CYP11B2 inhibitor dexfadrostat phosphate suppresses the aldosterone-to-renin ratio, an indicator of sodium retention, in healthy volunteers.

AIMS: High aldosterone is a key driver of hypertension and long-term negative sequelae. We evaluated the safety and efficacy of dexfadrostat phosphate (DP13), a novel aldosterone synthase (CYP11B2) inhibitor, in healthy participants. METHODS: This randomized, double-blind, placebo-controlled study was conducted in two parts. In part A, a single-ascending dose escalation, 16 participants received oral DP13 1-16 mg. Part B was a multiple-ascending dose, sequential group study in which 32 participants received oral DP13 4, 8 or 16 mg once daily for 8 days. Safety and tolerability were monitored throughout. An adrenocorticotropic hormone (ACTH) stimulation test at maximal blood drug concentrations defined the dose range for multiple dosing. RESULTS: DP13 was well tolerated at all doses, with no serious adverse events. In part B, all DP13 doses (4, 8 and 16 mg) over 8 days effectively suppressed aldosterone production, increased the urinary sodium/potassium ratio, decreased plasma sodium and increased plasma potassium and renin levels compared with placebo, resulting in potent suppression of the aldosterone-to-renin ratio (ARR). Endocrine counter-regulation resulted in the 4 mg dose no longer sustaining 24-h aldosterone suppression after 8 days of treatment, unlike the 8- and 16 mg doses. There was no evidence of drug-induced adrenal insufficiency (ACTH stress challenge). CONCLUSIONS: In patients with excess aldosterone and ensuing sodium retention driving hypertension, managing sodium balance is critical. A CYP11B2 inhibitor like DP13, whose effectiveness can be monitored by a reduction in ARR, may prove valuable in managing aldosterone-dependent hypertension and primary aldosteronism.

Parallel targeted and non-targeted quantitative analysis of steroids in human serum and peritoneal fluid by liquid chromatography high-resolution mass spectrometry.

We developed and validated a liquid chromatography high-resolution mass spectrometry method for the absolute quantification of 51 steroids for clinical analysis of human serum and, for the first time, peritoneal fluid. Data acquisition was performed in both targeted and untargeted mode simultaneously, thus allowing the accurate and precise quantification of the main components of the classical steroid pathways (17 steroids) as well as the analysis of 34 additional non-classical steroids. For targeted analysis, validation was performed according to FDA guidelines, resulting, among other parameters, in accuracy < 13% RSD and precision < 10% relative error, for both inter- and intra-day validation runs. By establishing steroid-specific response factors, the calibration curves of the targeted analytes can be extended to untargeted analytes. This approach opens novel possibilities for the post hoc analysis of clinical samples as the data can be examined for virtually any steroid even after data acquisition, enabling facile absolute quantification once a standard becomes available. We demonstrate the applicability of the approach to evaluate the differences in steroid content between peripheral serum and peritoneal fluid across the menstrual cycle phases, as well as the effect of the synthetic gestagen dienogest on the steroid metabolome.

Salivary cortisol in healthy dogs: a randomized cross-over study to evaluate different saliva stimulation methods and their effects on saliva volume and cortisol concentration.

BACKGROUND: Salivary cortisol collected at home is a useful test to diagnose and monitor Cushing's syndrome in humans. The main problem in dogs is to retrieve a sufficient amount of saliva. The aim of this study was to evaluate different salivary collection methods and compare their effects on volume, pH and cortisol concentration of saliva. Sixteen healthy Beagles were used in a 4 × 4 randomized crossover study with a washout period of 1 week between each of the following collection methods: 1. Salimetrics® cotton swab dipped in ginger powder (ginger group); 2. beef-flavored Salimetrics® (bouillon group); 3. Salivette® cotton swab with an enclosed treat (treat group); 4. plain Salimetrics® (control group). First, baseline saliva (plain cotton swab, S0) and, 2 min later, experimental saliva (according to group allocation above, SExp) were collected. Saliva was gathered by holding the swabs in the animal's mouth for 2 min. After the cross-over study, another saliva sample was collected from all dogs by the ginger method, using a 30 s sampling time (30s-ginger method). Cortisol concentrations were measured by liquid chromatography tandem mass spectrometry. RESULTS: All three stimulation methods increased saliva production significantly (S0 compared to SExp: ginger p = 0.0005; bouillon p = 0.009; treat p = 0.007). Only ginger stimulation, however, generated a significantly higher amount of saliva (SExp) compared to the control group (p = 0.00001; median (range) amount of saliva for SExp: ginger 1200 ul (600-1700), bouillon 650 ul (200-1900), treat 700 ul (300-1000), control 400 ul (0-1100)). The amount of saliva retrieved by the 30s-ginger method was still higher than that from the control group (p = 0.0004). Bouillon and treat stimulation led to decreased pH values (bouillon, p = 0.0028; treat, 0.0018). Excitement was higher in the ginger group (p = 0.01). Chewing was intensified in the ginger and treat group (ginger, p = 0.003; treat, 0.0009). The cortisol concentration SExp was higher compared to that of S0 in the ginger and treat group (p = 0.02, 0.003). The experimental cortisol concentrations (SExp) were not different between groups. CONCLUSIONS: The 30s-ginger method could prove useful in evaluating or monitoring dogs with Cushing's syndrome, as sampling at home for 30 s by the owner seems feasible.

Revisiting Steroidogenic Pathways in the Human Placenta and Primary Human Trophoblast Cells.

Steroid hormones play a crucial role in supporting a successful pregnancy and ensuring proper fetal development. The placenta is one of the principal tissues in steroid production and metabolism, expressing a vast range of steroidogenic enzymes. Nevertheless, a comprehensive characterization of steroidogenic pathways in the human placenta and potential developmental changes occurring during gestation are poorly understood. Furthermore, the specific contribution of trophoblast cells in steroid release is largely unknown. Thus, this study aimed to (i) identify gestational age-dependent changes in the gene expression of key steroidogenic enzymes and (ii) explore the role of trophoblast cells in steroid biosynthesis and metabolism. Quantitative and Droplet Digital PCR analysis of 12 selected enzymes was carried out in the first trimester (n = 13) and term (n = 20) human placentas. Primary trophoblast cells (n = 5) isolated from human term placentas and choriocarcinoma-derived cell lines (BeWo, BeWo b30 clone, and JEG-3) were further screened for gene expression of enzymes involved in placental synthesis/metabolism of steroids. Finally, de novo steroid synthesis by primary human trophoblasts was evaluated, highlighting the functional activity of steroidogenic enzymes in these cells. Collectively, we provide insights into the expression patterns of steroidogenic enzymes as a function of gestational age and delineate the cellular origin of steroidogenesis in the human placenta.

Recommendations for reporting ion mobility Mass Spectrometry measurements.

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0 ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

Rapid quantification of insulin degludec by immunopurification combined with liquid chromatography high-resolution mass spectrometry.

Insulin degludec is an ultra-long-acting insulin analogue that is increasingly being used in diabetes due to its favourable efficacy and safety profile. Thus, there is an increasing demand for a reliable and specific analytical method to quantify insulin degludec for research, pharmaceutical industry and clinical applications. We developed and validated an automated, high-throughput method for quantification of insulin degludec in human blood samples across the expected clinical range combining immunopurification with high-resolution mass spectrometry. Validation was performed according to the requirements of the US Food and Drug Administration. The method satisfyingly met the following parameters: lower limit of quantification (120 pM), linearity, accuracy (error < 5%), precision (CV < 7.7%), selectivity, carry-over, recovery (89.7-97.2%), stability and performance in the presence of other insulin analogues. The method was successfully applied to clinical samples of patients treated with insulin degludec showing a good correlation with the administered dose (r2 = 0.78). High usability of the method is supported by the small specimen volume, automated sample processing and short analysis time. In conclusion, this reliable, easy-to-use and specific mass spectrometric insulin degludec assay offers great promise to address the current unmet need for standardized insulin analytics in academic and industrial research. Graphical Abstract.

Characterization of Two Novel Variants of the Steroidogenic Acute Regulatory Protein Identified in a Girl with Classic Lipoid Congenital Adrenal Hyperplasia.

Congenital adrenal hyperplasia (CAH) consists of several autosomal recessive disorders that inhibit steroid biosynthesis. We describe a case report diagnosed with adrenal insufficiency due to low adrenal steroids and adrenocorticotropic hormone excess due to lack of cortisol negative feedback signaling to the pituary gland. Genetic work up revealed two missense variants, p.Thr204Arg and p.Leu260Arg in the STAR gene, inherited by both parents (non-consanguineous). The StAR protein supports CYP11A1 enzyme to cleave the side chain of cholesterol and synthesize pregnenolone which is metabolized to all steroid hormones. We used bioinformatics to predict the impact of the variants on StAR activity and then we performed functional tests to characterize the two novel variants. In a cell system we tested the ability of variants to support cholesterol conversion to pregnenolone and measured their mRNA and protein expression. For both variants, we observed loss of StAR function, reduced protein expression and categorized them as pathogenic variants according to guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. These results fit the phenotype of the girl during diagnosis. This study characterizes two novel variants and expands the list of missense variants that cause CAH.

Quantitative Fragmentation Model for Bottom-Up Shotgun Lipidomics.

Quantitative bottom-up shotgun lipidomics relies on molecular species-specific "signature" fragments consistently detectable in tandem mass spectra of analytes and standards. Molecular species of glycerophospholipids are typically quantified using carboxylate fragments of their fatty acid moieties produced by higher-energy collisional dissociation of their molecular anions. However, employing standards whose fatty acids moieties are similar, yet not identical, to the target lipids could severely compromise their quantification. We developed a generic and portable fragmentation model implemented in the open-source LipidXte software that harmonizes the abundances of carboxylate anion fragments originating from fatty acid moieties having different sn-1/2 positions at the glycerol backbone, length of the hydrocarbon chain, and number and location of double bonds. The postacquisition adjustment enables unbiased absolute (molar) quantification of glycerophospholipid species independent of instrument settings, collision energy, and employed internal standards.

High-Resolution Ion Mobility Spectrometry for Rapid Cannabis Potency Testing.

We report initial results from an ion mobility spectrometry (IMS)-based analysis of natural cannabis samples and explore the possibility of using this technique to distinguish medical marijuana from illegal forms of the drug, as defined by Swiss legislation. We analyzed cannabis extracts by electrospray ionization IMS-MS and found that high-resolution drift-tube IMS ( R > 150) can effectively isolate and quantify the controlled substance, Δ9-tetrahydrocannabinol (THC), even in the presence of other noncontrolled cannabinoid isomers including cannabidiol (CBD). We used this information to determine whether the THC content of a given sample surpassed the legal limit, which is 1% by weight in Switzerland. Our IMS-MS methodology produced equivalent quantification results to standard HPLC-based methods and offers the additional advantage of significantly shorter time requirements for the analysis. In addition, IMS-based analysis offers flexibility over HPLC in that it can be performed on portable devices. As such, these findings may have implications for cannabis testing in police laboratories.

A calibration framework for the determination of accurate collision cross sections of polyanions using polyoxometalate standards.

RATIONALE: Polyoxometalates (POMs) are remarkable oxo-clusters forming compact highly charged anions. We measured their collision cross sections (CCS) in N2 with drift tube ion mobility spectrometry (DTIMS). These values were then used to calibrate a traveling wave ion mobility spectrometry (TWIMS) device and the accuracy of the calibration was tested. METHODS: Six POM standards were analyzed by DTIM-MS (Tofwerk, Thun, Switzerland) at different voltages to determine absolute DT CCS (N2 ) values. Five POM compounds (Lindqvist TBA2 Mo6 O19; decatungstate TBA4 W10 O32; Keggin TBA3 PMo12 O40 ; TBA3 PW12 O40 and Dawson TBA6 P2 W18 O62 ) were used for the calibration of the TWIM-MS instrument (Synapt G2 HDMS, Waters, Manchester, UK) and a sixth Dawson POM, TBA9 P2 Nb3 W15 O62 , was used to compare the accuracy of the calibrations with POM or with polyalanine and dextran reference ions. RESULTS: We determined 45 DT CCS (N2 ) values at 30°C or 60°C. Fourteen DT CCS (N2 ) values at 30°C were used to perform calibration of the TWIMS instrument. Better correlations were observed than when DT CCS values in helium from the literature were used. The accuracy tests on six ions of Dawson POM TBA9 P2 Nb3 W15 O62 led to relative errors below 3.1% while relative errors of 3.6% to 10.1% were observed when calibration was performed with polyalanine and dextran reference ions. CONCLUSIONS: Our novel calibration strategy for determination of CCS values of multiply negatively charged ions on TWIM-MS devices based on DT CCS (N2 ) of standard POM structures covered a wider range of CCS and improved the accuracy to 2.1% relative error on average compared with 6.9% using polyalanine and dextran calibration.

A comprehensive urinary steroid analysis strategy using two-dimensional gas chromatography - time of flight mass spectrometry.

Steroids are key players in a high variety of physiological processes and are typically analyzed for the diagnosis of hormonal disorders. Due to their chemical and structural similarity many of these metabolites cannot be separated by conventional techniques such as liquid chromatography. Herein, we present an analysis strategy based on two dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry (TOF MS) which demonstrates superior separation power and enables comprehensive screening of steroids. We show absolute quantitation of 40 steroids in human urine over three orders of magnitude with limits of detection ≤50 nM and the tentative identification of additional 30 steroids based on accurate mass, isotopic pattern analysis and spectral similarity matching to known steroids. The method displays excellent inter- and intra-day stability, repeatability and recovery and was validated for clinical routine analysis. Additionally, we demonstrate the potential of the approach for untargeted analysis of urinary steroids in mouse and rat.

Response Profiling Using Shotgun Proteomics Enables Global Metallodrug Mechanisms of Action To Be Established.

Response profiling using shotgun proteomics for establishing global metallodrug mechanisms of action in two colon carcinoma cell lines, HCT116 and SW480, has been applied and evaluated with the clinically approved arsenic trioxide. Surprisingly, the complete established mechanism of action of arsenic trioxide was observed by protein regulations in SW480, but not HCT116 cells. Comparing the basal protein expression in the two cell lines revealed an 80 % convergence of protein identification, but with significant expression differences, which in turn seem to affect the extent of protein regulation. A clear-cut redox response was observed in SW480 cells upon treatment with arsenic, but hardly in HCT116 cells. Response profiling was then used to investigate four anti-cancer metallodrugs (KP46, KP772, KP1339 and KP1537). Proteome alterations were mapped to selected functional groups, including DNA repair, endocytosis, protection from oxidative stress, protection from endoplasmatic reticulum (ER) stress, cell adhesion and mitochondrial function. The present data suggest that knowledge of the mechanism of action of anti-cancer metallodrugs and improved patient stratification strategies are imperative for the design of clinical studies.

Highly Oxygenated Multifunctional Compounds in α-Pinene Secondary Organic Aerosol.

Highly oxygenated multifunctional organic compounds (HOMs) originating from biogenic emissions constitute a widespread source of organic aerosols in the pristine atmosphere. However, the molecular forms in which HOMs are present in the condensed phase upon gas-particle partitioning remain unclear. In this study, we show that highly oxygenated molecules that contain multiple peroxide functionalities are readily cationized by the attachment of Na+ during electrospray ionization operated in the positive ion mode. With this method, we present the first identification of HOMs characterized as C8-10H12-18O4-9 monomers and C16-20H24-36O8-14 dimers in α-pinene derived secondary organic aerosol (SOA). Simultaneous detection of these molecules in the gas phase provides direct evidence for their gas-to-particle conversion. Molecular properties of particulate HOMs generated from ozonolysis and OH oxidation of unsubstituted (C10H16) and deuterated (C10H13D3) α-pinene are investigated using coupled ion mobility spectrometry with mass spectrometry. The systematic shift in the mass of monomers in the deuterated system is consistent with the decomposition of isomeric vinylhydroperoxides to release vinoxy radical isotopologues, the precursors to a sequence of autoxidation reactions that ultimately yield HOMs in the gas phase. The remarkable difference observed in the dimer abundance under O3- versus OH-dominant environments underlines the competition between intramolecular hydrogen migration of peroxy radicals and their bimolecular termination reactions. Our results provide new and direct molecular-level information for a key component needed for achieving carbon mass closure of α-pinene SOA.

Simultaneous endo and exo†Complex Formation of Pyridine[4]arene Dimers with Neutral and Anionic Guests.

The formation of complexes between hexafluorophosphate (PF6- ) and tetraisobutyloctahydroxypyridine[4]arene has been thoroughly studied in the gas phase (ESI-QTOF-MS, IM-MS, DFT calculations), in the solid state (X-ray crystallography), and in chloroform solution (1 H, 19 F, and DOSY NMR spectroscopy). In all states of matter, simultaneous endo complexation of solvent molecules and exo complexation of a PF6- anion within a pyridine[4]arene dimer was observed. While similar ternary complexes are often observed in the solid state, this is a unique example of such behavior in the gas phase.

DOSY NMR, X-ray Structural and Ion-Mobility Mass Spectrometric Studies on Electron-Deficient and Electron-Rich M6L4 Coordination Cages.

A novel modular approach to electron-deficient and electron-rich M6L4 cages is presented. From the same starting compound, via a minor modulation of the synthesis route, two C3-symmetric ligands L1 and L2 with different electronic properties are obtained in good yield. The trifluoro-triethynylbenzene-based ligand L1 is more electron-deficient than the well-known 2,4,6-tri(4-pyridyl)-1,3,5-triazine, while the trimethoxy-triethynylbenzene-based ligand L2 is more electron-rich than the corresponding benzene analogue. Complexation of the ligands with cis-protected square-planar [(dppp)Pt(OTf)2] or [(dppp)Pd(OTf)2] corner-complexes yields two electron-deficient (1a and 1b) and two electron-rich (2a and 2b) M6L4 cages. The single crystal X-ray diffraction study of 1a and 2a confirms the expected octahedral shape with a ca. 2000 Å(3) cavity and ca. 11 Å wide apertures. The crystallographically determined diameters of 1a and 2a are 3.7 and 3.6 nm, respectively. The hydrodynamic diameters obtained from the DOSY NMR in CDCl3:CD3OD (4:1), and diameters calculated from collision cross sections (CCS) acquired by ion-mobility mass spectrometry (IM-MS) were for all four cages similar. In solution, the cage structures have diameters between 3.3 to 3.6 nm, while in the gas phase the corresponding diameters varied between 3.4 to 3.6 nm. In addition to the structural information the relative stabilities of the Pt6L4 and Pd6L4 cages were studied in the gas phase by collision-induced dissociation (CID) experiments, and the photophysical properties of the ligands L1 and L2 and cages 1a, 1b, 2a, and 2b were studied by UV-vis and fluorescence spectroscopy.

Superchiral Pd3 L6 Coordination Complex and Its Reversible Structural Conversion into Pd3 L3 Cl6 Metallocycles.

Large, non-symmetrical, inherently chiral bispyridyl ligand L derived from natural ursodeoxycholic bile acid was used for square-planar coordination of tetravalent Pd(II) , yielding the cationic single enantiomer of superchiral coordination complex 1 Pd3 L6 containing 60 well-defined chiral centers in its flower-like structure. Complex 1 can readily be transformed by addition of chloride into a smaller enantiomerically pure cyclic trimer 2 Pd3 L3 Cl6 containing 30 chiral centers. This transformation is reversible and can be restored by the addition of silver cations. Furthermore, a mixture of two constitutional isomers of trimer, 2 and 2', and dimer, 3 and 3', can be obtained directly from L by its coordination to trans- or cis-N-pyridyl-coordinating Pd(II) . These intriguing, water-resistant, stable supramolecular assemblies have been thoroughly described by (1) H DOSY NMR, mass spectrometry, circular dichroism, molecular modelling, and drift tube ion-mobility mass spectrometry.

Proteome profiling of breast cancer biopsies reveals a wound healing signature of cancer-associated fibroblasts.

Breast cancer is still the most common type of cancer in women; an important role in carcinogenesis is actually attributed to cancer-associated fibroblasts. In this study, we investigated whether it is possible to assess the functional state of cancer-associated fibroblasts through tumor tissue proteome profiling. Tissue proteomics was performed on tumor-central, tumor-near, and tumor-distant biopsy sections from breast adenocarcinoma patients, which allowed us to identify 2074 proteins. Data were interpreted referring to reference proteome profiles generated from primary human mammary fibroblasts comprising 4095 proteins. These cells were analyzed in quiescent cell state as well as after in vitro treatment with TGFß or IL-1ß, stimulating wound healing or inflammatory processes, respectively. Representative for cancer cells, we investigated the mammary carcinoma cell line ZR-75-1, identifying 5212 proteins. All mass analysis data have been made fully accessible via ProteomeXchange, DOI PXD001311 and PXD001323-8. Comparison of tissue proteomics data with all of those reference profiles revealed predominance of cancer cell-derived proteins within the tumor and fibroblast-derived proteins in the tumor-distant tissue sections. Remarkably, proteins characteristic for acute inflammation were hardly identified in the tissue samples. In contrast, several proteins found by us to be induced by TGFß in mammary fibroblasts, including fibulin-5, SLC2A1, and MUC18, were positively identified in all tissue samples, with relatively higher abundance in tumor neighboring tissue sections. These findings indicate a predominance of cancer-associated fibroblasts with wound healing activities localized around tumors.

Changes of several brain receptor complexes in the cerebral cortex of patients with Alzheimer disease: probable new potential pharmaceutical targets.

Although Alzheimer disease (AD) has been linked to defects in major brain receptors, studies thus far have been limited to the determination of receptor subunits or specific ligand binding studies. However, the availability of current technology enables the determination and quantification of brain receptor complexes. Thus, we examined levels of native receptor complexes in the brains of patients with AD. Cortical tissue was obtained from control subjects (n = 12 females and 12 males) and patients with AD (n = 12 females and 12 males) within a 3-h postmortem time period. The tissues were kept frozen until further biochemical analyses. Membrane proteins were extracted and subsequently enriched by ultracentrifugation using a sucrose gradient. Membrane proteins were then electrophoresed onto native gels and immunoblotted using antibodies against individual brain receptors. We found that the levels were comparable for complexes containing GluR2, GluR3 and GluR4 as well as 5-HT1A. Moreover, the levels of complexes containing muscarinic AChR M1, NR1 and GluR1 were significantly increased in male patients with AD. Nicotinic AChRs 4 and 7 as well as dopaminergic receptors D1 and D2 were also increased in males and females with AD. These findings reveal a pattern of altered receptor complex levels that may contribute to the deterioration of the concerted activity of these receptors and thus result in cognitive deficits observed in patients with AD. It should be emphasised that receptor complexes function as working units rather than individual subunits. Thus, the receptor deficits identified may be relevant for the design of experimental therapies. Therefore, specific pharmacological modulation of these receptors is within the pharmaceutical repertoire.

Application of mass spectrometric techniques to delineate the modes-of-action of anticancer metallodrugs.

Mass spectrometry (MS) has emerged as an important tool for studying anticancer metallodrugs in complex biological samples and for characterising their interactions with biomolecules and potential targets on a molecular level. The exact modes-of-action of these coordination compounds and especially of next generation drug candidates have not been fully elucidated. Due to the fact that DNA is considered a crucial target for platinum chemotherapeutics, metallodrug-DNA binding studies dominated the field for a long time. However, more recently, alternative targets were considered, including enzymes and proteins that may play a role in the overall pharmacological and toxicological profile of metallodrugs. This review focuses on MS-based techniques for studying anticancer metallodrugs in vivo, in vitro and in situ to delineate their modes-of-action.