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11β-Hydroxysteroid dehydrogenase 1 (11βHSD1) is a drug target to attenuate adverse effects of chronic glucocorticoid excess. It catalyses intracellular regeneration of active glucocorticoids in tissues including brain, liver and adipose tissue (coupled to hexose-6-phosphate dehydrogenase, H6PDH). 11βHSD1 activity in individual tissues is thought to contribute significantly to glucocorticoid levels at those sites, but its local contribution versus glucocorticoid delivery via the circulation is unknown. Here, we hypothesised that hepatic 11βHSD1 would contribute significantly to the circulating pool. This was studied in mice with Cre-mediated disruption of Hsd11b1 in liver (Alac-Cre) versus adipose tissue (aP2-Cre) or whole-body disruption of H6pdh. Regeneration of [9,12,12-2 46 H3]-cortisol (d3F) from [9,12,12-2 47 H3]-cortisone (d3E), measuring 11βHSD1 reductase activity was assessed at steady state following infusion of [9,11,12,12-2 48 H4]-cortisol (d4F) in male mice. Concentrations of steroids in plasma and amounts in liver, adipose tissue and brain were measured using mass spectrometry interfaced with matrix assisted laser desorption ionisation or liquid chromatography. Amounts of d3F were higher in liver, compared with brain and adipose tissue. Rates of appearance of d3F were ~6-fold slower in H6pdh-/- 52 mice, showing the importance of whole-body 11βHSD1 reductase activity. Disruption of liver 11βHSD1 reduced amounts of d3F in liver (by ~36%), without changes elsewhere. In contrast disruption of 11βHSD1 in adipose tissue reduced rates of appearance of circulating d3F (by ~67%) and also reduced regeneration of d3F in liver and brain (both by ~30%). Thus, the contribution of hepatic 11βHSD1 to circulating glucocorticoid levels and amounts in other tissues is less than that of adipose tissue.

Impaired hepatic glucose and lipid metabolism are hallmarks of type–2 diabetes. Increased sulfide production from cysteine, or sulfide–donor compounds, may beneficially regulate hepatic metabolism. Disposal of sulfide through the sulfide oxidation pathway (SOP) is critical for maintaining sulfide within a safe physiological range. We show that mice lacking the liver–enriched mitochondrial SOP enzyme thiosulfate sulfur–transferase ( Tst —/— mice) exhibit high circulating sulfide, increased gluconeogenesis, hypertriglyceridemia and fatty liver, despite whole–body insulin– sensitisation. Unexpectedly, hepatic sulfide levels were normal in Tst —/— mice, a result of homeostatic induction of mitochondrial sulfide disposal and glutathione excretion associated with net suppression of protein persulfidation and nuclear respiratory factor–2 target proteins. Proteomic and persulfidomic profiling converged on hepatic lipid metabolism, revealing a deficit in medium–chain fatty acid oxidation in Tst —/— mice. In conclusion our findings have implications for sulfide-donor strategies in the context of liver function and metabolic disease.

Estrogens and their bioactive metabolites play key roles in regulating diverse processes in health and disease. In particular, estrogen and estrogenic metabolites have shown both protective and non-protective effects on disease pathobiology, implicating the importance of this steroid pathway in disease diagnostics and monitoring. All estrogens circulate in a wide range of concentrations, which in some patient cohorts can be extremely low. However, elevated levels of E2 are also reported in disease. For example, in pulmonary arterial hypertension (PAH) levels are elevated in men with idiopathic PAH and in postmenopausal women with PAH. Conventional immunoassay techniques have been under scrutiny for some time with their selectivity, accuracy and precision coming into question. Analytical methodologies such as gas and liquid chromatography coupled to single and tandem mass spectrometric approaches (GC-MS, GC-MS/MS, LC-MS and LC-MS/MS) have been developed to quantify endogenous estrogens and in some cases their bioactive metabolites in biological fluids such as urine, serum, plasma and saliva. Liquid-liquid or solid-phase extraction approaches are favoured with derivatization remaining a necessity for lower volumes of sample. The limits of quantitation of individual assays vary but are commonly in the range of 0.5 - 5 pg/mL for estrone and estradiol, with limits of their bioactive metabolites being higher. This review provides an overview of current approaches for measurement of estrogens in biological matrices by MS, highlighting the advances in this field and the challenges remaining for routine use in the clinical and research environment. [Abstract copyright: Copyright © 2019. Published by Elsevier Ltd.]

Estrogens regulate many diverse biological processes in health and disease. They circulate at a wide range of concentrations in females generating several active metabolites (hydroxy and methoxyestrogens). The metabolites are assumed to be present in much lower levels and are thought to contribute to diseases such as pulmonary arterial hypertension (PAH). Estrogen metabolites are challenging to quantify in plasma and currently available immunoassays are non-specific. Here we have developed and validated a novel assay to simultaneously quantify parent estrogens and their metabolites by mass spectrometry (MS). Estrogens were extracted from human plasma using solid phase extraction and derivatized using 1-(5-fluoro-2, 4-dinitrophenyl)-4-methylpiperazine (PPZ) before quaternization by methylation ("MPPZ"). MPPZ derivatives were separated and quantified by liquid chromatography tandem MS (LC-MS/MS) in positive electrospray ionization mode, using a QTrap 6500 + coupled to a Shimadzu Nexera X2. Separation was achieved using an ACE Excel 2 C18-PFP column (2 μm, 2.1 mm × 150 mm). The limits of quantification (LOQ) were 0.43-2.17 pg on column with a linear range from 2 or 10 - 2000 pg mL . Intra and inter-day precision and accuracy were acceptable (

Analysis of catechol estrogens (2 & 4 hydroxy-estrone and estradiol) has proven troublesome by liquid chromatography tandem mass spectrometry due to their low concentrations, short half-lives and temperature-labile nature. Derivatization to methyl piperazine analogues has been reported for a panel of 9 estrogens in, "Derivatization enhances analysis of estrogens and their bioactive metabolites in human plasma by liquid chromatography tandem mass spectrometry" (Denver et al., 2019). Data show alteration of the base catalyst in this method was required to allow detection of catechol estrogens to low levels. Data also highlight the challenges faced in chromatographic separation of isomers and isotopologues, which were partially overcome by employing an extended column length and reduced oven temperature. In addition, data analysis displayed significant matrix effects during quantitation in plasma, following solid-phase extraction, despite efficient recoveries.

Estrogens circulate at concentrations less than 20 pg/mL in men and postmenopausal women, presenting analytical challenges. Quantitation by immunoassay is unreliable at these low concentrations. Liquid chromatography tandem mass spectrometry (LC–MS/MS) offers greater specificity and sometimes greater sensitivity, but ionization of estrogens is inefficient. Introduction of charged moieties may enhance ionization, but many such derivatives of estrogens generate non-specific product ions originating from the “reagent” group. Therefore an approach generating derivatives with product ions specific to individual estrogens was sought. Estrogens were extracted from human plasma and serum using solid phase extraction and derivatized using 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS). Electrospray in positive mode with multiple reaction monitoring using a QTrap 5500 mass spectrometer was used to quantify “FMP” derivatives of estrogens, following LC separation. Transitions for the FMP derivatives of estrone (E1) and estradiol (E2) were compound specific (m/z 362→238 and m/z 364→128, respectively). The limits of detection and quantitation were 0.2 pg on-column and the method was linear from 1–400 pg/sample. Measures of intra- and inter-assay variability, precision and accuracy were acceptable (, Graphical abstract fx1, Highlights • Quantitative analysis of low amounts of estrone and estradiol in plasma and serum. • Quantitation across physiological range in men and pre- and post-menopausal women. • Methylpyridinium ether derivatives improve analytical specificity and sensitivity.

Increased risk and severity of idiopathic pulmonary arterial hypertension (iPAH) is associated with elevated estradiol in men and postmenopausal women. Pulmonary arteries synthesise estradiol via aromatase and metabolise it via CYP1B1 to mitogenic metabolites; SNPs in aromatase and CYP1B1 have been associated with PAH. This suggests that estradiol metabolism could be altered in iPAH. This proof-of-concept study profiles estradiol and several metabolites of estradiol simultaneously in serum from iPAH patients and controls. We show that the estradiol and metabolite profile is altered in iPAH and that 16-hydroxyestrone and 16-hydroxyestradiol accumulate in iPAH patients with 16-hydroxyestrone levels relating to disease severity.