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Capillary dried blood spot (DBS) analysis coupled with multi-analyte steroid liquid chromatography mass spectrometry (LCMS) is attractive for field studies, home-based self-sampling as well as clinical trials by eliminating costly and laborious sample processing involving venipuncture and frozen storage/shipping while providing multiple steroid measurements from a single small sample. We investigated steroid measurements in DBS samples stored for four years at room temperature prior to analysis compared with the original venipuncture serum samples. Healthy women (n=12) provided paired DBS and blood samples over two weeks run-in before seven days treatment with daily transdermal T gel (12.5 mg) and after the end of treatment on days 0, 1, 2, 4, 7 and 14. Compliance with treatment and sampling was high and no adverse effects were reported. Testosterone (T), androstenedione (A 4), 17 hydroxyprogesterone (17OHP) and progesterone (P 4) were measured in extracted DBS samples as whole blood concentrations with and without adjustment for hematocrit. Using the same LCMS methods, DBS T and A 4 measurements had high correlation with minimal bias from prior serum measurements with DBS T displaying the same pattern as serum, with or without hematocrit adjustment. However, serial whole blood measurements of T without hematocrit adjustment provided the best fitting model compared with serum, urine, or hematocrit-adjusted whole blood T measurements. These finding facilitate and simplify DBS methodology for wider field and home-based self-sampling studies of reproductive steroids indicating the need for hematocrit adjustment may be superfluous. [ABSTRACT FROM AUTHOR]

17-β-estradiol, involved in mesothelioma pathogenesis, and its precursors were explored as potential biomarkers for the early diagnosis of mesothelioma. Using enzyme-linked immunosorbent assay(ELISA) for 17-β-estradiol and ultra-high performance liquid chromatography/tandem mass spectrometry(UHPLC-MS/MS) for 19 17-β-estradiol precursors, a comprehensive analysis of 20steroid hormones was conducted in the serum of mesothelioma patients(n=67), asbestos-exposed healthy subjects(n=39), and non-asbestos-exposed healthy subjects(n=35). Bioinformatics analysis explored three potential serum biomarkers: 17-β-estradiol, DHEA-S, and androstenedione. The results revealed significant differences in 17-β-estradiol levels between mesothelioma patients and both non-asbestos-exposed and asbestos-exposed healthy subjects. No significant variations in serum 17-β-estradiol levels were observed among mesothelioma patients at different stages, suggesting its potential as an early diagnostic marker. 17-β-estradiol levels were similar in mesothelioma patients with environmental and occupational asbestos exposure, while males with occupational asbestos exposure exhibited significantly higher levels of 17-β-estradiol compared to females. Significant reduction in androstenedione and an increase in DHEA-S were observed in asbestos-exposed individuals compared to non-asbestos-exposed individuals. The analysis of DHEA-S-androstenedione-17-β-estradiol signature score showed an increase in asbestos-exposed individuals and mesothelioma patients compared to non-asbestos-exposed individuals, and this score effectively distinguished between the groups. The Cancer Genome Atlas data was utilized to analyze the expression of 5-α-reductase1 and hydroxysteroid-17β-dehydrogenase2 genes. The findings indicated that mesothelioma patients with elevated gene values for 5-α-reductase1 and hydroxysteroid-17β-dehydrogenase2 have a worse or better prognosis on overall survival, respectively. In conclusion, this study suggests 17-β-estradiol, DHEA-S, and androstenedione as biomarkers for mesothelioma risk and early diagnosis of mesothelioma in asbestos-exposed individuals, aiding timely intervention and improved care. [Display omitted] • E2 increased in healthy asbestos-exposed subjects and MPM patients. • DHEA-S-androstenedione-E2 signature score increased in asbestos-exposed subjects. • The signature differentiates subjects exposed to asbestos from those not exposed. • DHEA-S-androstenedione-E2 as biomarkers of MPM risk and early diagnosis of MPM. [ABSTRACT FROM AUTHOR]

Androstenedione (ADD), an important precursor for steroid hormone drug synthesis, can be obtained through the transformation of phytosterols by members of the Mycobacteria genus. In this study, multidirectional pathway engineering of Mycobacterium neoaurum LY-2 was applied to improve ADD production from phytosterols. Initially, the nonessential gene KasB encoding a cell wall β-ketoacyl acyl carrier protein synthase in Mycobacterium neoaurum LY-2 was knocked out, and the production of ADD was increased by 21.0%. Then, the key enzymes involved in ADD synthetic metabolism were systematically explored. These assays revealed that propionyl coenzyme A carboxylase (PCC) and Hsd4A played important roles in the synthesis of ADD. In addition, the heterologous expression of Vitreoscilla hemoglobin (VHb), a protein with oxygen-binding capacity, was successfully realized in Mycobacterium neoaurum LY-2, and the ADD concentration reached 4.42 g/L. Finally, the recombinant strain LY-2- ΔKasB-hsd4A-pcc-vgb with high ADD production was constructed by combining the above strategies. Through optimization of conversion conditions, the recombinant strain LY-2-Δ KasB - hsd4A - pcc-vgb produced an ADD concentration of 11.12 g/L when adding 20 g/L phytosterols. [Display omitted] • Mycobacterium neoaurum LY-2 efficiently catalyzes phytosterols to produce ADD. • Deletion of KasB enhanced the productivity of M. neoaurum LY-2. • PCC, Hsd4A, and VHb were co-expressed in the recombinant strain LY-2-Δ KasB. • The recombinant strain LY-2-Δ KasB - hsd4A - pcc-vgb produced 11.12 g/L of ADD. [ABSTRACT FROM AUTHOR]

• A unified total synthetic route to 18- and 19-trideuteromethyl steroids is described. • The method can also be used to prepare 18,19-hexadeuteromethyl steroids. • Compounds are useful for preparing calibration curves for mass spec analysis of these steroid hormones. • Compounds are useful for distinguishing between these endogenous and exogenous steroids under various conditions. A unified total synthesis route has been used to prepare 18- and 19-trideuterated testosterone, androstenedione and progesterone. The 18-trideuterated steroid synthetic method starts with the synthesis of 2-(methyl- d 3)-1,3-cyclopentanedione from CD 3 I and 1,3-cyclopentanedione and is subsequently converted into the Hajos-Parrish ketone for synthesis of these trideuterated steroids. The 19-trideuterated steroid synthesis proceeds through non-deuterated Hajos-Parrish ketone with incorporation of the 19-methyl- d 3 group from CD 3 I at a later stage of the same synthetic route. Utilization of CD 3 I at both the initial and later stages of the synthesis provides a route to 18,19-hexadeuterated steroids. The deuterated steroids are useful for studies of steroid biosynthesis and metabolism. [ABSTRACT FROM AUTHOR]

Testosterone biosynthesis from its precursor androstenedione is thought to be exclusively catalysed by the 17β-hydroxysteroid dehydrogenases—HSD17B3 in testes, and AKR1C3 in the ovary, adrenal and peripheral tissues. Here we show for the first time that the glucocorticoid activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) can also catalyse the 17β-reduction of androstenedione to testosterone, using a combination of in vitro enzyme kinetic assays, mathematical modelling, and molecular docking analysis. Furthermore, we show that co-expression of HSD11B1 and AKR1C3 increases testosterone production several-fold compared to the rate observed with AKR1C3 only, and that HSD11B1 is likely to contribute significantly to testosterone production in peripheral tissues. [ABSTRACT FROM AUTHOR]

• Influence of ethanol consumption and food intake on serum concentrations of endogenous steroids. • Ethanol consumption increased testosterone/androstenedione ratio in blood. • Meal-induced ACTH and cortisol peaks coincided with pulsatile androstenedione peaks. • Female testosterone levels are more susceptible to perturbations by ethanol or ACTH. Steroid biosynthesis and biotransformation are based on a cascade of enzymatic processes being highly sensitive to various external influences. Amongst those, ethanol was shown to affect testosterone metabolism. For doping analyses, athlete steroid profiles comprise seven urinary steroid metabolites, of which relevant ratios are significantly increased following ethanol consumption. This effect is presumably based on the lack of hepatic NAD+-coenzyme as a consequence of ethanol oxidation. Only recently, testosterone (T) and androstenedione (A4) blood profiles have been introduced as additional approach for doping control. However, a potential influence of ethanol intake on testosterone biosynthesis and thus on blood steroid profiles has not been investigated so far. Therefore, steroid concentrations from 10 males and 10 females receiving an ethanol infusion up to a breath alcohol concentration of 0.5 mg/L which was hold as a plateau for two hours were conducted. Blood samples were drawn every 15 min for steroid quantification. An ethanol-dependent T/A4 increase up to 385 % resulting from A4 suppression was observed in 14 volunteers. In addition, we observed sporadic A4 increases coinciding with cortisol and ACTH pulses pointing to a meal-induced adrenal stimulation. While testosterone levels in males showed diurnal variation solely, testosterone levels in some females were found to be susceptible to ethanol- and ACTH-dependent perturbations, which is thought to be due to its predominant adrenal synthesis in females. In conclusion, the results of the present study emphasize the importance of blood sampling at a sufficient time interval from food and ethanol intake. This is of interest if T and A4 are used for diagnostics in doping control. [ABSTRACT FROM AUTHOR]