Detection of 18-methyl steroids: Case report on a forensic urine sample and corresponding dietary supplements.

The detection of a putative 18-methyl-19-nortestosterone metabolite in a forensic bodybuilder's urine sample collected as part of a criminal proceeding has triggered a follow-up investigation. Four different dietary supplements in the possession of the suspect were examined with regard to possible precursor steroids. This led to the detection of the declared ingredient methoxydienone, which was confirmed by both, GC-MSMS and LC-HRMSMS. As neither 18-methyl-testosterone, nor 18-methyl-19-nortestosterone were detectable in the supplements, the possibility that the metabolite originates from methoxydienone was investigated. For this purpose, the metabolic fate of methoxydienone was studied in vitro using human HepG2 cells and in vivo by a single oral administration. While the 18-methyl-19-nortestosterone metabolite was not generated by HepG2 cells incubated with methoxydienone, it was observed in the urine samples collected at 2, 6, 10 and 24 h after methoxydienone administration. Moreover, the potential binding of methoxydienone as ligand to the human androgen receptor was modelled in silico in comparison with 18-methylnandrolone, for which androgen receptor activation had been shown in an in vitro approach before. In conclusion, we could ascribe the presence of the 18-methyl-19-nortestosterone metabolite in a forensic urine sample to originate from methoxydienone present in dietary supplements. Methoxydienone was observed to slowly degrade by demethylation of the methoxy substituent in liquid solutions. While no compound-specific intermediates were identified that allowed differentiation from other 18-methyl steroids, the 18-methyl-19-nortestosterone metabolite proved to be a suitable marker for reliable detection in doping analysis.

Androgenic properties of the dietary supplement 5α-hydroxy-laxogenin.

Dietary supplements sold for anabolic benefits or performance enhancement often contain substances, which are non-approved and might lack quality controls. With regard to athletes, the inclusion of substances or methods in the prohibited list of the World Anti-Doping Agency is based on medical or scientific evidence. 5α-hydroxy-laxogenin is a synthetic spirostane-type steroid, which is contained in dietary supplements and advertised as anabolic agent. To date, evidence is missing on anabolic or androgenic activity of 5α-hydroxy-laxogenin. We investigated its androgenic potential in two in vitro bioassays. While no activity was observed in the yeast androgen screen, 5α-hydroxy-laxogenin was able to trans-activate the androgen receptor in human prostate cells in a dose-dependent manner. Interestingly, a biphasic response was observed with antagonistic properties at lower concentrations and agonistic effects at higher concentrations tested. The demonstrated androgenic properties of the higher concentrations demonstrate that further investigations should focus on the safety as well as on potential anabolic effects of 5α-hydroxy-laxogenin. This is of interest with regard to abuse for doping purposes.

B-Cell Homeostasis Is Maintained During Two Months of Head-Down Tilt Bed Rest With or Without Antioxidant Supplementation.

Alterations of the immune system could seriously impair the ability to combat infections during future long-duration space missions. However, little is known about the effects of spaceflight on the B-cell compartment. Given the limited access to astronaut samples, we addressed this question using blood samples collected from 20 healthy male volunteers subjected to long-duration bed rest, an Earth-based analog of spaceflight. Hematopoietic progenitors, white blood cells, total lymphocytes and B-cells, four B-cell subsets, immunoglobulin isotypes, six cytokines involved in inflammation, cortisone and cortisol were quantified at five time points. Tibia microarchitecture was also studied. Moreover, we investigated the efficiency of antioxidant supplementation with a cocktail including polyphenols, omega 3, vitamin E and selenium. Our results show that circulating hematopoietic progenitors, white blood cells, total lymphocytes and B-cells, and B-cell subsets were not affected by bed rest. Cytokine quantification suggested a lower systemic inflammatory status, supported by an increase in serum cortisone, during bed rest. These data confirm the in vivo hormonal dysregulation of immunity observed in astronauts and show that bed rest does not alter B-cell homeostasis. This lack of an impact of long-term bed rest on B-cell homeostasis can, at least partially, be explained by limited bone remodeling. None of the evaluated parameters were affected by the administration of the antioxidant supplement. The non-effectiveness of the supplement may be because the diet provided to the non-supplemented and supplemented volunteers already contained sufficient antioxidants. Given the limitations of this model, further studies will be required to determine whether B-cell homeostasis is affected, especially during future deep-space exploration missions that will be of unprecedented durations.

Evaluation of estrogenic potency of a standardized hops extract on mammary gland biology and on MNU-induced mammary tumor growth in rats.

Supplements with estrogenic activities are intensively investigated as potential alternatives for the treatment of menopausal symptoms. These investigations include studies on their safety regarding potential breast cancer risks. Therefore, the aim of this study was to assess whether or not a standardized hops (Humulus lupulus) extract, containing 0.42% of the estrogenic flavanone, 8-prenylnaringenin, would stimulate growth of methyl-nitrosourea (MNU) induced mammary cancer in ovariectomized (OVX) Sprague-Dawley (SD) rats or would impact on the proliferative activity within the normal mammary gland of Wistar rats. To induce tumorigenesis SD-rats received an intraperitoneal injection of 50mg/kg body weight of MNU on postnatal days PND 50 and 52. 28days later animals were OVX or were SHAM operated (positive control) and randomly allocated and maintained for 140days on either a phytoestrogen-free placebo diet (SHAM and negative control) or on the hops fortified diet. For the investigations in the normal mammary gland young adult Wistar rats were bilaterally OVX and randomly allocated to a control group fed to a phytoestrogen-free diet, or to a diet supplemented either with E2-benzoate or the hops extract. As a major result, the tumor incidence was 15% (3 tumors totally) in OVX controls, whereas it was 85% (39 tumors totally) in SHAM operated positive controls. No tumors were detectable in the hops group. In addition, no estrogenic activity of the hops extract was detectable in uterus and liver of these animals. In investigations on the normal mammary gland, no impact of hops extract on the expression of estrogen dependent proliferation markers or of progesterone receptor became apparent. In conclusion, the lack of growth stimulation of MNU-induced breast cancer in OVX SD-rats and the lack of stimulation proliferative events in the normal mammary gland of OVX Wistar rats by standardized hops extracts provides an important piece of evidence regarding the safety of these extracts in the management of menopausal symptoms.

A standardized Humulus lupulus (L.) ethanol extract partially prevents ovariectomy-induced bone loss in the rat without induction of adverse effects in the uterus.

BACKGROUND: Hops (Humulus lupulus (L.)) dietary supplements are of interest as herbal remedies to alleviate menopausal symptoms, such as hot flushes, depression and anxiety. So far, the evidence regarding estrogenic and related properties of hops preparations has been considered insufficient for a market authorization for menopausal indications. PURPOSE: The study aims to investigate a chemically standardized hops extract regarding its safety in the uterus, as wells as its efficacy to prevent bone loss in the ovariectomized rat model. STUDY DESIGN/METHODS: Female Wistar rats were ovariectomized and divided into a control group receiving phytoestrogen-free diet, a group treated with E2benzoate (0.93 mg/kg body weight/d) and a group treated with the standardized hops extract (60 mg/kg body weight/d) for 8 weeks. Micro-computed tomography of the tibiae and vertebrae, as wells as histological changes in the uterus and tibia were analyzed. RESULTS: Neither uterotrophic nor proliferative effects were observed in the endometrium in response to the oral 8-week administration of the hops extract. However, site-dependent skeletal effects were observed. The hops extract significantly decreased the number of osteoclasts in the tibial metaphysis and prevented reduction of the trabecular thickness that resulted from estradiol depletion. In contrast, the hops extract did not prevent the ovariectomy-induced micro-architectural changes in the lumbar vertebra. Certain parameters (e.g. thickness and number of trabeculae) were even found to be below the values determined in the ovariectomized control group. CONCLUSION: Taken together, the results provide evidence for the safety of the standardized hops extract and point to a weak bone type-specific, protective effect on bone loss following estradiol depletion.

Hop extracts and hop substances in treatment of menopausal complaints.

Hop extract is a long used medicinal product and, regarding hormonal activities, in 1999 a number of prenylflavanones have been identified as its major constituents with 8-prenylnaringenin (8-PN) being the main active estrogenic compound. There have been several in vivo studies performed that demonstrate the potential of hop extract and the single compound 8-PN to alleviate climacteric symptoms like osteoporosis, vasomotoric complaints, and sexual motivation. On the other hand, only a few clinical studies have been performed so far, and these mainly focused on menopausal discomforts, especially hot flushes, yielding rather inconclusive results. Despite preferentially activating estrogen receptor α, 8-PN is only slightly uterotrophic, but it also elucidates estrogenic effects on the mammary gland. In conclusion, although hop extract and especially 8-PN are promising candidates as a relief for climacteric symptoms, data on the safety and efficacy is still scarce.