A liquid chromatography-tandem mass spectrometry method for the analysis of dehydroepiandrosterone sulphate (DHEAs) in serum and plasma with comparison to an immunoassay method in a neonatal population.

The measurement of dehydroepiandrosterone-sulphate (DHEAs) is an important second-line test to aid in the diagnosis of premature adrenarche, peripubertal gynaecomastia in males and in identifying the source of elevated androgens in females. Historically, DHEAs has been measured by immunoassay platforms which are prone to poor sensitivity and more importantly poor specificity. The aim was to develop an LC-MSMS method for the measurement of DHEAs in human plasma and serum, develop an in-house paediatric (<6 year old) reference limit and compare the performance against the Abbott Alinity DHEAs immunoassay method. Following pre-treatment with an internal standard, samples were loaded onto EVOLUTE® EXPRESS ABN plate. Analytes were separated with reverse-phase chromatography using ACQUITY® UPLC® HSS T3 2.1 mm × 50 mm, 1.8 µm column. Mass spectrometry detection was performed using a Waters® Xevo TQ-XS in electrospray negative mode. For the paediatric reference range, samples were collected from an inpatient setting (age ≤ 6 years old) with no evidence of adrenal dysfunction or history of/current steroid use. The method comparison was performed using samples from this cohort aged between 0 and 52 weeks. The assay demonstrated linearity up to 15 µmol/L (r2 > 0.99) with a functional sensitivity of 0.1 µmol/L. Accuracy results revealed a mean bias of 0.7% (-14% to 15%) when compared against the NEQAS EQA LC-MSMS consensus mean (n = 48). The paediatric reference limit was calculated as ≤ 2.3 µmol/L (95% C.I. 1.4 to 3.8 µmol/L) for ≤ 6 year olds (n = 38). Comparison of neonatal (<52 weeks) DHEAs with the Abbott Alinity revealed that the immunoassay ran at a 166% positive bias (n = 24) which appeared to lessen with increasing age. Described is a robust LC-MSMS method for the measurement of plasma or serum DHEAs validated against internationally recognised protocols. Comparison of paediatric samples of <52 weeks against an immunoassay platform demonstrated that in the immediate new-born period results generated from the LC-MSMS method offer superior specificity than an immunoassay platform.

A rapid liquid chromatography-tandem mass spectrometry method for the analysis of urinary 5-hydroxyindoleacetic acid (5-HIAA) that incorporates a 13C-labelled internal standard.

BACKGROUND: Urinary 5-hydroxyindoleacetic acid (5-HIAA) is a first-line investigation for gastrointestinal neuroendocrine tumours that secrete serotonin. It also has clinical utility for monitoring disease progression and therapeutic response. AIM: To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for urinary 5-hydroxyindoleacetic acid that incorporates a supported liquid extraction and 13C-labelled internal standard. METHODS: Samples were diluted in ammonium acetate containing a 13C-labelled internal standard (5-hydroxyindole-3a,4,5,6,7,7a-13C6-3-acetic acid). Supported liquid extraction was performed followed by chromatographic separation using the 2.1 × 30 mm CORTECS® UPLC® T3 column. Mass spectrometry detection (Waters Xevo TQ-XS) was performed in electrospray positive mode using the transitions 192.3 > 146.4 m/z (quantifier) and 192.3 > 118.4 m/z (qualifier) for 5-hydroxyindoleacetic acid and 198.2 > 152.4 m/z for 13C-5-HIAA. RESULTS: A well-defined 5-hydroxyindoleacetic acid peak was observed at 0.8 min with a run time of 2.4 min. The assay was linear (r2 > 0.99) to 382 µmol/L, with a lower limit of quantification of 5.3 µmol/L (CV <15%). Analysis of 29 external quality assurance samples showed good agreement between our method and the UKNEQAS method mean (4.7% positive bias). The intra- and inter-assay precision was within acceptable limits, and the assay was stable up to 96 h postextraction with minimal carryover. CONCLUSION: We have developed a robust LC-MS/MS method with semi-automated extraction that offers an improved run time and performance over the existing, labour-intensive, HPLC method. The method was quick, precise, showed good agreement with UKNEQAS external quality assurance material and is in routine service for clinical samples.

Regulation of Corticosteroidogenic Genes by MicroRNAs.

The loss of normal regulation of corticosteroid secretion is important in the development of cardiovascular disease. We previously showed that microRNAs regulate the terminal stages of corticosteroid biosynthesis. Here, we assess microRNA regulation across the whole corticosteroid pathway. Knockdown of microRNA using Dicer1 siRNA in H295R adrenocortical cells increased levels of CYP11A1, CYP21A1, and CYP17A1 mRNA and the secretion of cortisol, corticosterone, 11-deoxycorticosterone, 18-hydroxycorticosterone, and aldosterone. Bioinformatic analysis of genes involved in corticosteroid biosynthesis or metabolism identified many putative microRNA-binding sites, and some were selected for further study. Manipulation of individual microRNA levels demonstrated a direct effect of miR-125a-5p and miR-125b-5p on CYP11B2 and of miR-320a-3p levels on CYP11A1 and CYP17A1 mRNA. Finally, comparison of microRNA expression profiles from human aldosterone-producing adenoma and normal adrenal tissue showed levels of various microRNAs, including miR-125a-5p to be significantly different. This study demonstrates that corticosteroidogenesis is regulated at multiple points by several microRNAs and that certain of these microRNAs are differentially expressed in tumorous adrenal tissue, which may contribute to dysregulation of corticosteroid secretion. These findings provide new insights into the regulation of corticosteroid production and have implications for understanding the pathology of disease states where abnormal hormone secretion is a feature.

PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling.

cAMP/PKA signalling is compartmentalised with tight spatial and temporal control of signal propagation underpinning specificity of response. The cAMP-degrading enzymes, phosphodiesterases (PDEs), localise to specific subcellular domains within which they control local cAMP levels and are key regulators of signal compartmentalisation. Several components of the cAMP/PKA cascade are located to different mitochondrial sub-compartments, suggesting the presence of multiple cAMP/PKA signalling domains within the organelle. The function and regulation of these domains remain largely unknown. Here, we describe a novel cAMP/PKA signalling domain localised at mitochondrial membranes and regulated by PDE2A2. Using pharmacological and genetic approaches combined with real-time FRET imaging and high resolution microscopy, we demonstrate that in rat cardiac myocytes and other cell types mitochondrial PDE2A2 regulates local cAMP levels and PKA-dependent phosphorylation of Drp1. We further demonstrate that inhibition of PDE2A, by enhancing the hormone-dependent cAMP response locally, affects mitochondria dynamics and protects from apoptotic cell death.

Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2.

RATIONALE: Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodeling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A signaling seems to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signaling microdomains. OBJECTIVE: How individual cAMP microdomains affect cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. METHODS AND RESULTS: Using pharmacological and genetic manipulation of PDE activity, we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy, whereas increasing cAMP levels via PDE2 inhibition is antihypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of protein kinase A isoforms, we demonstrate that the antihypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a protein kinase A type II subset, leading to phosphorylation of the nuclear factor of activated T cells. CONCLUSIONS: Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo, and its inhibition may have therapeutic applications.