Data for analysis of catechol estrogen metabolites in human plasma by liquid chromatography tandem mass spectrometry

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

ABCC1 modulates negative feedback control of the hypothalamic-pituitary-adrenal axis in vivo in humans

BACKGROUND: Cortisol and corticosterone both circulate in human plasma and, due to differing export by ATP-binding cassette (ABC) transporters, may exert differential cellular effects. ABCB1 (expressed in brain) exports cortisol not corticosterone while ABCC1 (expressed in adipose and skeletal muscle) exports corticosterone not cortisol. We hypothesised that ABCC1 inhibition increases corticosteroid receptor occupancy by corticosterone but not cortisol in humans. METHODS: A randomised double-blind crossover study was conducted in 14 healthy men comparing placebo and ABCC1 inhibitor probenecid. Blood sampling, including from veins draining adipose and muscle, was undertaken before and after administration of mineralocorticoid receptor antagonist potassium canrenoate and glucocorticoid receptor antagonist mifepristone (RU486). RESULTS: During placebo, systemic plasma cortisol and corticosterone concentrations increased promptly after canrenoate. Cortisol uptake was detected from adipose but not muscle following canrenoate + RU486. Probenecid significantly increased systemic cortisol concentrations, and tended to increase corticosterone and ACTH concentrations, after combined receptor antagonism but had no effects on net glucocorticoid balance in either adipose or muscle. Using quantitative PCR in brain bank tissue, ABCC1 expression was 5-fold higher in human pituitary than hypothalamus and hippocampus. ABCB1 was more highly expressed in hypothalamus compared to pituitary. CONCLUSIONS: Although displacement of corticosterone and/or cortisol from receptors in adipose and skeletal muscle could not be measured with sufficient precision to detect effects of probenecid, ABCC1 inhibition induced a greater incremental activation of the hypothalamic-pituitary-adrenal axis after combined receptor blockade, consistent with ABCC1 exporting corticosterone from the pituitary and adding to the evidence that ABC transporters modulate tissue glucocorticoid sensitivity

Aromatase Inhibition Reduces Insulin Sensitivity in Healthy Men

CONTEXT: Deficiency of aromatase, the enzyme that catalyzes the conversion of androgens to estrogens, is associated with insulin resistance in humans and mice. OBJECTIVE: We hypothesized that pharmacological aromatase inhibition results in peripheral insulin resistance in humans. DESIGN: This was a double-blind, randomized, controlled, crossover study. SETTING: The study was conducted at a clinical research facility. PARTICIPANTS: Seventeen healthy male volunteers (18–50 y) participated in the study. INTERVENTION: The intervention included oral anastrozole (1 mg daily) and placebo, each for 6 weeks with a 2-week washout period. MAIN OUTCOME MEASURE: Glucose disposal and rates of lipolysis were measured during a stepwise hyperinsulinemic euglycemic clamp. Data are mean (SEM). RESULTS: Anastrozole therapy resulted in significant estradiol suppression (59.9 ± 3.6 vs 102.0 ± 5.7 pmol/L, P = < .001) and a more modest elevation of total T (25.8 ± 1.2 vs 21.4 ± 0.7 nmol/L, P = .003). Glucose infusion rate, during the low-dose insulin infusion, was lower after anastrozole administration (12.16 ± 1.33 vs 14.15 ± 1.55 μmol/kg·min, P = .024). No differences in hepatic glucose production or rate of lipolysis were observed. CONCLUSION: Aromatase inhibition reduces insulin sensitivity, with respect to peripheral glucose disposal, in healthy men. Local generation and action of estradiol, at the level of skeletal muscle, is likely to be an important determinant of insulin sensitivity

De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone-metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1 in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lung metastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts in vitro but does not alter osteoclast-specific gene expression. Instead, tumor-derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB

Simultaneous quantification of estrogens and glucocorticoids in human adipose tissue by liquid-chromatography-tandem mass spectrometry

The presence of estrogens, androgens and glucocorticoids as well as their receptors and steroid converting enzymes in adipose tissue has been established. Their contribution to diseases such as obesity, diabetes and hormone-dependent cancers is an active area of research. Our objective was to develop a LC–MS/MS method to quantify bioactive estrogens and glucocorticoids simultaneously in human adipose tissue. Estrogens and glucocorticoids were extracted from adipose tissue samples using solid-phase extraction. Estrogens were derivatized using 1-(2,4-dinitro-5-fluorophenyl)-4-methylpiperazine (PPZ) and methyl iodide to generate a permanently charged molecule (MPPZ). Steroids were separated and quantified by LC–MS/MS. The limit of quantitation for the steroids was between 15 and 100 pg per sample. Accuracy and precision were acceptable (<20%). Using this method, estradiol, estrone, cortisone and cortisol were quantified in adipose tissue from women with and without breast cancer. This novel assay of estrogens and glucocorticoids by LC–MS/MS coupled with derivatization allowed simultaneous quantification of a panel of steroids in human adipose tissue across the endogenous range of concentrations encountered in health and disease

Measurement of tamsulosin in human serum by liquid chromatography-tandem mass spectrometry

AbstractA simple, sensitive and robust method to extract tamsulosin from human serum, and quantify by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated and is applicable as a measure of compliance in clinical research. Tamsulosin was extracted from human serum (100μL) via liquid–liquid extraction with methyl tert-butyl ether (2mL) following dilution with 0.1M ammonium hydroxide (100μL), achieving 99.9% analyte recovery. Internal standard, d9-finasteride, was synthesised in-house. Analyte and internal standard were separated on an Ascentis® Express C18 (100mm×3mm, 2.7μm) column using a gradient elution with mobile phases methanol and 2mM aqueous ammonium acetate (5:95, v/v). Total run-time was 6min. Tamsulosin was quantified using a triple quadrupole mass spectrometer operated in multi-reaction-monitoring (MRM) mode using positive electrospray ionisation. Mass transitions monitored for quantitation were: tamsulosin m/z 409→228 and d9-finasteride m/z 382→318, with the structural formulae of ions confirmed by Fourier transform ion cyclotron resonance mass spectrometry (within 10ppm). The limit of quantitation was 0.2ng/mL, and the method was validated in the linear range 0.2–50ng/mL with acceptable inter- and intra-assay precision and accuracy and stability suitable for routine laboratory practice. The method was successfully applied to samples taken from research volunteers in a clinical study of benign prostatic hyperplasia

ATP-binding cassette family C member 1 constrains metabolic responses to high-fat diet in male mice

Glucocorticoids modulate glucose homeostasis, acting on metabolically active tissues such as liver, skeletal muscle, and adipose tissue. Intracellular regulation of glucocorticoid action in adipose tissue impacts metabolic responses to obesity. ATP-binding cassette family C member 1 (ABCC1) is a transmembrane glucocorticoid transporter known to limit the accumulation of exogenously administered corticosterone in adipose tissue. However, the role of ABCC1 in the regulation of endogenous glucocorticoid action and its impact on fuel metabolism has not been studied. Here, we investigate the impact of Abcc1 deficiency on glucocorticoid action and high-fat-diet (HFD)-induced obesity. In lean male mice, deficiency of Abcc1 increased endogenous corticosterone levels in skeletal muscle and adipose tissue but did not impact insulin sensitivity. In contrast, Abcc1-deficient male mice on HFD displayed impaired glucose and insulin tolerance, and fasting hyperinsulinaemia, without alterations in tissue corticosterone levels. Proteomics and bulk RNA sequencing revealed that Abcc1 deficiency amplified the transcriptional response to an obesogenic diet in adipose tissue but not in skeletal muscle. Moreover, Abcc1 deficiency impairs key signalling pathways related to glucose metabolism in both skeletal muscle and adipose tissue, in particular those related to OXPHOS machinery and Glut4. Together, our results highlight a role for ABCC1 in regulating glucose homeostasis, demonstrating diet-dependent effects that are not associated with altered tissue glucocorticoid concentrations.</p

ATP-binding cassette family C member 1 constrains metabolic responses to high-fat diet in male mice

Glucocorticoids modulate glucose homeostasis, acting on metabolically active tissues such as liver, skeletal muscle, and adipose tissue. Intracellular regulation of glucocorticoid action in adipose tissue impacts metabolic responses to obesity. ATP-binding cassette family C member 1 (ABCC1) is a transmembrane glucocorticoid transporter known to limit the accumulation of exogenously administered corticosterone in adipose tissue. However, the role of ABCC1 in the regulation of endogenous glucocorticoid action and its impact on fuel metabolism has not been studied. Here, we investigate the impact of Abcc1 deficiency on glucocorticoid action and high-fat-diet (HFD)-induced obesity. In lean male mice, deficiency of Abcc1 increased endogenous corticosterone levels in skeletal muscle and adipose tissue but did not impact insulin sensitivity. In contrast, Abcc1-deficient male mice on HFD displayed impaired glucose and insulin tolerance, and fasting hyperinsulinaemia, without alterations in tissue corticosterone levels. Proteomics and bulk RNA sequencing revealed that Abcc1 deficiency amplified the transcriptional response to an obesogenic diet in adipose tissue but not in skeletal muscle. Moreover, Abcc1 deficiency impairs key signalling pathways related to glucose metabolism in both skeletal muscle and adipose tissue, in particular those related to OXPHOS machinery and Glut4. Together, our results highlight a role for ABCC1 in regulating glucose homeostasis, demonstrating diet-dependent effects that are not associated with altered tissue glucocorticoid concentrations.</p

Kynurenine monooxygenase blockade reduces endometriosis-like lesions, improves visceral hyperalgesia, and rescues mice from a negative behavioural phenotype in experimental endometriosis.

Endometriosis is a common and debilitating neuro-inflammatory disorder that is associated with chronic pain. Definitive diagnosis is based on the presence of endometrial-like tissue (lesions) in sites outside the uterus. Kynurenine monooxygenase (KMO) is a mitochondrial enzyme of tryptophan metabolism that regulates inflammation and immunity. Here, we show that KMO is expressed in epithelial cells in human endometriosis tissue lesions and in corresponding lesions in a mouse model of endometriosis. In mice, oral treatment with the potent KMO inhibitor KNS898 induced a biochemical state of KMO blockade with accumulation of kynurenine, diversion to kynurenic acid and ablation of 3-hydroxykynurenine production. In the mouse model of endometriosis, KMO inhibition improved histological outcomes and endometriosis pain-like behaviours, even when KNS898 treatment commenced one week after initiation of lesions. Taken together, these results suggest that KMO blockade is a promising new non-hormonal therapeutic modality for endometriosis