A practical approach to supported liquid extraction and measurement of 18 steroids in plasma and serum by targeted liquid chromatography tandem mass spectrometry.

Chromatography combined with mass spectrometry is a gold standard technique for steroid measurement, however the type of sample preparation, the dynamic range and reliability of the calibration curve, the chromatographic separation and mass spectrometry settings ultimately determine the success of the method. The steroid biosynthetic pathway is conserved in higher mammals and literature demonstrates that the concentration ranges of different steroid groups are relatively comparable across species. We sought to develop a robust and reliable multi steroid targeted analysis method for blood that would have wide application across higher mammals. The method was developed following bioanalytical method validation guidelines to standards typically applied to human clinical studies, including isotopically labelled internal standards where at all possible. Here we describe the practical approach to a 96-well supported liquid extraction (SLE) method of extraction from plasma (200 µL) using an Extrahera liquid handling robot (Biotage, Sweden), including quality control samples, followed by a comprehensive separation and targeted LC-MS/MS analysis of 18 steroids in plasma (pregnenolone, progesterone, 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, aldosterone, 11-deoxycortisol, 21-deoxycortisol, cortisol, cortisone, androstenedione, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, estrone, 17ß-estradiol and estriol). •SLE in a 96-well format of up to 74 biological plasma samples, enriched with multiple isotopically labelled internal standards, a 12-point aqueous calibration curve, and 6 serum quality controls, designed to monitor long-term performance of the method•Chromatographic separation of multiple steroids along the gradient, with ammonium fluoride mobile phase additive to improve sensitivity, followed by electrospray ionisation and constant polarity switching•Aqueous calibration standards that cover physiologically relevant ranges - high nanomolar glucocorticoids, low nanomolar androgens and picomolar ranges for estrogens and steroid intermediates.

The ATP-binding cassette proteins ABCB1 and ABCC1 as modulators of glucocorticoid action.

Responses to hormones that act through nuclear receptors are controlled by modulating hormone concentrations not only in the circulation but also within target tissues. The role of enzymes that amplify or reduce local hormone concentrations is well established for glucocorticoid and other lipophilic hormones; moreover, transmembrane transporters have proven critical in determining tissue responses to thyroid hormones. However, there has been less consideration of the role of transmembrane transport for steroid hormones. ATP-binding cassette (ABC) proteins were first shown to influence the accumulation of glucocorticoids in cells almost three decades ago, but observations over the past 10 years suggest that differential transport propensities of both exogenous and endogenous glucocorticoids by ABCB1 and ABCC1 transporters provide a mechanism whereby different tissues are preferentially sensitive to different steroids. This Review summarizes this evidence and the new insights provided for the physiology and pharmacology of glucocorticoid action, including new approaches to glucocorticoid replacement.

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.

GDF15 Is Elevated in Conditions of Glucocorticoid Deficiency and Is Modulated by Glucocorticoid Replacement.

CONTEXT: GDF15 is a stress-induced hormone acting in the hindbrain that activates neural circuitry involved in establishing aversive responses and reducing food intake and body weight in animal models. Anorexia, weight loss, nausea and vomiting are common manifestations of glucocorticoid deficiency, and we hypothesized that glucocorticoid deficiency may be associated with elevated levels of GDF15. OBJECTIVE: To determine the impact of primary adrenal insufficiency (PAI) and glucocorticoid replacement on circulating GDF15 levels. METHODS AND RESULTS: We measured circulating concentrations of GDF15 in a cohort of healthy volunteers and Addison's disease patients following steroid withdrawal. Significantly higher GDF15 (mean ± standard deviation [SD]) was observed in the Addison's cohort, 739.1 ± 225.8 pg/mL compared to healthy controls, 497.9 ± 167.7 pg/mL (P = 0.01). The effect of hydrocortisone replacement on GDF15 was assessed in 3 independent PAI cohorts with classical congenital adrenal hyperplasia or Addison's disease; intravenous hydrocortisone replacement reduced GDF15 in all groups. We examined the response of GDF15 to increasing doses of glucocorticoid replacement in healthy volunteers with pharmacologically mediated cortisol deficiency. A dose-dependent difference in GDF15 (mean ± SD) was observed between the groups with values of 491.0 ± 157.7 pg/mL, 427.0 ± 152.1 pg/mL and 360 ± 143.1 pg/mL, in the low, medium and high glucocorticoid replacement groups, respectively, P < .0001. CONCLUSIONS: GDF15 is increased in states of glucocorticoid deficiency and restored by glucocorticoid replacement. Given the site of action of GDF15 in the hindbrain and its effects on appetite, further study is required to determine the effect of GDF15 in mediating the anorexia and nausea that is a common feature of glucocorticoid deficiency.

Substantial Metabolic Activity of Human Brown Adipose Tissue during Warm Conditions and Cold-Induced Lipolysis of Local Triglycerides.

Current understanding of in vivo human brown adipose tissue (BAT) physiology is limited by a reliance on positron emission tomography (PET)/computed tomography (CT) scanning, which has measured exogenous glucose and fatty acid uptake but not quantified endogenous substrate utilization by BAT. Six lean, healthy men underwent 18fluorodeoxyglucose-PET/CT scanning to localize BAT so microdialysis catheters could be inserted in supraclavicular BAT under CT guidance and in abdominal subcutaneous white adipose tissue (WAT). Arterial and dialysate samples were collected during warm (∼25°C) and cold exposure (∼17°C), and blood flow was measured by 133xenon washout. During warm conditions, there was increased glucose uptake and lactate release and decreased glycerol release by BAT compared with WAT. Cold exposure increased blood flow, glycerol release, and glucose and glutamate uptake only by BAT. This novel use of microdialysis reveals that human BAT is metabolically active during warm conditions. BAT activation substantially increases local lipolysis but also utilization of other substrates such as glutamate.