The recent onslaught of mass spectrometry (MS) to measurements of steroid hormones, including demands that they should be the only acceptable method, has confused clinicians and scientists who have ...relied for more than 40 years on a variety of immunoassay (IA) methods in steroid hormone measurements. There is little doubt that MS methods with their superior specificity will be the future method of choice in many clinical and research applications of steroid hormone measurement. However, the majority of steroid measurements are currently, and will continue to be, carried out using various types of IAs for several reasons, including their technical ease, cost and availability of commercial reagents. Speedy replacement of all IAs with MS is an unrealistic and unnecessary goal, because the availability of MS measurements is limited by cost, need of expensive equipment, technical demands and lack of commercial applications. Furthermore, IAs have multiple well-known advantages that vindicate their continuing use. The purpose of this article is to elucidate the advantages and limitations of the MS and IA techniques from two angles, i.e. promotion of MS and defence of IA. The purpose of the text is to give the reader an unbiased view about the current state and future trends of steroid analysis and to help him/her choose the correct assay method to serve his/her diagnostic and research needs.
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•The serum metabolome does not reflect steroids activated in an intracrine manner.•The urinary steroid metabolome reflects steroid biosynthesis and metabolism.•Defined pathways link ...the circulating and urinary steroid metabolomes.•Modern mass spectrometry techniques allow for comprehensive steroid profiling.
Advances in technology have allowed for the sensitive, specific, and simultaneous quantitative profiling of steroid precursors, bioactive steroids and inactive metabolites, facilitating comprehensive characterization of the serum and urine steroid metabolomes. The quantification of steroid panels is therefore gaining favor over quantification of single marker metabolites in the clinical and research laboratories. However, although the biochemical pathways for the biosynthesis and metabolism of steroid hormones are now well defined, a gulf still exists between this knowledge and its application to the measured steroid profiles. In this review, we present an overview of steroid hormone biosynthesis and metabolism by the liver and peripheral tissues, specifically highlighting the pathways linking and differentiating the serum and urine steroid metabolomes. A brief overview of the methodology used in steroid profiling is also provided.
Context:Androgen excess is a defining feature of polycystic ovary syndrome (PCOS) but the exact origin of hyperandrogenemia remains a matter of debate. Recent studies have highlighted the importance ...of the 11-oxygenated C19 steroid pathway to androgen metabolism in humans. In this study, we analyzed the contribution of 11-oxygenated androgens to the androgen excess phenotype in women with PCOS.Methods:114 women with PCOS and 49 healthy controls underwent measurement of serum androgens by liquid chromatography-tandem mass spectrometry. 24-h urinary androgen excretion was analyzed by gas chromatography-mass spectrometry. Fasting plasma insulin and glucose were measured for calculation of homeostatic model assessment of insulin resistance (HOMA-IR). Baseline demographic data including body mass index were recorded.Results:As expected, serum concentrations of the classic androgens testosterone (p<0.001), androstenedione (p<0.001), and DHEA (p<0.01) were significantly increased in PCOS. Mirroring this, serum concentrations of the 11-oxygenated androgens 11β-hydroxyandrostenedione, 11-ketoandrostenedione, 11β-hydroxytestosterone and 11-ketotestosterone were all significantly higher in PCOS women than controls, as was the urinary excretion of the 11-oxygenated androgen metabolite 11β-hydroxyandrosterone. The proportionate contribution of 11-oxygenated to total serum androgens was significantly higher in PCOS compared to controls 53.0% (IQR 48.7-60.3) vs. 44.0% (IQR 32.9-54.9), p<0.0001. Both obese (n=51) and non-obese (n=63) PCOS patients had significantly increased 11-oxygenated androgens. Serum 11β-hydroxyandrostenedione and 11-ketoandrostenedione correlated significantly with markers of insulin resistance.Conclusions:For the first time, we show that 11-oxygenated androgens represent the majority of circulating androgens in women with PCOS, with close correlation to markers of metabolic risk.
Abstract
Context
Patients with adrenal insufficiency require increased hydrocortisone cover during major stress to avoid a life-threatening adrenal crisis. However, current treatment recommendations ...are not evidence-based.
Objective
To identify the most appropriate mode of hydrocortisone delivery in patients with adrenal insufficiency who are exposed to major stress.
Design and Participants
Cross-sectional study: 122 unstressed healthy subjects and 288 subjects exposed to different stressors (major trauma N = 83, sepsis N = 100, and combat stress N = 105). Longitudinal study: 22 patients with preserved adrenal function undergoing elective surgery. Pharmacokinetic study: 10 patients with primary adrenal insufficiency undergoing administration of 200 mg hydrocortisone over 24 hours in 4 different delivery modes (continuous intravenous infusion; 6-hourly oral, intramuscular or intravenous bolus administration).
Main Outcome Measure
We measured total serum cortisol and cortisone, free serum cortisol, and urinary glucocorticoid metabolite excretion by mass spectrometry. Linear pharmacokinetic modeling was used to determine the most appropriate mode and dose of hydrocortisone administration in patients with adrenal insufficiency exposed to major stress.
Results
Serum cortisol was increased in all stress conditions, with the highest values observed in surgery and sepsis. Continuous intravenous hydrocortisone was the only administration mode persistently achieving median cortisol concentrations in the range observed during major stress. Linear pharmacokinetic modeling identified continuous intravenous infusion of 200 mg hydrocortisone over 24 hours, preceded by an initial bolus of 50–100 mg hydrocortisone, as best suited for maintaining cortisol concentrations in the required range.
Conclusions
Continuous intravenous hydrocortisone infusion should be favored over intermittent bolus administration in the prevention and treatment of adrenal crisis during major stress.
Objective- Three distinct human monocyte subsets have been identified based on the surface marker expression of CD14 and CD16. We hypothesized that monocytes were likely more heterogeneous in ...composition. Approach and Results- We used the high dimensionality of mass cytometry together with the FlowSOM clustering algorithm to accurately identify and define monocyte subsets in blood of healthy human subjects and those with coronary artery disease (CAD). To study the behavior and functionality of the newly defined monocyte subsets, we performed RNA sequencing, transwell migration, and efferocytosis assays. Here, we identify 8 human monocyte subsets based on their surface marker phenotype. We found that 3 of these subsets fall within the CD16
nonclassical monocyte population and 4 subsets belong to the CD14
classical monocytes, illustrating significant monocyte heterogeneity in humans. As nonclassical monocytes are important in modulating atherosclerosis in mice, we studied the functions of our 3 newly identified nonclassical monocytes in subjects with CAD. We found a marked expansion of a Slan
CXCR6
nonclassical monocyte subset in CAD subjects, which was positively correlated with CAD severity. This nonclassical subset can migrate towards CXCL16 and shows an increased efferocytosis capacity, indicating it may play an atheroprotective role. Conclusions- Our data demonstrate that human nonclassical monocytes are a heterogeneous population, existing of several subsets with functional differences. These subsets have changed frequencies in the setting of severe CAD. Understanding how these newly identified subsets modulate CAD will be important for CAD-based therapies that target myeloid cells.
Triple A syndrome is caused by mutations in AAAS encoding the protein ALADIN. We investigated the role of ALADIN in the human adrenocortical cell line NCI-H295R1 by either over-expression or ...down-regulation of ALADIN. Our findings indicate that AAAS knock-down induces a down-regulation of genes coding for type II microsomal cytochrome P450 hydroxylases CYP17A1 and CYP21A2 and their electron donor enzyme cytochrome P450 oxidoreductase, thereby decreasing biosynthesis of precursor metabolites required for glucocorticoid and androgen production. Furthermore we demonstrate that ALADIN deficiency leads to increased susceptibility to oxidative stress and alteration in redox homeostasis after paraquat treatment. Finally, we show significantly impaired nuclear import of DNA ligase 1, aprataxin and ferritin heavy chain 1 in ALADIN knock-down cells. We conclude that down-regulating ALADIN results in decreased oxidative stress response leading to alteration in steroidogenesis, highlighting our knock-down cell model as an important in-vitro tool for studying the adrenal phenotype in triple A syndrome.
T helper 2 (Th2) cells regulate helminth infections, allergic disorders, tumor immunity, and pregnancy by secreting various cytokines. It is likely that there are undiscovered Th2 signaling ...molecules. Although steroids are known to be immunoregulators, de novo steroid production from immune cells has not been previously characterized. Here, we demonstrate production of the steroid pregnenolone by Th2 cells in vitro and in vivo in a helminth infection model. Single-cell RNA sequencing and quantitative PCR analysis suggest that pregnenolone synthesis in Th2 cells is related to immunosuppression. In support of this, we show that pregnenolone inhibits Th cell proliferation and B cell immunoglobulin class switching. We also show that steroidogenic Th2 cells inhibit Th cell proliferation in a Cyp11a1 enzyme-dependent manner. We propose pregnenolone as a “lymphosteroid,” a steroid produced by lymphocytes. We speculate that this de novo steroid production may be an intrinsic phenomenon of Th2-mediated immune responses to actively restore immune homeostasis.
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•Differential upregulation of the steroid biosynthetic pathway during Th2 differentiation•T helper cells produce the steroid pregnenolone in vitro and in vivo•Steroidogenic Th2 cells suppress Th cell proliferation in a Cyp11a1-dependent manner•Pregnenolone inhibits B cell immunoglobulin class switching in vitro
Immune cells signal by secreting cytokines and by direct cell-to-cell contact. Although steroids are known to be immunoregulators, immune cells are not generally thought to be steroidogenic. Here, Mahata et al. demonstrate that T helper (Th) cells synthesize and secrete the steroid pregnenolone in vitro and in vivo. Pregnenolone inhibits Th cell proliferation and B cell immunoglobulin class switching, and steroidogenic Th cells inhibit Th cell proliferation. This evidence suggests that steroidogenic Th cells contribute to immune homeostasis.
ABSTRACT
RF system-on-chip (RFSoC) devices provide the potential for implementing a complete radio astronomy receiver on a single board, but performance of the integrated analogue-to-digital ...converters (ADCs) is critical. We have evaluated the performance of the data converters in the Xilinx ZU28DR RFSoC, which are 12-bit, 8-fold interleaved converters with a maximum sample speed of 4.096 Giga-sample per second (GSPS). We measured the spurious-free dynamic range (SFDR), signal-to-noise and distortion (SINAD), effective number of bits (ENOB), intermodulation distortion (IMD), and cross-talk between adjacent channels over the bandwidth of 2.048 GHz. We both captured data for off-line analysis with floating-point arithmetic, and implemented a real-time integer arithmetic spectrometer on the RFSoC. The performance of the ADCs is sufficient for radio astronomy applications and close to the vendor specifications in most of the scenarios. We have carried out spectral integrations of up to 100 s and stability tests over tens of hours and find thermal noise-limited performance over these time-scales.
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.
Regulatory T (Treg) cells contribute to the anti-inflammatory response during atherogenesis. Here we show that during atherogenesis Treg cells lose Foxp3 expression and their immunosuppressive ...function, leading to the conversion of a fraction of these cells into T follicular helper (Tfh) cells. We show that Tfh cells are pro-atherogenic and that their depletion reduces atherosclerosis. Mechanistically, the conversion of Treg cells to Tfh cells correlates with reduced expression of IL-2Rα and pSTAT5 levels and increased expression of IL-6Rα. In vitro, incubation of naive T cells with oxLDL prevents their differentiation into Treg cells. Furthermore, injection of lipid-free Apolipoprotein AI (ApoAI) into ApoE
mice reduces intracellular cholesterol levels in Treg cells and prevents their conversion into Tfh cells. Together our results suggest that ApoAI, the main protein in high-density lipoprotein particles, modulates the cellular fate of Treg cells and thus influences the immune response during atherosclerosis.
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