11-oxygenated estrogens are a novel class of human estrogens but do not contribute to the circulating estrogen pool

Androgens are the obligatory precursors of estrogens. In humans, classic androgen biosynthesis yields testosterone, thought to represent the predominant circulating active androgen in both men and women. However, recent work has shown that 11-ketotestosterone, derived from the newly described 11-oxygenated androgen biosynthesis pathway, makes a substantial contribution to the active androgen pool in women. Considering that classic androgens are the obligatory substrates for estrogen biosynthesis catalyzed by cytochrome P450 aromatase, we hypothesized that 11-oxygenated androgens are aromatizable. Here we utilize steroid analysis by tandem mass spectrometry to demonstrate that human aromatase generates 11-oxygenated estrogens from 11-oxygenated androgens in three different cell-based aromatase expression systems and in human ex vivo placenta explant cultures. We also show that 11-oxygenated estrogens are generated as a byproduct of the aromatization of classic androgens. We show that 11β-hydroxy-17β-estradiol binds and activates estrogen receptors α and β and that 11β-hydroxy-17β-estradiol and the classic androgen pathway-derived active estrogen, 17β-estradiol, are equipotent in stimulating breast cancer cell line proliferation and expression of estrogen-responsive genes. 11-oxygenated estrogens were, however, not detectable in serum from individuals with high aromatase levels (pregnant women) and elevated 11-oxygenated androgen levels (patients with congenital adrenal hyperplasia or adrenocortical carcinoma). Our data shows that while 11-oxygenated androgens are aromatizable in vitro and ex vivo, the resulting 11-oxygenated estrogens are not detectable in circulation, suggesting that 11-oxygenated androgens function primarily as androgens in vivo.

Automated Supported Liquid Extraction for the analysis of a panel of 12 endogenous steroids in human plasma by LC-MS/MS

Steroid analysis is important in the clinical assessment of endocrine function in health and disease. Although tandem mass spectrometry methods coupled with chromatographic separation are considered the gold standard analytical technique in this setting, enabling profiling of multiple steroids in a single sample, sample processing can be labour-intensive. Here we present a simple, efficient automated 96-well Supported Liquid Extraction method with dichloromethane/isopropanol as organic solvent, carried out on a Extrahera automated sample handler (Biotage), which completes sample preparation of 80 plasma samples (200µL) in 90 minutes. Compounds were separated on a Kinetex C18 column (150x3mm;2.6um) using a mobile phase of methanol and water (0.1% formic acid). The run time was 16 minutes on a Nexera uHPLC system (Shimadzu) with a QTrap 6500+ linear ion trap mass spectrometer (AB Sciex). Precisions ranged 8.1 to 18.1% RSD, bias -10.1-5.8%, and extraction recoveries 73.5-111.9%. LOQs ranged between 0.025–0.500 ng/mL.

Recent Applications of Supercritical Fluid Chromatography in Modern Analysis: Updates from 2017 to 2020

Background: Supercritical fluid chromatography (SFC) is one of the powerful analytical techniques of the modern time. Recently, extensive analytical work has been reported and is in progress by utilizing the advanced features of SFC. Low solvent consumption, high sensitivity and solvent recovery make it advantageous over the traditional liquid chromatographic techniques. It utilizes supercritical fluids having properties of both liquid and gases making the applicability of this technique to wider range of analytes. Methods: Various research reports were collected from search engines like Sciencedirect, Pubmed, Researchgate and Google Scholar. Further upon through study of these reports, significant findings/data was collected and compiled under suitable headings. Important parameters/conditions utilized in methodologies were depicted with the help of tables. Results: It was found in various reports that SFC and its hyphenation with mass spectroscopy (MS), ultra violet spectroscopy (UV), high resolution mass spectrometry (HRMS), photo diode array detector (PDA), nuclear magnetic resonance (NMR), charged aerosol detector (CAD) etc. have made possible to quantify analytes even in ultra small concentrations in complex matrices. These techniques have been successfully employed for the quantification of a wide variety of analytes with excellent accuracy, selectivity and sensitivity. Conclusion: The present review highlights the recent applications of SFC techniques in various analytical fields such as pesticide analysis, vitamin analysis, enantioseparation, lipid analysis, drug metabolite estimation, polycyclic aromatic hydrocarbons and steroid analysis. Reports from 2017 to 2020 have been included in this compilation.

Isolated 17, 20 Lyase Deficiency Secondary to a Novel CYB5A Variant: Comparison of Steroid Metabolomic Findings with Published Cases Provides Diagnostic Guidelines and Greater Insight into Its Biological Role

Objective: The objective of this study was to report CYB5A deficiency, to discuss the contribution of steroid metabolomics to diagnosis and interpretation, and to highlight the presence of testicular microlithiasis. Methods: Two siblings with ambiguous genitalia at birth were later found to carry novel CYB5A variants, with resulting isolated 17, 20 lyase deficiency. We compared urine steroid data obtained between birth and adulthood with that from other cases. Results: Neonatal urine steroid profiles show a relative increase of 16-hydroxylated pregnenolone metabolites. Thereafter, there are no distinguishing features until puberty, when sex steroid deficiency drives gonadotrophin production, resulting in marked increases of 17-hydroxyprogesterone metabolites derived from the gonads. This excess may be revealed pre-pubertally by gonadotrophin stimulation testing. Novel findings are first, a considerable capacity for DHEA synthesis in the neonatal period compared to childhood and adulthood, suggesting that DHEAS production is much less dependent on CYB5A at birth; second, no consistent change in “backdoor pathway” intermediates; third, side chain cleavage of cortisol is largely unaffected, supporting the existence of a different lyase not dependent on CYB5A; fourth, increased 17-hydroxyprogesterone metabolites and very low androgen metabolites are diagnostic post-pubertally. Conclusion: This is the fourth disease-causing variant in CYB5A in isolated 17, 20 lyase deficiency and the first associated with testicular microlithiasis. Establishing a biochemical diagnosis pre-pubertally should now be possible using urine steroid profiling, supported by synacthen and gonadotrophin stimulation testing. We recommend liquid chromatography-mass spectrometry/mass spectrometry rather than immunoassay for serum steroid analysis, early methaemoglobin measurement and surveillance should testicular microlithiasis be detected.

Steroid Metabolome Analysis in Disorders of Adrenal Steroid Biosynthesis and Metabolism

Steroid biosynthesis and metabolism are reflected by the serum steroid metabolome and, in even more detail, by the 24-hour urine steroid metabolome, which can provide unique insights into alterations of steroid flow and output indicative of underlying conditions. Mass spectrometry–based steroid metabolome profiling has allowed for the identification of unique multisteroid signatures associated with disorders of steroid biosynthesis and metabolism that can be used for personalized approaches to diagnosis, differential diagnosis, and prognostic prediction. Additionally, steroid metabolome analysis has been used successfully as a discovery tool, for the identification of novel steroidogenic disorders and pathways as well as revealing insights into the pathophysiology of adrenal disease. Increased availability and technological advances in mass spectrometry–based methodologies have refocused attention on steroid metabolome profiling and facilitated the development of high-throughput steroid profiling methods soon to reach clinical practice. Furthermore, steroid metabolomics, the combination of mass spectrometry–based steroid analysis with machine learning–based approaches, has facilitated the development of powerful customized diagnostic approaches. In this review, we provide a comprehensive up-to-date overview of the utility of steroid metabolome analysis for the diagnosis and management of inborn disorders of steroidogenesis and autonomous adrenal steroid excess in the context of adrenal tumors.

100 Reproductive cycle and pregnancy monitoring in the common hippopotamus (Hippopotamus amphibius) through salivary steroid analyses and transabdominal ultrasonography

The common hippopotamus (Hippopotamus amphibius) is listed as vulnerable to extinction by the IUCN due to a significant decrease in population size, caused by habitat loss and poaching. Ex situ populations can help ensure against species loss, but careful reproductive management is essential to maintain sustainable populations. Hormone monitoring allows for characterisation of the reproductive cycle and gestation, offering insight into timing of receptivity and conception and facilitating pregnancy diagnosis and estimation of parturition date. Fecal steroid analysis has been validated for measuring progestogens in hippos. However, hippos are often housed in groups and frequently defecate in the water, making sample collection and source identification difficult. Salivary steroid analysis has been employed for monitoring reproductive activity in several species, but has not been tested in hippos. Additionally, transabdominal ultrasonography has proven valuable in diagnosing and monitoring pregnancy in many large mammals, but efficacy in the common hippo is unknown. The goals of this project were to (1) validate the use of an enzyme immunoassay to monitor progestogens in hippo saliva, (2) confirm that salivary progestogen profiles accurately reflect reproductive activity, (3) determine if transabdominal ultrasonography can be used to diagnose pregnancy, and, if so, (4) monitor and characterise fetal development via weekly examinations. Saliva (4-7 per week) and fecal (2-7 per week) samples were collected from 7 adult female hippos housed at 3 USA facilities over 3-7 months. Saliva and fecal samples were extracted in ethanol and extracts diluted (1:2 to 1:10 and 1:25 to 1:500, respectively) before evaluation by enzyme immunoassay (Progesterone mini-kit; Arbor Assays). Parallelism was confirmed between serially diluted fecal (r2=0.993) and saliva (r2=0.990) samples and the standard curve. Inter- and intra-assay coefficients of variation were maintained at <10%. Comparison of fecal and saliva progestogen concentrations revealed a strong correlation between the 2 sample types (r2=0.848) and suggested that saliva offers a comparable alternative. Both fecal and saliva extracts exhibited elevated progestogens during luteal phases and gestation. One nulliparous female housed at the Cincinnati Zoo & Botanical Garden (Cincinnati, OH, USA) was trained for voluntary transabdominal ultrasound exams. An Ibex Pro portable ultrasound machine (E.I. Medical Imaging, Loveland, CO, USA) with curvilinear probe (5-2.5MHz) was used at a scanning depth of 17.8 and 23.4cm. Intrauterine fluid and possible fetal tissue were observed 79 days following the last confirmed mating. Spine, rib cage, and beating heart were clearly visible at ~156 days of gestation. Ultrasound procedures were continued until the premature birth of a calf at ~181 days (normal hippo gestation ~231 days). Salivary progestogen monitoring and transabdominal ultrasonography appear suitable for tracking reproductive activity and diagnosing and monitoring pregnancy in the common hippo.

Direct and rapid analysis of trace levels steroids in water by thermal desorption atmospheric pressure photoionization mass spectrometry

A novel TD-APPI technique for steroid analysis at trace levels from a 10 μL water sample without derivatization and chromatographic separation.