-pregnane-3α,17,20β-triol and 5β-pregnane-3β,17,20β-triol (which are putative metabolites of 17,20β-dihydroxy-4-pregnen-3-one, the oocyte maturation-inducing steroid) increased significantly at 4 days and reached a peak between 12 and 16 days. Concentrations were still very elevated on day 20. Plasma concentrations of sulphated 3α,17,21-trihydroxy-5β-pregnan-20-one showed a slight increase on day 4 but did not change thereafter. There was a highly significant difference in the amounts of GnRHa released into the bloodstream by the two methods of administration on day 4. However, this was not matched by significant differences in the concentrations of any of the steroids.     

Steroids from Euphorbia and other latex-bearing plants

Steroids in latices isolated from 15 Euphorbia species and four other latex-bearing plants were analysed (in terms of their chemotaxonomy) using computerized GC-MS. Steroid patterns divided these plants into six groupings.     

The effects of steroids on the granulosa cells in the domestic fowl

Summary The fine structure of granulosa cells of the domestic fowl as seen after administration of steroids is described. Diaethylstilboestrol, estradiol and hydroxy-progesterone were given as intramuscular injections for a 28-days period. The main cytoplasmic changes of the granulosa cells were an increase in the number of mitochondria and dense bodies. The Golgi apparatus became enlarged, and occupied a large portion of the cell. The nucleus was found located adjacent to the oocyte, and there was an increase in number of the annular desmosomes. The investigation has demonstrated that even if steroids in high dosages induce atrophic alterations of the ovary, they stimulated proliferation of the granulosa cells of the small follicles. Apparently the small follicles with the granulosa cells retain the ability to regain development, which may be to some importance when steroids are used therapeutically (gynecologic disorders, contra-ception).     

Catabolisme des steroides chez les Nocardia

Nocardia restrictus and N. corallina oxidize the A ring of 4-hydroxy-4-cholesten-3-one and a 3,5-seco-4-nor-3-keto-5-oic acid is formed. The enzymes necessary to this reaction are induced and their biosynthesis is suppressed by chloramphenicol. The catabolism of the aliphatic side chain at C-17 involves a cleavage between C-24 and C-25 and the liberation of propionic acid.     

Comparison of sensitivity between gas chromatography–low-resolution mass spectrometry and gas chromatography–high-resolution mass spectrometry for determining metandienone metabolites in urine

In doping control laboratories the misuse of anabolic androgenic steroids is commonly investigated in urine by gas chromatography–low-resolution mass spectrometry with selected ion monitoring (GC–LRMS–SIM). By using high-resolution mass spectrometry (HRMS) detection sensitivity is improved due to reduction of biological background. In our study HRMS and LRMS methods were compared to each other. Two different sets were measured both with HRMS and LRMS. In the first set metandienone (I) metabolites 17α-methyl-5β-androstan-3α,17β-diol (II), 17-epimetandienone (III), 17β-methyl-5β-androst-1-ene-3α,17α-diol (IV) and 6β-hydroxymetandienone (V) were spiked in urine extract prepared by solid-phase extraction, hydrolysis with β-glucuronidase from Escherichia coli and liquid–liquid extraction. In the second set the metabolites were first spiked in blank urine samples of four male persons before pretreatment. Concentration range of the spiked metabolites was 0.1–10 ng/ml in both sets. With HRMS (resolution of 5000) detection limits were 2–10 times lower than with LRMS. However, also with the HRMS method the biological background hampered detection and compounds from matrix were coeluted with some metabolites. For this reason the S/N values of the metabolites spiked had to be first compared to S/N values of coeluted matrix compounds to get any idea of detection limits. At trace concentrations selective isolation procedures should be implemented in order to confirm a positive result. The results suggest that metandienone misuse can be detected by HRMS for a prolonged period after stopping the intake of metandienone.