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

Urinary Excretion of Ecdysterone and Its Metabolites Following Spinach Consumption

Scope The phytosteroid ecdysterone is present in spinach. In this study, the urinary elimination of ecdysterone and its metabolites in humans is investigated following spinach consumption of two different culinary preparations. Methods and results Eight participants (four males, four females) ingested 950 (27.1) g sautéed spinach (average [±standard deviation (SD)]) and 912 (70.6) g spinach smoothie as second intervention after washout. Post-administration urines are analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). After intake of both preparations, ecdysterone and two metabolites, 14-deoxy-ecdysterone, and 14-deoxy-poststerone, are excreted in urine. The maximum concentration of ecdysterone is ranging from 0.09 to 0.41 µg mL−1 after sautéed spinach and 0.08–0.74 µg mL−1 after smoothie ingestion. The total excreted amount (mean% [±SD]) in the urine as a parent drug plus the metabolites is only 1.4 (1.0) for both sautéed spinach and smoothie. The apparent sex related differences in 14-deoxy-poststerone excretion will need further investigations. Conclusion Only a small proportion of ecdysterone from spinach is excreted into urine. No significant differences are found in concentration and recovered amount (%) of ecdysterone, 14-deoxy-ecdysterone, and 14-deoxy-poststerone in urine between sautéed spinach and smoothie ingestion. A discrimination between ecdysterone from food or preparations will be challenging based on urinary concentrations only, at least for later post-administration samples

The dietary flavonol quercetin ameliorates angiotensin II-induced redox signaling imbalance in a human unbilical vein endothelial cell model of endothelial dysfunction via ablation of p47phox expression

YesQuercetin is reported to reduce blood pressure in hypertensive but not normotensive humans, but the role of endothelial redox signaling in this phenomenon has not been assessed. This study investigated the effects of physiologically obtainable quercetin concentrations in a human primary cell model of endothelial dysfunction in order to elucidate the mechanism of action of its antihypertensive effects. Angiotensin II (100 nM, 8 h) induced dysfunction, characterized by suppressed nitric oxide availability (85 ± 4% p<0.05) and increased superoxide production (136 ± 5 %, p<0.001). These effects were ablated by an NADPH oxidase inhibitor. Quercetin (3 μM, 8 h) prevented angiotensin II induced changes in nitric oxide and superoxide levels, but no effect upon nitric oxide or superoxide in control cells. The NADPH oxidase subunit p47(phox) was increased at the mRNA and protein levels in angiotensin II-treated cells (130 ± 14% of control, p<0.05), which was ablated by quercetin co-treatment. Protein kinase C activity was increased after angiotensin II treatment (136 ± 51%), however this was unaffected by quercetin co-treatment. Physiologically obtainable quercetin concentrations are capable of ameliorating angiotensin II-induced endothelial nitric oxide and superoxide imbalance via protein kinase C-independent restoration of p47(phox) gene and protein expression.Innovate UK and Boots Pharmaceutical

New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone (“oral turinabol”), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5β-metabolite was detected. Additionally, 3α,5β-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring

The Helicobacter pylori Genome Project : insights into H. pylori population structure from analysis of a worldwide collection of complete genomes

Helicobacter pylori, a dominant member of the gastric microbiota, shares co-evolutionary history with humans. This has led to the development of genetically distinct H. pylori subpopulations associated with the geographic origin of the host and with differential gastric disease risk. Here, we provide insights into H. pylori population structure as a part of the Helicobacter pylori Genome Project (HpGP), a multi-disciplinary initiative aimed at elucidating H. pylori pathogenesis and identifying new therapeutic targets. We collected 1011 well-characterized clinical strains from 50 countries and generated high-quality genome sequences. We analysed core genome diversity and population structure of the HpGP dataset and 255 worldwide reference genomes to outline the ancestral contribution to Eurasian, African, and American populations. We found evidence of substantial contribution of population hpNorthAsia and subpopulation hspUral in Northern European H. pylori. The genomes of H. pylori isolated from northern and southern Indigenous Americans differed in that bacteria isolated in northern Indigenous communities were more similar to North Asian H. pylori while the southern had higher relatedness to hpEastAsia. Notably, we also found a highly clonal yet geographically dispersed North American subpopulation, which is negative for the cag pathogenicity island, and present in 7% of sequenced US genomes. We expect the HpGP dataset and the corresponding strains to become a major asset for H. pylori genomics

Optimierung des Prozesses der Einführung neuer Metabolite anabol-androgener Steroide - Methodenevaluation, Synthese, Verifikation

The presented thesis concentrates on the metabolism of three AAS in the human body and the synthesis of potential new long-term metabolites. It demonstrates an effective way of introducing new markers to anti-doping analysis and finally confirming the proposed structures. First, the substrate specificity of a human CYP (21A2) was elucidated to use its stereoselective hydroxylation capacity for the targeted synthesis of new metabolites of several AAS. Second, new metabolites of MD and MT were synthesized chemically, characterized, and detected in urine samples. Third, a controlled administration study with DHCMT was performed to reveal the metabolism of this doping substance and provide kinetic data of the metabolic products. Different synthesis methods were used depending on the structure: either a full chemical or a combined chemical and biotechnological approach. Several analysis techniques were used to provide sufficient information during the particular project steps. To verify intermediate products in the synthesis, gas chromatography coupled by electron ionization to single quadrupole-mass spectrometry (GC-EI-MS) was used. NMR experiments together with GC-EI-MS and gas chromatography coupled by electron ionization to quadrupole time-of-flight-mass spectrometry (GC-EI-QTOF-MS) were used for the structure elucidation of the synthesized reference material. Post administration samples were analyzed by GC-EI- QQQ-MS and LC-ESI-QTOF-MS. The results of this thesis show the ideal way of providing reference material to the anti-doping community or every other research field that is dealing with the metabolism of endogenous and exogenous substances in case that the concentrations are too low for other means of comprehensive identification. In a first step, the synthesis of reference material must be planned, and the used compounds, e.g., enzymes or reagents, must be evaluated for their potential use. Subsequently, the synthesis has to be performed, and the product needs to be fully characterized so that there is no doubt of its structure. Finally, an administration of the parent compound and subsequent analysis of the excreted metabolites confirm the previous work. In summary, this work shows how new ideas about metabolism should be dealt with. It helps to dispel the doubts that have existed for years about postulated results. At the same time, it shows that steroid metabolism still has various blind spots, whose elucidation must be the goal of further research. Although anti-doping was the all-dominant topic, the gained results are also relevant for fields in physiology and pharmacology.Die vorliegende Arbeit konzentriert sich auf den Metabolismus von drei AAS im menschlichen Körper und die Synthese von potentiellen neuen Langzeitmetaboliten. Es wird ein effektiver Weg aufgezeigt, um neue Marker in die Anti-Doping-Analyse einzuführen und schließlich die vorgeschlagenen Strukturen zu bestätigen. Zuerst wurde die Substratspezifität eines menschlichen CYP (21A2) aufgeklärt, um dessen stereoselektive Hydroxylierungskapazität für die gezielte Synthese neuer Metaboliten mehrerer AAS zu nutzen. Folgend wurden neue Metaboliten von MD und MT chemisch synthetisiert, mittels Kernspinresonanzspektroskopie (NMR) charakterisiert und in Urinproben nachgewiesen. Um den Metabolismus dieser Dopingsubstanz aufzudecken und kinetische Daten der Metabolite zu erhalten, erfolgte letztlich eine kontrollierte Ausscheidungsstudie mit DHCMT. Je nach Struktur wurden unterschiedliche Synthesemethoden verwendet: entweder ein vollchemischer oder ein kombinierter chemisch-biotechnologischer Ansatz. Um in den einzelnen Projektschritten ausreichend Informationen zu erhalten, wurden verschiedene Analysetechniken eingesetzt. Um die Zwischenprodukte in der Synthese zu verifizieren, wurde Gaschromatographie gekoppelt mit Elektronenionisations-Single- Quadrupol-Massenspektrometrie (GC-EI-MS) verwendet. Für die Strukturaufklärung des synthetisierten Referenzmaterials wurden NMR-Experimente zusammen mit GC-MS und Gaschromatographie gekoppelt an Elektronenionisations-Quadrupol-Flugzeit- Massenspektrometrie (GC-EI-QTOF-MS) verwendet. Die Proben der Ausscheidungsstudie wurden mittels GC-EI-QQQ-MS und LC-ESI-QTOF-MS analysiert. Die Ergebnisse dieser Arbeit zeigen den idealen Weg, der Anti-Doping-Gemeinschaft oder anderen Forschungsbereichen, die sich mit dem Metabolismus von endogenen und exogenen Substanzen beschäftigen, Referenzmaterial zur Verfügung zu stellen, falls die Konzentrationen zu gering sind, um sie mit anderen Mitteln umfassend charakterisieren zu können. In einem ersten Schritt muss die Synthese von Referenzmaterial geplant und die verwendeten Verbindungen, z.B. Enzyme oder Reagenzien, auf ihre mögliche Verwendung hin evaluiert werden. Anschließend muss die Synthese durchgeführt und das Produkt vollständig charakterisiert werden, so dass kein Zweifel an seiner Struktur besteht. Schließlich bestätigen eine Verabreichung der Ausgangsverbindung und die anschließende Analyse der ausgeschiedenen Metaboliten die bisherige Arbeit. Zusammenfassend zeigt diese Arbeit, wie mit neuen Ideen zum Metabolismus umgegangen werden sollte. Sie hilft, die seit Jahren bestehenden Zweifel an postulierten Ergebnissen auszuräumen. Gleichzeitig zeigt sie, dass der Steroidstoffwechsel noch verschiedene blinde Flecken hat, deren Aufklärung das Ziel weiterer Forschung sein muss. Obwohl Anti-Doping das alles beherrschende Thema war, sind die gewonnenen Ergebnisse auch für Bereiche der Physiologie und Pharmakologie relevant

Controlled administration of dehydrochloromethyltestosterone in humans: Urinary excretion and long-term detection of metabolites for anti-doping purpose

Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol (R). It is prohibited in sports at all times; nevertheless, there are several findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/ 12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail. Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4 alpha-chloro-17 beta-hydroxymethyl-17 alpha-methyl-18-nor-5 alpha-androstan-13-en-3 alpha-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite. The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17 alpha-hydroxymethyl-17 beta-methyl-18-nor-androsta-4,13-dien-3 beta-ol. It may serve as additional confirmatory metabolite. It is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated, to improve identification of cheating athletes. This study also offers some deeper insights into the metabolism of DHCMT and of 17 alpha-methyl steroids in general

New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17 alpha-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17 xi-hydroxymethyl-17 xi-methyl-18-nor-5 xi-androst-13-en-3 xi-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone (oral turinabol), in this study we investigated the formation of similar metabolites of metandienone and 17 alpha-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17 alpha-hydroxymethyl-17 beta-methyl-18-nor-5 beta-androst-13-en-3 alpha-ol and its 5 alpha-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5 beta-metabolite was detected. Additionally, 3 alpha,5 beta-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17 alpha-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring

Cardiovascular disease risk biomarkers and liver and kidney function are not altered in postmenopausal women after ingesting an elderberry extract rich in anthocyanins for 12 weeks

Growing evidence supports a cardio-protective role for anthocyanins; however, there is limited evidence on their efficacy and safety following the consumption of relatively high but dietarily achievable doses in humans. We conducted a parallel-designed, randomized, placebo-controlled study to examine the effect of chronic consumption of anthocyanins on biomarkers of cardiovascular disease (CVD) risk and liver and kidney function in 52 healthy postmenopausal women (n = 26 in treatment and placebo groups). Volunteers (BMI, 24.7 +/- 3.6 kg/m(2); age, 58.2 +/- 5.6 y) consumed 500 mg/d anthocyanins as cyanidin glycosides (from elderberry) or placebo for 12 wk (2 capsules twice/d). At the beginning (wk 0) and end of the 12-wk intervention, levels of anthocyanins and biomarkers of CVD (inflammatory biomarkers, platelet reactivity, lipids, and glucose) and liver and kidney function (total bilirubin, albumin, urea, creatinine, alkaline phosphatase, alanine aminotransferase, and gamma-glutyl transferase) were assessed in fasted blood. Anthropometric, blood pressure, and pulse measurements were also taken. In addition, postprandial plasma anthocyanins were measured (t = 1, 2, 3 h) following a 500-mg oral bolus dose. After 12 wk of chronic exposure to anthocyanins, there was no significant change in biomarkers of CVD risk and liver and kidney function remained within clinically acceptable ranges. We observed no plasma accumulation of anthocyanins; however, postprandial metabolism increased (P = 0.02). In conclusion, these data suggest that chronic consumption of 500 mg/d of elderberry extract for 12 wk is apparently safe, but ineffective in altering biomarkers of CVD risk in healthy postmenopausal women