Investigations on Phase I Metabolism of Anabolic Androgenic Steroids and Its Influenceability as Tool to Refine Steroid Detection and Evaluation

This thesis addresses the steroid metabolism and its role in the detection of steroids. The results were achieved by performing metabolic studies in combination with different analytical techniques. Three main outcomes were achieved. First, the generation of reference material for a long-term metabolite of the synthetic steroid Oral-Turinabol (OT) was presented. The obtained results enhance unambiguous detection and quantification of this metabolite and hence help to uncover the prohibited OT administration in sports doping. Second, structural requirements and tolerated functionalities for substrates of the enzyme Cytochrome P450 subtype 21A2 (CYP21A2) were evaluated. These insights help to extrapolate known metabolic reactions to new and potentially unknown compounds. Third, investigations on steroid profile changes caused by the intake of non-steroidal anti-inflammatory drugs were described. These findings provide knowledge on substances influencing steroid metabolism, which is of high relevance for accurate steroid determination and hence the correct interpretation of analytical results. All results were obtained using mainly three models to study steroid metabolism and its influenceability. First, incubations with Schizosaccharomyces pombe strains recombinantly expressing human CYPs were used to generate reference material in small amounts and to evaluate metabolic reactions. Second, incubations with isolated recombinantly expressed human enzymes were used to examine metabolic reactions and their influenceability by other drugs. Third, in vivo drug administration was used to study steroid metabolism in humans and to investigate whether the intake of non-steroidal anti-inflammatory drugs leads to changes in steroid concentrations and concentration ratios. To subsequently evaluate the samples generated by the methods described above different analytical techniques were utilized. High-resolution mass spectrometry (HRMS) was used for quantification and tentative structure identification. Liquid chromatography and gas chromatography were both combined with HRMS. Additionally, gas chromatography coupled to single or triple quadrupole mass spectrometry was employed to identify and accurately quantify compounds where reference material was already available. Finally, fluorometric measurements carried out in real time were used to determine kinetic characteristics of enzymatic reactions. The results presented in this thesis build a solid base for the refinement of steroid detection. It was shown that combinations of in vitro and in vivo metabolic studies and investigations on metabolic reactions together with adequate analytical techniques are of high relevance for the improvement of steroid detection methods. Furthermore, the results are of relevance to enhance methods for generation of reference compounds, to identify potentially new drugs and to re-evaluate the risk potential of drugs. Further areas that can benefit from the results of this work include clinical and toxicological analysis, analysis of environmental and biological samples and endocrinology. Finally, the results are of high importance for anti-doping analysis. This field shall be mentioned separately as the results presented particularly contribute to tackle challenges relevant in this research area.Diese Arbeit befasst sich mit dem Steroidmetabolismus und dessen Rolle in der Steroidbestimmung. Die Ergebnisse wurden mit Hilfe von Stoffwechselstudien in Kombination mit unterschiedlichen analytischen Techniken erzielt. Drei Hauptergebnisse können formuliert werden. Ein erstes Ergebnis ist die Beschreibung der Erzeugung von Referenzmaterial eines Langzeitmetaboliten von Oral-Turinabol (OT). Die dabei gewonnenen Erkenntnisse vereinfachen den eindeutigen Nachweis und die Quantifizierung dieses Metaboliten und tragen somit dazu bei, die verbotene Einnahme von OT als Doping im Sport zu enthüllen. Weiterhin wurden strukturelle Voraussetzungen und tolerierte Funktionalitäten von Substraten des Enzyms Cytochrom P450 Subtyp 21A2 (CYP21A2) untersucht. Die daraus resultierenden Erkenntnisse ermöglichen die Extrapolation bekannter Stoffwechselreaktionen auf neue und möglicherweise unbekannte Verbindungen. Als drittes Hauptergebnis wurden Untersuchungen beschrieben, die sich mit Steroidprofilveränderungen befassen, die durch die Einnahme von Nichtsteroidalen Antirheumatika hervorgerufen werden. Dabei wurden Erkenntnisse gewonnen, die einen besseren Überblick über Substanzen, welche den Steroidstoffwechsel beeinflussen, ermöglichen. Dieses Wissen ist von großer Bedeutung für den eindeutigen Steroidnachweis und somit die korrekte Interpretation von Analysenergebnissen. Alle Ergebnisse wurden größtenteils durch die Verwendung von drei Modellen zur Untersuchung des Steroidstoffwechsels und seiner Beeinflussbarkeit generiert. Als erstes Modell wurden Schizosaccharomyces pombe Stämme genutzt, welche rekombinant humane CYP Enzyme exprimieren. Einerseits wurden sie verwendet, um Referenzsubstanzen in kleineren Mengen zu erzeugen, andererseits um Stoffwechselreaktionen zu bewerten. Als zweites Modell wurden Inkubationen mit isolierten rekombinanten humanen Enzymen durchgeführt, um Stoffwechselreaktionen und deren Beeinflussbarkeit durch andere Arzneistoffe zu untersuchen. Als drittes Modell wurden in vivo Arzneimittelanwendungen genutzt, um den Steroidstoffwechsel im Menschen zu untersuchen sowie die Auswirkungen der Einnahme von Nichtsteroidalen Antirheumatika auf Steroidkonzentrationen und -konzentrationsverhältnisse zu beleuchten. Um die durch die oben genannten Methoden erzeugten Proben anschließend zu analysieren, wurden verschiedene analytische Techniken verwendet. Hochauflösende Massenspektrometrie (HRMS) wurde zur Quantifizierung und vorläufigen Strukturaufklärung genutzt. Dabei wurden Flüssigchromatographie und Gaschromatographie gekoppelt mit HRMS angewendet. Zusätzlich wurde eine Kombination aus Gaschromatographie gekoppelt mit Single- oder Triple-Quadrupol Massenspektrometrie verwendet um Verbindungen, für welche Referenzmaterialien bereits verfügbar waren, nachzuweisen und exakt zu quantifizieren. Abschließend wurden fluorometrische Messungen in Echtzeit durchgeführt, um kinetische Kenndaten von Enzymreaktionen zu bestimmen. Die im Rahmen dieser Arbeit vorgestellten Erkenntnisse bilden eine zuverlässige Grundlage für die Verbesserung des Steroidnachweises. Es wurde gezeigt, dass eine Kombination aus in vitro und in vivo Stoffwechselstudien und Untersuchungen zu Stoffwechselreaktion in Verbindung mit geeigneten analytischen Techniken von hohem Stellenwert für die Verbesserung von Steroidnachweismethoden ist. Außerdem sind die Erkenntnisse von Bedeutung, um Methoden zur Erzeugung von Referenzmaterialien zu verbessern, potenzielle neue Arzneistoffe zu erkennen und das Risikopotenzial von Arzneistoffen neu zu evaluieren. Weitere Gebiete, die von den Erkenntnissen dieser Arbeit profitieren können, sind unter Anderem, die klinische und toxikologische Analytik, die Umweltanalytik, die Untersuchung von biologischen Proben und die Endokrinologie. Weiterhin sind die Ergebnisse von hoher Bedeutung für die Anti-Doping Analytik. Dieses Wissenschaftsfeld soll gesondert genannt werden, da die vorgestellten Ergebnisse dazu beitragen, Herausforderungen, die in diesem Gebiet von besonderer Relevanz sind, zu überwinden

Music and Clowning in Europe, 20th-21st centuries

This research project explores the interactions between music and clowning from two perspectives: first, by investigating the uses and functions of music and sound in European clowning traditions, and second, by revisiting the notion of musical humor and introducing the category of the ‘clownesque’ – taking Gustav Mahler’s Seventh Symphony as a case study. Clowning practices can inform modern instrumental music, I suggest, because of the cross-fertilization of ‘cultivated’ and ‘popular’ genres characteristic of this era. My musicological perspective on clowning will throw new light on this tradition; and, in turn, interpreting musical humor in the 20th century through the lens of clowning practices will emphasize the physicality of musical gestures

Experimentelle in-vitro-Versuche zur Viskosität von Embolisations-Emulsionen

Arbeit zur Untersuchung des Viskositätsverhalten des Chemotherapeutikum Epirubicin und des Embolisat Lipiodol® Ultra-Fluid in Abhängigkeit vonMischungsverhältnis, Mischungsmethode und Mischungsreihenfolgen zur Anwendung im Bereich der interventionelle Radiologie.Work on the investigation of the viscosity behavior of the chemotherapeutic agent Epirubicin and the Embolisat Lipiodol® Ultra-Fluid depending on the mixing ratio, mixing method and mixing sequence for use in the field of interventional radiology

Benefit of visual speech information for word comprehension in post-stroke aphasia

Aphasia is a language disorder that often involves speech comprehension impairments affecting communication. In face-to-face settings, speech is accompanied by mouth and facial movements, but little is known about the extent to which they benefit aphasic comprehension. This study investigated the benefit of visual information accompanying speech for word comprehension in people with aphasia (PWA) and the neuroanatomic substrates of any benefit. Thirty-six PWA and 13 neurotypical matched control participants performed a picture-word verification task in which they indicated whether a picture of an animate/inanimate object matched a subsequent word produced by an actress in a video. Stimuli were either audiovisual (with visible mouth and facial movements) or auditory-only (still picture of a silhouette) with audio being clear (unedited) or degraded (6-band noise-vocoding). We found that visual speech information was more beneficial for neurotypical participants than PWA, and more beneficial for both groups when speech was degraded. A multivariate lesion-symptom mapping analysis for the degraded speech condition showed that lesions to superior temporal gyrus, underlying insula, primary and secondary somatosensory cortices, and inferior frontal gyrus were associated with reduced benefit of audiovisual compared to auditory-only speech, suggesting that the integrity of these fronto-temporo-parietal regions may facilitate cross-modal mapping. These findings provide initial insights into our understanding of the impact of audiovisual information on comprehension in aphasia and the brain regions mediating any benefit

No Direct Hydroxylation of Pregnenolone by Steroid 21-Hydroxylase

Cytochrome P450s (CYPs) are an essential family of enzymes in the human body. They play a crucial role in metabolism, especially in human steroid biosynthesis. Reactions catalyzed by these enzymes are highly stereo- and regio-specific. Lack or severe malfunctions of CYPs can cause severe diseases and even shorten life. Hence, investigations on metabolic reactions and structural requirements of substrates are crucial to gain further knowledge on the relevance of different enzymes in the human body functions and the origin of diseases. One key enzyme in the biosynthesis of gluco- and mineralocorticoids is CYP21A2, also known as steroid 21-hydroxylase. To investigate the steric and regional requirements of substrates for this enzyme, we performed whole-cell biotransformation assays using a strain of fission yeast Schizosaccharomyces pombe recombinantly expressing CYP21A2. The progestogens progesterone, pregnenolone, and their 17α-hydroxy-derivatives were used as substrates. After incubation, samples were analyzed using gas chromatography coupled to mass spectrometry. For progesterone and 17α-hydroxyprogesterone, their corresponding 21-hydroxylated metabolites 11-deoxycorticosterone and 11-deoxycortisol were detected, while after incubation of pregnenolone and 17α-hydroxypregnenolone, no hydroxylated product was observed. Findings were confirmed with authentic reference material. Molecular docking experiments agree with these results and suggest that interaction between the 3-oxo group and arginine-234 of the enzyme is a strict requirement. The presented results demonstrate once more that the presence of an oxo-group in position 3 of the steroid is indispensable, while a 3-hydroxy group prevents hydroxylation in position C-21 by CYP21A2. This knowledge may be transferred to other CYP21A2 substrates and hence help to gain essential insights into steroid metabolism

A two-center pilot study on the effects of clinical ethics support on coercive measures in psychiatry.

BACKGROUND The use of formal coercion such as seclusion, mechanical restraint, and forced medication is one of the most challenging and complex issues in mental health care, on the clinical, the legal, and the ethical level. Clinical ethics support aims at assisting healthcare practitioners in determining the morally most justifiable course of action in these situations. However, the effectiveness of clinical ethics support has hardly been studied so far. METHODS Monthly moral case deliberation (MCD) was implemented in two acute wards of two different psychiatric hospitals in Switzerland. Frequency and intensity of coercion was measured on ward level (npatients = 405), and the Moral Attentiveness Scale, Knowledge on Coercion Scale, and Staff Attitudes towards Coercion Scale were applied on healthcare practitioner level (nHP = 46). Pre-post-comparisons were conducted using multi-level modeling where appropriate. RESULTS After implementation of MCD, formal coercion was less frequent (particularly seclusion, small effect size; 9.6 vs. 16.7%, p = .034, Cramér's V = .105) and less intense (particularly mechanical restraint, large effect size; 86.8 ± 45.3 vs. 14.5 ± 12.1 h, exact p = .019, r = -.74), and approval for coercive measures among healthcare practitioners was lower when controlling for the number of MCD sessions attended. CONCLUSIONS Clinical ethics support such as MCD may be a hitherto underutilized service for the reduction of coercion, complementing existing strategies and programs. Implementing clinical ethics support may help improve quality of care for persons suffering from severe mental illness

Influence of Indomethacin on Steroid Metabolism: Endocrine Disruption and Confounding Effects in Urinary Steroid Profiling of Anti-Doping Analyses

Anabolic androgenic steroids (AAS) are prohibited as doping substances in sports by the World Anti-Doping Agency. Concentrations and concentration ratios of endogenous AAS (steroid profile markers) in urine samples collected from athletes are used to detect their administration. Certain (non-prohibited) drugs have been shown to influence the steroid profile and thereby sophisticate anti-doping analysis. It was shown in vitro that the non-steroidal anti-inflammatory drug (NSAID) indomethacin inhibits selected steroid-biotransformations catalyzed by the aldo-keto reductase (AKR) 1C3, which plays a key role in the endogenous steroid metabolism. Kinetic parameters for the indomethacin-mediated inhibition of the AKR1C3 catalyzed reduction in etiocholanolone were determined in vitro using two comparing methods. As NSAIDs are very frequently used (not only) by athletes, the inhibitory impact of indomethacin intake on the steroid metabolism was evaluated, and steroid profile alterations were detected in vivo (one male and one female volunteer). Significant differences between samples collected before, during or after the intake of indomethacin for selected steroid profile markers were observed. The presented results are of relevance for the interpretation of results from doping control analysis. Additionally, the administration of NSAIDs should be carefully reconsidered due to their potential as endocrine disruptors

Blueprint from nature: Multi-omics comparison of CHO and plasma cells unveils novel cell engineering targets to improve productivity

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