Ocular steroidome in human eyes and in eyes with complex central serous chorioretinopathy (CSCR).

The exact link between systemic and ocular endogenous corticoids (steroidome) is unclear and whether the ocular steroidome is altered in CSCR eyes is unknown. The aims of this study were to analyze the human steroidome in the aqueous humor as a function of age, sex and time of the day, to correlate systemic and ocular steroidome and to analyze the ocular steroidome in long lasting complex inactive CSCR. Based on our results, we present two CSCR cases treated by the combination of oral mineralocorticoid antagonist and glucocorticoids drops. In a cross-sectional study, aqueous humor (AH) was collected between 8am and 6 pm from 50 unaffected individuals (25 men and 25 women) and from 14 patients with chronic CSCR, during cataract surgery. In addition, simultaneous serum and AH were collected from 27 individuals undergoing cataract surgery and, simultaneous AH and vitreous were collected from 9 patients undergoing cataract and vitrectomy to estimate corticoids levels in the different compartments. The steroidome was determined using a LC-MS/MS method that quantifies 13 endogenous corticoids from the gluco, mineralocorticoid and androgen pathways. In AH and vitreous, the highest corticoid level is reached by cortisol (F), that represents less than 10% of F serum level. The cortisol levels in the serum did not correlate with ocular cortisol levels. Serum and ocular cortisone (E) levels correlate, although less than 5% of circulating E reaches the eye. The only mineralocorticoids measured in the AH were corticosterone (B) and its inactive form, the 11-desoxycorticosterone (A). There was no influence of circadian rhythm on cortisol ocular levels and there was no correlation between the age or the sex and the level of F, E, A, and B. In eyes with chronic inactive CSCR, the levels of the active glucocorticoid form F was lower than in control eyes and the F/E ratio was reduced by 50% but the B/A ratio was higher indicating imbalance towards active mineralocorticoids. Base on this observation, we propose to combine an antagonist of the mineralocorticoid receptor together with topical glucocorticoids in two CSCR patients, resistant to all other treatments, with favorable outcome. Our results indicate that the ocular psteroidome is highly regulated suggesting a local metabolism of ocular corticoids. In eyes with long-lasting complex inactive CSCR, the steroidome analysis shows lower active glucocorticoids and higher active mineralocorticoids.

Loss of SUMO-specific protease 2 causes isolated glucocorticoid deficiency by blocking adrenal cortex zonal transdifferentiation in mice.

SUMOylation is a dynamic posttranslational modification, that provides fine-tuning of protein function involved in the cellular response to stress, differentiation, and tissue development. In the adrenal cortex, an emblematic endocrine organ that mediates adaptation to physiological demands, the SUMOylation gradient is inversely correlated with the gradient of cellular differentiation raising important questions about its role in functional zonation and the response to stress. Considering that SUMO-specific protease 2 (SENP2), a deSUMOylating enzyme, is upregulated by Adrenocorticotropic Hormone (ACTH)/cAMP-dependent Protein Kinase (PKA) signalling within the zona fasciculata, we generated mice with adrenal-specific Senp2 loss to address these questions. Disruption of SENP2 activity in steroidogenic cells leads to specific hypoplasia of the zona fasciculata, a blunted reponse to ACTH and isolated glucocorticoid deficiency. Mechanistically, overSUMOylation resulting from SENP2 loss shifts the balance between ACTH/PKA and WNT/ß-catenin signalling leading to repression of PKA activity and ectopic activation of ß-catenin. At the cellular level, this blocks transdifferentiation of ß-catenin-positive zona glomerulosa cells into fasciculata cells and sensitises them to premature apoptosis. Our findings indicate that the SUMO pathway is critical for adrenal homeostasis and stress responsiveness.

Pathogenic Effects of Mineralocorticoid Pathway Activation in Retinal Pigment Epithelium.

Glucocorticoids are amongst the most used drugs to treat retinal diseases of various origins. Yet, the transcriptional regulations induced by glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation in retinal pigment epithelium cells (RPE) that form the outer blood-retina barrier are unknown. Levels of endogenous corticoids, ligands for MR and GR, were measured in human ocular media. Human RPE cells derived from induced pluripotent stem cells (iRPE) were used to analyze the pan-transcriptional regulations induced by aldosterone-an MR-specific agonist, or cortisol or cortisol + RU486-a GR antagonist. The retinal phenotype of transgenic mice that overexpress the human MR (P1.hMR) was analyzed. In the human eye, the main ligand for GR and MR is cortisol. The iRPE cells express functional GR and MR. The subset of genes regulated by aldosterone and by cortisol + RU-486, and not by cortisol alone, mimics an imbalance toward MR activation. They are involved in extracellular matrix remodeling (CNN1, MGP, AMTN), epithelial-mesenchymal transition, RPE cell proliferation and migration (ITGB3, PLAUR and FOSL1) and immune balance (TNFSF18 and PTX3). The P1.hMR mice showed choroidal vasodilation, focal alteration of the RPE/choroid interface and migration of RPE cells together with RPE barrier function alteration, similar to human retinal diseases within the pachychoroid spectrum. RPE is a corticosteroid-sensitive epithelium. MR pathway activation in the RPE regulates genes involved in barrier function, extracellular matrix, neural regulation and epithelial differentiation, which could contribute to retinal pathology.

Adrenomedullin: new inhibitory regulator for cortisol synthesis and secretion.

Preterm birth is associated with immaturity of several crucial physiological functions notably those prevailing in the lung and kidney. Recently, a steroid secretion deficiency was identified in very preterm neonates, associated with a partial yet transient deficiency in 11ß-hydroxylase activity, sustaining cortisol synthesis. However, the P450c11ß enzyme is expressed in preterm adrenal glands, we hypothesized an inhibition of cortisol production by adrenomedullin (ADM), a peptide highly produced in neonates and whose effect on steroidogenesis remains poorly known. We studied the effects of ADM on three models: 104 cord-blood samples of the PREMALDO neonate cohort, genetically targeted mice overexpressing ADM, and two human adrenocortical cell lines (H295R and HAC15 cells). Mid-regional-proADM (MR-proADM) quantification in cord-blood samples showed strong negative correlation with gestational age (P = 0.0004), cortisol production (P < 0.0001), and 11ß-hydroxylase activity index (P < 0.0001). Mean MR-proADM was higher in very preterm than in term neonates (1.12 vs 0.60 nmol/L, P < 0.0001). ADM-overexpression mice revealed a lower 11ß-hydroxylase activity index (P < 0.05). Otherwise, aldosterone levels measured by LC-MS/MS were higher in ADM-overexpression mice (0.83 vs 0.46 ng/mL, P < 0.05). More importantly, the negative relationship between adrenal ADM expression and aldosterone production found in control was lacking in the ADM-overexpression mice. Finally, LC-MS/MS and gene expression studies on H295R and HAC15 cells revealed an ADM-induced inhibition of both cortisol secretion in cell supernatants and CYP11B1 expression. Collectively, our results converge toward an inhibitory effect of ADM on glucocorticoid synthesis in humans and should be considered to explain the steroid secretion deficiency observed at birth in premature newborns.

Mineralocorticoid Receptor Pathway and Its Antagonism in a Model of Diabetic Retinopathy.

Diabetic retinopathy remains a major cause of vision loss worldwide. Mineralocorticoid receptor (MR) pathway activation contributes to diabetic nephropathy, but its role in retinopathy is unknown. In this study, we show that MR is overexpressed in the retina of type 2 diabetic Goto-Kakizaki (GK) rats and humans and that cortisol is the MR ligand in human eyes. Lipocalin 2 and galectin 3, two biomarkers of diabetes complications regulated by MR, are increased in GK and human retina. The sustained intraocular delivery of spironolactone, a steroidal mineralocorticoid antagonist, decreased the early and late pathogenic features of retinopathy in GK rats, such as retinal inflammation, vascular leakage, and retinal edema, through the upregulation of genes encoding proteins known to intervene in vascular permeability such as Hey1, Vldlr, Pten, Slc7a1, Tjp1, Dlg1, and Sesn2 but did not decrease VEGF. Spironolactone also normalized the distribution of ion and water channels in macroglial cells. These results indicate that MR is activated in GK and human diabetic retina and that local MR antagonism could be a novel therapeutic option for diabetic retinopathy.

The invention of aldosterone, how the past resurfaces in pediatric endocrinology.

Sodium and water homeostasis are drastically modified at birth, in mammals, by the transition from aquatic life to terrestrial life. Accumulating evidence during the past ten years underscores the central role for the mineralocorticoid signaling pathway, in the fine regulation of this equilibrium, at this critical period of development. Interestingly, regarding evolution, while the mineralocorticoid receptor is expressed in fish, the appearance of its related ligand, aldosterone, coincides with terrestrial life, as it is first detected in lungfish and amphibian. Thus, aldosterone is likely one of the main hormones regulating the transition from an aquatic environment to an air environment. This review will focus on the different actors of the mineralocorticoid signaling pathway from aldosterone secretion in the adrenal gland, to mineralocorticoid receptor expression in the kidney, summarizing their regulation and roles throughout fetal and neonatal development, in the light of evolution.

Sexual Dimorphism of Corticosteroid Signaling during Kidney Development.

Sexual dimorphism involves differences between biological sexes that go beyond sexual characteristics. In mammals, differences between sexes have been demonstrated regarding various biological processes, including blood pressure and predisposition to develop hypertension early in adulthood, which may rely on early events during development and in the neonatal period. Recent studies suggest that corticosteroid signaling pathways (comprising glucocorticoid and mineralocorticoid signaling pathways) have distinct tissue-specific expression and regulation during this specific temporal window in a sex-dependent manner, most notably in the kidney. This review outlines the evidence for a gender differential expression and activation of renal corticosteroid signaling pathways in the mammalian fetus and neonate, from mouse to human, that may favor mineralocorticoid signaling in females and glucocorticoid signaling in males. Determining the effects of such differences may shed light on short term and long term pathophysiological consequences, markedly for males.

Preterm birth is associated with epigenetic programming of transgenerational hypertension in mice.

Renal and cardiovascular complications of prematurity are well established, notably the development of hypertension in adulthood. However, the underlying molecular mechanisms remain poorly understood. Our objective was to investigate the impact of prematurity on the ontogenesis of renal corticosteroid pathways, to evaluate its implication in perinatal renal complications and in the emergence of hypertension in adulthood. Swiss CD1 pregnant mice were injected with lipopolysaccharides at 18 days of gestation (E18) to induce prematurity at E18.5. Pups were sacrificed at birth, 7 days and 6 months of life. Second (F2) and third (F3) generations, established by mating prematurely born adult females with wild-type males, were also analyzed. Former preterm males developed hypertension at M6 (P < 0.0001). We found robust activation of renal corticosteroid target gene transcription at birth in preterm mice (αENaC (+45%), Gilz (+85%)), independent of any change in mineralocorticoid or glucocorticoid receptor expression. The offspring of the preterm group displayed increased blood pressure in F2 and F3, associated with increased renal Gilz mRNA expression, despite similar MR or GR expression and plasma corticosteroid levels measured by LC-MS/MS. Gilz promoter methylation measured by methylated DNA immunoprecipitation-qPCR was reduced with a negative correlation between methylation and expression (P = 0.0106). Our study demonstrates prematurity-related alterations in renal corticosteroid signaling pathways, with transgenerational inheritance of blood pressure dysregulation and epigenetic Gilz regulation up to the third generation. This study provides a better understanding of the molecular mechanisms involved in essential hypertension, which could partly be due to perinatal epigenetic programming from previous generations.

Urinary steroidomic profiles by LC-MS/MS to monitor classic 21-Hydroxylase deficiency.

21-hydroxylase deficiency, the most common enzyme defect associated with congenital adrenal hyperplasia (CAH) is characterized by an impairment of both aldosterone and cortisol biosynthesis. Close clinical and biological monitoring of Hydrocortisone (HC) and 9α-Fludrocortisone (FDR) replacement therapies is required to achieve an optimal treatment. As frequent and repeated reassessments of plasma steroids, 17-hydroxyprogesterone (17-OHP), androstenedione (Δ4-A) and testosterone (TESTO) is needed in childhood, urine steroid profiling could represent an interesting non-invasive alternative. We developed and validated a LC-MS/MS method for the measurement of 23-urinary mineralocorticoids, glucocorticoids and adrenal androgens. The usefulness of steroid profiling was investigated on single 08h00 am-collected spot urine for discriminating between 61 CAH patients and their age- and sex-matched controls. CAH patients were split into two groups according to their 08h00 am-plasma concentrations of 17-OHP: below (controlled patients, n = 26) and above 20 ng/mL (uncontrolled patients, n = 35). The lower limit of quantification and the wide analytical range allows to assay both free and total concentrations of the main urinary adreno-corticoids and their tetra-hydrometabolites. Extraction recoveries higher than 75% and intra-assay precision below 20% were found for most steroids. Urinary steroids upstream of the 21-hydroxylase defect were higher in uncontrolled CAH patients. Among CAH patients, plasma and urinary 17-OHP were closely correlated. As compared to controls, steroids downstream of the enzyme defect collapsed in CAH patients. This fall was more pronounced in controlled than in uncontrolled patients. Androgens (Δ4-A, TESTO and the sum etiocholanolone + androsterone) accumulated in uncontrolled CAH patients. A strong relationship was observed between plasma and urinary levels of androstenedione. Daily doses and urinary excretion of both FDR and HC were similar in both CAH groups. Urinary FDR was inversely related to the sodium-to-potassium ratio in urine. A partial least squares discriminant analysis model allowed to classify the patient's classes unaffected, controlled and un-controlled CAH patients based on urinary steroidomic profiles. Our LC-MS/MS method successfully established steroid profiling in urine and represents a useful and non-invasive tool for discriminating CAH patients according to treatment efficiency.

Interaction between accumulated 21-deoxysteroids and mineralocorticoid signaling in 21-hydroxylase deficiency.

21-Hydroxylase deficiency (21OHD) is a rare genetic disorder in which salt-wasting syndrome occurs in 75% of cases, due to inability to synthesize cortisol and aldosterone. Recent mass spectrometry progress allowed identification of 21-deoxysteroids, i.e., 17-hydroxyprogesterone (17OHP), 21-deoxycortisol (21DF), and 21-deoxycorticosterone (21DB). We hypothesized that they may interfere with mineralocorticoid signaling and fludrocortisone therapy in patients with congenital adrenal hyperplasia (CAH) without effective glucocorticoid replacement and ACTH suppression. Our goal was to quantify circulating 21-deoxysteroids in a pediatric cohort with CAH related to 21OHD and to examine their impact on mineralocorticoid receptor (MR) activation. Twenty-nine patients with salt-wasting phenotype were classified in two groups according to their therapeutic control. During routine follow-up, 17OHP, 21DF, 21DB, and cortisol levels were quantified by liquid chromatography with tandem mass spectrometry before hydrocortisone intake and 1 and 2.5 h following treatment administration. Luciferase reporter gene assays were performed on transfected HEK293T cells while in silico modeling examined structural interactions between these steroids within ligand-binding domain of MR. Plasma 17OHP, 21DF, and 21DB accumulate in uncontrolled patients reaching micromolar concentrations even after hydrocortisone intake. 21DF and 21DB act as partial MR agonists with antagonist features similar to 17OHP, consistent with altered anchoring to Asn770 and unfavorable contact with Ala773 in ligand-binding pocket of MR. Our results demonstrate a complex interaction between all accumulating 21-deoxysteroids in uncontrolled 21OHD patients and mineralocorticoid signaling and suggest that appropriate steroid profiling should optimize management and follow-up of such patients, as keeping those steroids to low plasma levels should attest therapeutic efficacy and prevent interference with MR signaling.

Impaired 11ß-Hydroxysteroid Dehydrogenase Type 2 in Glucocorticoid-Resistant Patients.

CONTEXT: Six patients carrying heterozygous loss-of-function mutations of glucocorticoid (GC) receptor (GR) presented with hypercortisolism, associated with low kalemia, low plasma renin, and aldosterone levels, with or without hypertension, suggesting a pseudohypermineralocorticism whose mechanisms remain unclear. We hypothesize that an impaired activity of the 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2; encoded by the HSD11B2 gene), catalyzing cortisol (F) inactivation, may account for an inappropriate activation of a renal mineralocorticoid signaling pathway in these GC-resistant patients. OBJECTIVE: We aim at studying the GR-mediated regulation of HSD11B2. DESIGN: The HSD11B2 promoter was subcloned and luciferase reporter assays evaluated GR-dependent HSD11B2 regulation, and 11ß-HSD2 expression/activity was studied in human breast cancer MCF7 cells, endogenously expressing this enzyme. RESULTS: Transfection assays revealed that GR transactivated the long (2.1-kbp) HSD11B2 promoter construct, whereas a defective 501H GR mutant was unable to stimulate luciferase activity. GR-mediated transactivation of the HSD11B2 gene was inhibited by the GR antagonist RU486. A threefold increase in HSD11B2 mRNA levels was observed after dexamethasone (DXM) treatment of MCF7 cells, inhibited by RU486 or by actinomycin, supporting a GR-dependent transcription. Chromatin immunoprecipitation further demonstrated a DXM-dependent GR recruitment onto the HSD11B2 promoter. 11ß-HSD2 activity, evaluated by the cortisone/F ratio, quantified by liquid chromatography/tandem mass spectrometry, was 10-fold higher in the supernatant of DXM-treated cells than controls, consistent with a GR-dependent stimulation of 11ß-HSD2 catalytic activity. CONCLUSION: Collectively, we demonstrate that 11ß-HSD2 expression and activity are transcriptionally regulated by GR. In the context of GR haploinsufficiency, these findings provide evidence that defective GR signaling may account for apparent mineralocorticoid excess in GC-resistant patients.

Potentiation of mitotane action by rosuvastatin: New insights for adrenocortical carcinoma management.

Mitotane (also termed o,p'­DDD) is the most effective therapy for advanced adrenocortical carcinoma (ACC). Mitotane­induced dyslipidemia is treated with statins. Mitotane and statins are known to exert anti­proliferative effects in vitro; however, the effects of statins have never been directly evaluated in patients with ACC and ACC cells, at least to the best of our knowledge. Thus, in this study, we aimed to examine the effects of the rosuvastatin on ACC cells. It has been shown that the combined use of mitotane and statins significantly increases the tumor control rate in patients with ACC; however, it would be of interest to elucidate the molecular mechanisms involved in this potentiation. In this study, we examined the effects of mitotane, rosuvastatin and their combination in NCI­H295R human ACC cells using proliferation assays, gene expression analyses and free intracellular cholesterol measurements. The results revealed that mitotane dose­dependently reduced cell viability, induced apoptosis and increased intracellular free cholesterol levels, considered as one of the key features of mitotane action, while rosuvastatin alone reduced cell viability and increased apoptosis at high concentrations. We also demonstrated that rosuvastatin potentiated the effects of mitotane by reducing cell viability, inducing apoptosis, increasing intracellular free cholesterol levels, and by decreasing the expression of 3­hydroxy­3­methylglutaryl­CoA reductase (HMGCR) and ATP binding cassette subfamily a member 1 (ABCA1), genes involved in cholesterol metabolism, and inhibiting steroidogenesis. Collectively, potentiating the effects of mitotane with the use of rosuvastatin may provide novel therapeutic strategies for ACC, given that the combination of these drugs, pending clinical validation, may lead to the better management of ACC.

Corticosteroid receptors adopt distinct cyclical transcriptional signatures.

Mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) are two closely related hormone-activated transcription factors that regulate major pathophysiologic functions. High homology between these receptors accounts for the crossbinding of their corresponding ligands, MR being activated by both aldosterone and cortisol and GR essentially activated by cortisol. Their coexpression and ability to bind similar DNA motifs highlight the need to investigate their respective contributions to overall corticosteroid signaling. Here, we decipher the transcriptional regulatory mechanisms that underlie selective effects of MRs and GRs on shared genomic targets in a human renal cellular model. Kinetic, serial, and sequential chromatin immunoprecipitation approaches were performed on the period circadian protein 1 ( PER1) target gene, providing evidence that both receptors dynamically and cyclically interact at the same target promoter in a specific and distinct transcriptional signature. During this process, both receptors regulate PER1 gene by binding as homo- or heterodimers to the same promoter region. Our results suggest a novel level of MR-GR target gene regulation, which should be considered for a better and integrated understanding of corticosteroid-related pathophysiology.-Le Billan, F., Amazit, L., Bleakley, K., Xue, Q.-Y., Pussard, E., Lhadj, C., Kolkhof, P., Viengchareun, S., Fagart, J., Lombès, M. Corticosteroid receptors adopt distinct cyclical transcriptional signatures.

Comparative profiling of adrenal steroids in maternal and umbilical cord blood.

Fetal steroidome in late pregnancy receives multiple contributions from both maternal and fetal adrenals as well as from placenta. Depressed glucocorticoid levels have been reported in fetal blood at birth, yet studies on mineralocorticoid pathways are sparse. To investigate biosynthesis pathways at birth, adrenal steroids profiles were established in paired mothers and neonates. Forty-six paired healthy term newborns and their mothers from the Aldo cohort were assessed. Steroidomic profiles of mineralocorticoids, glucocorticoids and adrenal androgens were established from umbilical cord and maternal blood at birth using a highly sensitive and specific LC-MS/MS methodology. As compared to maternal blood, umbilical cord blood exhibited high levels of steroids precursors (progesterone and 11-deoxycorticosterone) contrasting with a collapse in corticosterone levels. Consecutively, 18-hydroxycorticosterone and aldosterone levels were also depressed in neonates. Similarly, umbilical cord blood levels of both 17-hydroxyprogesterone and 11-deoxycortisol were higher while cortisol levels sharply decreased. The product-to-substrate ratios evaluating the 11-hydroxylation step (corticosterone/11-deoxycorticosterone and cortisol/11-deoxycortisol) fell for both pathways. As expected, cortisone and 11-dehydrocorticosterone levels exceed those of cortisol and corticosterone in umbilical cord blood reflecting the strong placental 11-ß-hydroxysteroid-dehydrogenase type 2 (11ßHSD2) activity. Dehydroepiandrosterone-sulphate levels are higher in neonates, while both androstenedione and testosterone levels sharply fell. No significant difference in steroid levels could be observed according the gender except higher testosterone concentrations in umbilical cord of boys. Moreover, a strong and negative relationship between testosterone and progesterone levels was recorded in umbilical cord of boys. These adrenal steroidomic profiling demonstrate a deficit in mineralocorticoids (aldosterone, 18-hydroxycorticosterone and corticosterone) and glucocorticoids (cortisol) in term neonates, reflecting either a relative defect in 11-hydroxylase activity or more likely the strong placental 11-ß-HSD2 activity. Collectively, these findings should be taken into account for a better understanding of regulatory interactions between placenta and fetal adrenal.

Alterations of adrenal steroidomic profiles in preterm infants at birth.

OBJECTIVE: Preterm infants have relative adrenal and kidney immaturity. Recently, we linked their urine sodium loss to a hypoaldosteronism at variance with an appropriate stimulation of the renin-angiotensin system. To investigate this defective aldosterone secretion, we analyse the biosynthesis pathways of adrenal steroids in neonates according to gestational age (GA). DESIGN: Multicentre study (Premaldo) including 152 neonates classified into three groups: group 1 (very preterm (VPT)): <33 gestational weeks (GW); group 2 (preterm (PT)): 33-36 GW and group 3 (term (T)): ≥GW. METHOD: Steroidomic profiles of mineralocorticoids, glucocorticoids and adrenal androgens were established from umbilical cord at birth (n=152) and peripheral blood at day 3 (n=70) using a recently developed liquid chromatography mass spectrometry method (LC-MS/MS). The enzymatic activity of each biosynthesis step was estimated by the product-to-substrate ratio. RESULTS: At birth, VPT infants exhibit a global defect in adrenal steroid synthesis pathways leading to lower levels of aldosterone, cortisol and androstenedione than in term infants. This defect was strongly related to GA. On day 3, steroid precursors (progesterone, 11-deoxycorticosterone (DOC), 17-hydroxyprogesterone(17-OH-P) and 11-deoxycortisol (S)) were higher in VPT and negatively correlated with GA. Despite of precursors' accumulation, aldosterone and cortisol were similar in the three groups. At birth and day 3, a low cortisol/11-deoxycortisol ratio was found in preterm infants, suggesting an 11-beta-hydroxylase activity (CYP11B1) deficiency. CONCLUSIONS: At birth, VPT infants exhibit a global deficit in mineralocorticoids, glucocorticoids and adrenal androgens that attenuates on day 3 of life. Steroid profiling using LC-MS/MS provides evidence for a partial defect in 11-hydroxylase along with prematurity.

Multiplexed steroid profiling of gluco- and mineralocorticoids pathways using a liquid chromatography tandem mass spectrometry method.

Serum steroid assays are major tools in the clinical evaluation of adrenal disorders. The main adrenal steroids are routinely measured with immunoassays. However, chromatographic methods are known to offer better specificity. We report a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of 15 adrenal steroids targeting the mineralo- and gluco-corticosteroid pathways. Serum steroids combined with deuterated internal standards were extracted using successive protein precipitation and solid phase extraction steps. Cortisol, cortisone, 11-deoxycortisol, 17-hydroxyprogesterone, 21-deoxycortisol, progesterone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, 18-hydroxycorticosterone, 18-hydroxy-11-deoxycorticosterone, aldosterone, dehydroepiandrosterone sulfate, testosterone and androstenedione were resolved in fourteen minutes using a BEH C18 column coupled to a methanol-ammonium formate gradient. Detection was performed using multiple reaction monitoring quantitation. Routinely determined steroid levels by immunoassays were compared to those measured by LC-MS/MS. This method was applied to assess steroid profiles in congenital adrenal hyperplasia (CAH) patients with 21-hydroxylase deficiency. Low quantification limits depending on each steroid (ranging from 0.015ng/mL for aldosterone to 20ng/mL for DHEAS) are adapted to the clinical use. Recoveries of steroids range from 64% for 21-deoxycortisol to 101% for cortisol and are fully corrected by internal standards. A good linearity with R>0.989 is obtained for each compound. The inter-day variation coefficients ranged from 4.7% for cortisol to 16.3% for 11-deoxycorticosterone. The immunoassay for cortisol (Immulite 2000, Siemens) showed acceptable agreement with LC-MS/MS (bias +7.2%). However, Bland-Altman plots revealed large negative bias for aldosterone (-33.4%, AldoCT, CisBio international), for 17-hydroxyprogesterone at concentrations below 2ng/mL (-74.1%, OHP-CT MP Biomedical), for androstenedione (-80.3%, RIA D4, Beckman Coulter) and for 11-deoxycortisol (-125.3%, Diasource Immunoassays). Finally, the analysis of samples from 21-hydroxylase defective patients demonstrated the potential usefulness of multiplexed steroid profiling for the diagnosis and/or monitoring of different forms of congenital adrenal hyperplasia. This LC-MS/MS method provides highly sensitive and specific assessments of mineralo- and glucocorticoids pathways from a small volume sample and is therefore a promising potent tool for clinical and experimental endocrine studies.

Mid-regional pro-adrenomedullin (MR-proADM), a marker of positive fluid balance in critically ill patients: results of the ENVOL study.

BACKGROUND: The optimal control of blood volume without fluid overload is a main challenge in the daily care of intensive care unit (ICU) patients. Accordingly this study focused on the identification of biomarkers to help characterize fluid overload status. METHODS: Sixty-seven patients were studied from ICU admission to day 7 (D7). Blood and urine samples were taken daily and sodium and water balance strictly calculated resulting in a total cumulative assessment of ∆Na+ and ∆H2O. Furthermore, plasmatic biomarkers (cortisol, epinephrine, norepinephrine, renin, angiotensin II, aldosterone, pro-endothelin, copeptine, atrial natriuretic peptide, erythropoietin, mid-regional pro-adrenomedullin (MR-proADM)) and Sequential Organ Failure Assessment (SOFA) scores were measured at D2, D5 and D7. Blood volumes were measured with 51Cr fixed on red blood cells at D2 and D7. RESULTS: The ∆Na+ or ∆H2O were increased in all patients but never related to blood volumes at D2 nor D7. Total blood volumes were at normal values with constantly low red blood cell volumes and normal or decreased plasmatic volume. Weight, plasmatic proteins, and hemoglobin were weakly related to ∆Na+ or ∆H2O. Amongst all tested biomarkers, only MR-proADM was related to sodium and fluid overload. This biomarker was also a predictor of SOFA scores. CONCLUSIONS: Plasmatic concentration in MR-proADM seems to be a good surrogate for evaluation of ∆Na+ or ∆H2O and predicts sodium and extracellular fluid overload. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01858675 in May 13, 2013.

High Mortality Risk in Hypoglycemic and Dysglycemic Children Admitted at a Referral Hospital in a Non Malaria Tropical Setting of a Low Income Country.

INTRODUCTION: Hypoglycemia is a recognized feature of severe malaria but its diagnosis and management remain problematic in resource-limited settings. There is limited data on the burden and prognosis associated with glycemia dysregulation in non-neonate children in non-malaria areas. We prospectively assessed the abnormal blood glucose prevalence and the outcome and risk factors of deaths in critically ill children admitted to a national referral hospital in Laos. METHODS: Consecutive children (1 month-15 years) admitted to the pediatric ward of Mahosot hospital, were categorized using the integrated management of childhood illness (IMCI). Blood glucose was assessed once on admission through a finger prick using a bedside glucometer. Glycemia levels: hypoglycemia: < 2.2 mmol/L (< 40 mg/ dl), low glycemia: 2.2-4.4 mmol/L (40-79 mg/ dl), euglycemia: 4.4-8.3 mmol/L (80-149 mg/ dl), and hyperglycemia: > 8.3 mmol/L (≥150 mg/ dl), were related to the IMCI algorithm and case fatality using univariate and multivariate analysis. RESULTS: Of 350 children, 62.2% (n = 218) were severely ill and 49.1% (n = 172) had at least one IMCI danger sign. A total of 15 (4.2%, 95%CI: 2.4-6.9) had hypoglycemia, 99 (28.2%, 95%CI: 23.6-33.3) low glycemia, 201 (57.4%, 95% CI: 52.0-62.6) euglycemia and 35 (10.0%, 95% CI: 7.0-13.6) hyperglycemia. Hypoglycemia was associated with longer fasting (p = 0.001) and limited treatment before admission (p = 0.09). Hypoglycemia and hyperglycemia were associated with hypoxemia (SaO2) (p = 0.001). A total of 21 (6.0%) of the children died: 66.6% with hypoglycemic, 6.0% with low glycemic, 5.7% with hyperglycemic and 1.4% with euglycemic groups. A total of 9 (2.5%) deaths occurred during the first 24 hours of admission and 5 (1.7%) within 3 days of hospital discharge. Compared to euglycemic children, hypoglycemic and low glycemic children had a higher rate of early death (20%, p<0.001 and 5%, p = 0.008; respectively). They also had a higher risk of death (OR: 132; 95%CI: 29.0-596.5; p = 0.001; and OR: 4.2; 95%CI: 1.1-15.6; p = 0.02; respectively). In multivariate analyses, hypoglycemia (OR: 197; 95%CI: 33-1173.9), hypoxemia (OR: 5.3; 95%CI: 1.4-20), presence of hepatomegaly (OR: 8.7; 95%CI: 2.0-37.6) and having an illiterate mother (OR: 25.9; 95%CI: 4.2-160.6) were associated with increased risk of death. CONCLUSION: Hypoglycemia is linked with a high risk of mortality for children in non malaria tropical settings. Blood sugar should be monitored and treatment provided for sick children, especially with danger signs and prolonged fasting. Further evaluations of intervention using thresholds including low glycemia is recommended in resource-limited settings. Research is also needed to determine the significance, prognosis and care of hyperglycemia.

Aldosterone-Signaling Defect Exacerbates Sodium Wasting in Very Preterm Neonates: The Premaldo Study.

CONTEXT: The neonatal period, notably in preterm infants, is characterized by high sodium wasting, implying that aldosterone, the main hormone regulating sodium reabsorption, is unable to maintain sodium homeostasis. OBJECTIVE: This study sought to assess aldosterone secretion and action in neonates according to gestational age (GA). DESIGN AND SETTING: This was a multicenter prospective study (NCT01176162) conducted between 2011 and 2014 at five neonatology departments in France. Infants were followed during their first 3 months. PARTICIPANTS: The 155 newborns included were classified into three groups: Group 1 (n = 46 patients), <33 gestational weeks (GW); Group 2 (n = 67 patients), 33-36 GW; and Group 3 (n = 42 patients), ≥37 GW. MAIN OUTCOME MEASURES: Plasma aldosterone was measured in umbilical cord blood. Urinary aldosterone (UAldo) was assessed at day 0, day 3, month 1, and month 3 postnatal. The correlation between UAldo and the urinary Na/K ratio was determined as an index of renal aldosterone sensitivity. RESULTS: UAldo significantly increased with GA: from 8.8 ± 7.5 µg/mmol of creatinine (Group 1) to 21.1 ± 21.0 (Group 3) in correlation with plasma aldosterone levels in all groups (P < .001), demonstrating its reliability. The aldosterone/renin ratio significantly increased with GA, suggesting an aldosterone secretion defect in preterm infants. UAldo and urinary Na/K were correlated in very preterm but not in term neonates, consistent with very preterm neonates being renal-aldosterone sensitive and term neonates being aldosterone resistant. CONCLUSIONS: Very preterm infants have a previously unrecognized defective aldosterone secretion but conserved renal aldosterone sensitivity in the neonatal period, which modifies the current view of sodium balance in these infants and suggests alternative management approaches.

Improving the management of dysglycemia in children in the developing world.

Improving the availability of point-of-care (POC) diagnostics for glucose is crucial in resource-constrained settings (RCS). Both hypo and hyperglycemia have an appreciable frequency in the tropics and have been associated with increased risk of deaths in pediatrics units. However, causes of dysglycemia, including hyperglycemia, are numerous and insufficiently documented in RCS. Effective glycemic control with glucose infusion and/or intensive insulin therapy can improve clinical outcomes in western settings. A non-invasive way for insulin administration is not yet available for hyperglycemia. We documented a few causes and developed simple POC treatment of hypoglycemia in RCS. We showed the efficacy of sublingual sugar in two clinical trials. Dextrose gel has been recently tested for neonate mortality. This represents an interesting alternative that should be compared with sublingual sugar in RCS. New studies had to be done to document dysglycemia mechanism, frequency and morbid-mortality, and safe POC treatment in the tropics.