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According to animal studies, saffron and its main volatile compound safranal may reduce biological and behavioral signs of acute stress. However, little is known about its impact in humans. This study investigated the acute effect of a saffron extract and safranal on the biological and psychological stress responses in healthy men experiencing a laboratory stress procedure. In this double-blind, placebo-controlled, randomized, cross-over study, 19 volunteers aged 18–25 received a single dose of 30 mg saffron extract (Safr’InsideTM), 0.06 mg synthetic safranal, or a placebo on three visits separated by a 28-day washout. Thirteen minutes after administration, participants were exposed to the Maastricht acute stress test (MAST). Salivary cortisol and cortisone were collected from 15 min before the MAST (and pre-dose), 3 min before the MAST, and then 15, 30, 45, 60, and 75 min after the MAST, and stress and anxiety were measured using visual analogic scales. Compared to the placebo, stress and anxiety were significantly toned down after Safranal and Safr’InsideTM administration and coupled with a delay in the times to peak salivary cortisol and cortisone concentrations (p < 0.05). Safr’InsideTM and its volatile compound seem to improve psychological stress response in healthy men after exposure to a lab-based stressor and may modulate the biological stress response.

Context Cortisol-lowering drugs may not restore a normal cortisol secretion in Cushing disease (CD). Objective This work aimed to assess the long-term cortisol exposure in medically treated CD patients using hair-cortisol (HF) and hair-cortisone (HE) measurement. Methods This multicenter prospective study included 3 groups of female patients: CushMed = 16 treated with a stable cortisol-lowering drug dosage and normal urinary free cortisol (UFC); CushSurg = 13 cured by pituitary surgery; CushBla = 15 receiving stable recommended doses of hydrocortisone following bilateral adrenalectomy. Patients were evaluated for 3 months with their usual treatments. Two late-night saliva and 24-hour urine samples were collected monthly in CushMed, and at study end in CushSurg and CushBla patients. A 3-cm hair sample was collected at study end from all patients. Main outcome measures included clinical score and centralized measurement of UFC, late-night salivary cortisol (LNSF), late-night salivary cortisone (LNSE), HE, HF. Results Despite having almost all UFCs normalized, CushMed patients exhibited increased HE as compared to CushSurg controls (P = .003). CushMed patients also had increased clinical score (P = .001), UFC (P = .03), LNSF, LNSE (P = .0001), and variability in the latter parameters (P = .004). CushBla patients had increased HF and HE, contrasting with LNSEs similar to CushSurg patients. Six of 15 CushMed patients exhibited increased HE concentrations and had increased antihypertensive drug dosage compared to CushMed patients with normal HE (P = .05). Conclusion Despite normalized UFCs, a subset of medically treated CD patients displays an altered circadian rhythm of serum cortisol. A single HE measurement identifies chronic mild persistent hypercortisolism and could replace multiple saliva analyzes to monitor medical treatments in CD patients once UFC is normalized.

Objective Hair cortisol (HF) and cortisone (HE) measurements reflect tissular exposure to cortisol over months and are increased in overt Cushing’s syndrome (CS). No data is available in mild CS. We compared the diagnostic performance of HF and HE between patients with overt or mild CS. Design Single centre retrospective study. Methods HF&HE were measured by LC-MS/MS in 48 consecutive adult females with Cushing’s disease (CD), ectopic ACTH syndrome, secreting adenomas and carcinomas, and adrenal incidentalomas. All had impaired dexamethasone suppression tests. Overt CS (n = 25) was diagnosed in front of specific symptoms, a mean UFC (>1.5 ULN) and increased midnight serum cortisol or salivary cortisol. Mild CS (n = 23) was diagnosed in patients lacking specific symptoms and displaying at least one additional biological abnormality including mildly increased UFC (≤1.5 ULN), increased midnight serum cortisol or salivary cortisol and suppressed plasma ACTH in patients with adrenal tumours. In this study, 84 healthy subjects and obese patients served as controls. Results HF and HE showed roughly similar performance in overt CS (92 and 100% sensitivity, 91 and 99% specificity, respectively). HF and HE were lower in mild CS but higher than in controls (P < 0.01). HE was correlated with midnight serum cortisol (P < 0.02) and volume of adrenal incidentalomas (P < 0.04) but not with UFC. HF and HE had 59% and 68% sensitivity, and 79 and 94% specificity, respectively, for the diagnosis of mild CS. Contrary to UFC, both HF and HE were in the range of overt CS in 11/23 patients with mild CS. Patients with mild CS and increased HE required more antihypertensive treatments and showed worser lipid profiles than patients with normal HE. Conclusions HF and HE measurement performed better in overt than in mild CS but is a useful adjunct to diagnose mild CS and to identify adrenocortical incidentalomas responsible for excessive cortisol exposure.

An 18-year-old woman was referred by her GP to the endocrinology department of the University Hospital of Bordeaux on suspicion of premature ovarian failure because of a disorder of the menstrual cycle and pathological results of biological exploration of the gonadotropic axis. Repeatedly-found elevated concentrations of FSH contrasted with a normal concentration of LH leading to a hypothesis of ovarian failure. However, different investigations favoured an analytical interference. The presence of heterophilic antibodies or anti-mouse antibodies (HAMA) was unlikely but, finally, a complex combining FSH and autoantibody (called macro-FSH) was evidenced.

Primary aldosteronism is responsible for a major cardiovascular risk that can be avoided by specific treatment. A better characterization of the hypertensive population with primary aldosteronism would not only improve the overall diagnosis but also allows a better selection of patients requiring adrenal vein sampling (AVS).Creation of a prospective single-center Bordeaux ABORDAGE study of hypertensive patients with primary aldosteronism who underwent AVS. Primary aldosteronism was diagnosed according to the recommendations of the SFE/SFHTA. Peripheral and central blood pressure measurements were performed with mercury sphygmomanometer, SphygmoCor applanation tonometer and ambulatory blood pressure measurement. An adrenal computed tomography and an unstimulated AVS were performed in each patient.One hundred and eighty-eight patients were included in our study. They were mostly men (61.7%), with a mean age of 48.7 ± 10.5 years, BMI of 29.7 ± 5 kg/ m2 and duration of hypertension of 101.5 ± 84 months. AVS was selective in 82.3% of patients and lateralization was concordant with CT in only 35.4% of patients. Lateralized secretion was significantly associated with a marked biological primary aldosteronism and hypertension. In multivariate analysis, no variable specifically differentiated patients with aldosterone lateralization.The ABORDAGE population description is consistent with the data found in the literature. These characteristics are ultimately those expected in essential hypertension population, which therefore, could explain part of the underdiagnosis of primary aldosteronism. Only AVS is able to predict the lateralization of secretion with a post adrenalectomy recovery of about 90% in case of lateralization. The generalization of AVS would, therefore, increase the proportion of patients with primary aldosteronism cured.

Identifying analytical interference is a challenge for the medical biologist in providing advice to the prescriber. Indeed, these analytical interferences often have deleterious consequences on the care of patients. Understanding their mechanisms and mastering corrective procedures is essential to limit these management errors. Faced with the many questions from clinicians in current practice, we propose an algorithm for managing a sample when interference is suspected.

The bone marrow (BM) niche impacts the progression of acute myeloid leukemia (AML) by favoring the chemoresistance of AML cells. Intimate interactions between leukemic cells and BM mesenchymal stromal cells (BM-MSCs) play key roles in this process. Direct intercellular communications between hematopoietic cells and BM-MSCs involve connexins, components of gap junctions. We postulated that blocking gap junction assembly could modify cell–cell interactions in the leukemic niche and consequently the chemoresistance. The comparison of BM-MSCs from AML patients and healthy donors revealed a specific profile of connexins in BM-MSCs of the leukemic niche and the effects of carbenoxolone (CBX), a gap junction disruptor, were evaluated on AML cells. CBX presents an antileukemic effect without affecting normal BM-CD34+ progenitor cells. The proapoptotic effect of CBX on AML cells is in line with the extinction of energy metabolism. CBX acts synergistically with cytarabine (Ara-C) in vitro and in vivo. Coculture experiments of AML cells with BM-MSCs revealed that CBX neutralizes the protective effect of the niche against the Ara-C-induced apoptosis of leukemic cells. Altogether, these results suggest that CBX could be of therapeutic interest to reduce the chemoresistance favored by the leukemic niche, by targeting gap junctions, without affecting normal hematopoiesis.

Anxiety, stress, and low mood are closely related and may contribute to depressive symptoms. Among non-pharmacological solutions to improve subclinical mood symptoms and resilience to stress, natural products such as saffron—identified as promising following preliminary beneficial effects in major depressive disorder—represent a relevant strategy. This study aimed to assess the efficacy of 8 weeks' supplementation with 30 mg standardized saffron extract on emotional well-being in healthy adults with subclinical feelings of low mood and anxiety and/or stress and evaluate the acute effect of saffron in response to a lab-based psychosocial stressor. The study adopted a double-blind, randomized, parallel groups design in which 56 healthy male and female individuals (18–54 years) received either a saffron extract or a placebo for 8 weeks. Chronic effects of saffron on subjective anxiety, stress, and depressive feelings were assessed using a questionnaire battery [including Profile of Mood State-2, (POMS)] and acute effects in response to a lab-based psychosocial stressor were measured through psychological and physiological parameters. Urinary crocetin levels were quantified. Participants who received the saffron extract reported reduced depression scores and improved social relationships at the end of the study. Urinary crocetin levels increased significantly with saffron supplementation and were correlated with change in depression scores. The typical stress-induced decrease in heart rate variability (HRV) during exposure to the stressor was attenuated following acute saffron intake. Saffron extract appears to improve subclinical depressive symptoms in healthy individuals and may contribute to increased resilience against the development of stress-related psychiatric disorders.Clinical trials number: NCT03639831.

Background Early consumption of obesogenic diets, rich in saturated fat and added sugar, is associated with a plethora of biological dysfunctions, at both peripheral and brain levels. Obesity is also linked to decreased vitamin A bioavailability, an essential molecule for brain plasticity and memory function. Methods Here we investigated in mice whether dietary vitamin A supplementation (VAS) could prevent some of the metabolic, microbiota, neuronal and cognitive alterations induced by obesogenic, high-fat and high-sugar diet (HFSD) exposure from weaning to adulthood, i.e. covering periadolescent period. Results As expected, VAS was effective in enhancing peripheral vitamin A levels as well as hippocampal retinoic acid levels, the active metabolite of vitamin A, regardless of the diet. VAS attenuated HFSD-induced excessive weight gain, without affecting metabolic changes, and prevented alterations of gut microbiota α-diversity. In HFSD-fed mice, VAS prevented recognition memory deficits but had no effect on aversive memory enhancement. Interestingly, VAS alleviated both HFSD-induced higher neuronal activation and lower glucocorticoid receptor phosphorylation in the hippocampus after training. Conclusion Dietary VAS was protective against the deleterious effects of early obesogenic diet consumption on hippocampal function, possibly through modulation of the gut-brain axis.

In routine hormonology, liquid chromatography mass spectrometry (LCMS) is now an established technique for androgen, urinary cortisol and metanephrine assay. It has the undeniable advantage of great analytical specificity, but with sensitivity that clearly depends on financial investment in a very high-end spectrometer. We describe the general principles of LCMS and the routine applications so far developed in hormonology. The purpose is to familiarise endocrinologists with the techniques under development and their pros and cons.

Background: Twenty-four hour urinary free cortisol (UFC) determination can be used for screening and follow-up of Cushing syndrome (CS). As immunoassay methods lack specificity for UFC measurement, the use of high-performance liquid chromatography coupled to mass spectrometer (LC-MSMS) is recommended. The aim of our study was to compare UFC results using four LC-MSMS methods performed in four independent laboratories in order to evaluate interlaboratory agreement. Methods: Frozen aliquots of 24-h urine samples (78 healthy volunteers and 20 patients with CS) were sent to four different laboratories for analysis. Following liquid-liquid or solid-liquid extraction, UFC were determined using four different LC-MSMS assay. Results: UFC intra- and interassays variation coefficients were lower than 10% for each centre. External quality control results were not significantly different. UFC normal ranges (established from healthy volunteers) were 17–126, 15–134, 12–118 and 27–157 nmol/day, respectively. Classification of UFC from healthy volunteers and patients with CS using a 95th percentile threshold was similar. However, for extreme UFC values (270 nmol/day), negative or positive bias was noted. Conclusions: Even for highly specific methods such as LC-MSMS, variations of results can be found depending on analytical process. Validation of LC-MSMS methods including determination of the reference range is essential.

International audience; Vitamin A (retinol) is a micronutrient critical for cell proliferation and differentiation. In adults, vitamin A and metabolites such as retinoic acid (RA) play major roles in vision, immune and brain functions and tissue remodelling and metabolism. This review presents the physiological interactions of retinoids and endocrine tissues and hormonal systems. Two endocrine systems have been particularly studied. In the pituitary, retinoids target the corticotrophs with a possible therapeutic use in corticotropinomas. In the thyroid, retinoids interfere with iodine metabolism and vitamin A deficiency aggravates thyroid dysfunction caused by iodine-deficient diets. Retinoids use in thyroid cancer appears less promising than expected. Recent and still controversial studies investigated the relations between retinoids and metabolic syndrome. Indeed, retinoids contribute to pancreatic development and modify fat and glucose metabolism. However, more detailed studies are needed before planning any therapeutic use. Finally, retinoids probably play more minor roles in adrenal and gonads development and function apart from their major effects on spermatogenesis.

International audience; The diagnosis of Type 1 Diabetes (T1D) in ever younger children led us to question the impact of insulin deficiency or chronic hyperglycemia on cerebral development and memory performances. Here, we sought abnormalities in these traits in a model of streptozotocin-induced diabetes in juvenile rats treated or not by insulin. We made the assumption that such alterations would be related, at least in part, to excessive glucocorticoid exposition in hippocampal neurons. We have compared 3 groups of juvenile rats: controls, untreated diabetics and insulin-treated diabetics. Diabetes was induced by streptozotocin (65 mg/kg IP/day, 2 consecutive days), at postnatal days 21 and 22 and a subcutaneous pellet delivering 2 U of insulin/day was implanted in treated diabetic rats 3 days later. Three weeks after diabetes induction, cognitive performances (Y maze, object location and recognition tests), in vivo brain structure (brain volume and water diffusion by structural magnetic resonance imaging), and hippocampal neurogenesis (immunohistochemical labeling) measurements were undertaken. Corticosterone levels were evaluated in plasma under basal and stress conditions, and within hippocampus together with 11β-dehydrocorticosterone to assess 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activity. The comparison of the three experimental groups revealed that, compared to controls, untreated diabetic rats showed decreased cognitive performances in Y-maze and object location test (p

L’hypercorticisme pathologique entraine une rupture du rythme nycthemeral possiblement exploree par la determination de la cortisolemie a minuit cotee NABM a la difference de celle du cortisol salivaire pourtant recommandee. Nous en proposons ici des valeurs de references non fournies par les fabricants de trousses d’immunodosages. La population etudiee retrospectivement etait composee de : –sujets (214 F/121 H) (57 [25–75] ans, mediane [5e–95e] centiles) sans hypercorticisme (explores pour diabete, HTA, obesite) avec 421 couples de cortisolemies 8 h/0 h ; –56 sujets (49 [29–71] ans) presentant un hypercortisolisme (5 syndromes paraneoplasiques, 7 hyperplasies surrenaliennes, 8 corticosurrenalomes, 9 adenomes surrenaliens ou 27 corticotropes). Le cortisol serique a ete dose sur Dxi800 (Beckman–Coulter), la cortisolurie en LCMS. La cortisolemie 8 h etait a 337 [189–619] nmol/L (Vref [185–624] nmol/L) ; la cortisolurie 17 [5–55] μg/j (Vref 10–60 μg/j). La cortisolemie 0 h etait a 96 [36–257] nmol/L ; Vref superieure (CLSIC28-A3) 261 nmol/L (IC90 % : 243–278). Treize pour cent etaient prelevees le jour de l’hospitalisation et 79 % le lendemain. Vingt sujets presentaient des cortisolemie > 95e % (une acromegalie, une osteoporose avec un cancer thyroidien, 1 pheochromocytome, 1 pseudo syndrome de Cushing, 1 syndrome anxieux, 1 syndrome coronarien aigu, une tumeur neuroendocrine d’origine inconnue, 3 cancers, 3 diabetes avec HTA, 7 incidentalome surrenaliens non secretants). Les sujets avec un hypercortisolisme presentaient une cortisolurie a 104 [24–365] μg/j, des cortisolemies 8 h a 463 [274–738] nmol/L et 0 h a 417 [109–755] nmol/L. Toutes ces valeurs sont significativement differentes de celles des sujets sans hypercortisolisme.

The bone marrow (BM) niche impacts the progression of acute myeloid leukemia (AML) by favoring the chemoresistance of AML cells. Intimate interactions between leukemic cells and BM mesenchymal stromal cells (BM-MSCs) play key roles in this process. Direct intercellular communications between hematopoietic cells and BM-MSCs involve connexins, components of gap junctions. We postulated that blocking gap junction assembly could modify cell-cell interactions in the leukemic niche and consequently the chemoresistance. The comparison of BM-MSCs from AML patients and healthy donors revealed a specific profile of connexins in BM-MSCs of the leukemic niche and the effects of carbenoxolone (CBX), a gap junction disruptor, were evaluated on AML cells. CBX presents an antileukemic effect without affecting normal BM-CD34

Glucocorticoid receptor (GR) function is modulated by phosphorylation. As retinoic acid (RA) can activate some cytoplasmic kinases able to phosphorylate GR, we investigated whether RA could modulate GR phosphorylation in neuronal cells in a context of long-term glucocorticoid exposure. A 4-day treatment of dexamethasone (Dex) plus RA, showed that RA potentiated the (Dex)-induced phosphorylation on GR Serine 220 (pSer220GR) in the nucleus of a hippocampal HT22 cell line. This treatment increased the cytoplasmic ratio of p35/p25 proteins, which are major CDK5 cofactors. Roscovitine, a pharmacological CDK5 inhibitor, or a siRNA against CDK5 prevented RA potentiation of GR phosphorylation. Furthermore, roscovitine counter-acted the effect of RA on GR sensitive target proteins such as BDNF or tissue-transglutaminase. These data help understanding the interaction between RA- and glucocorticoid-signalling pathways, both of which have strong influences on the adult brain.

Context Biogenic amines such as 5-hydroxy-indole acetic acid (5HIAA) the main metabolite of serotonin or metanephrines (catecholamines metabolites) are used as biomarkers of neuroendocrine tumours. Objective To re-evaluate the recommendations for urinary sampling (preservatives, diet, drugs, etc.) as many of the reported analytical interferences supporting these recommendations are related to obsolete assays. Methods Bibliographic analysis of old and modern assays concerning preservation, extraction, assay and interferences. Results 5HIAA may degrade as soon as urine is excreted. Thus, acids as preservatives (hydrochloric or acetic acid) have to be immediately added. Care should be taken not to decrease the pH under 2. Urine preservative for metanephrine assays is not mandatory. Diets including serotonin-, tryptophan- and dopamine-rich foods have to be avoided depending on the biomarkers investigated (bananas, plantain, nuts, etc.). Tryptophan-rich over-the-counter formulas have to be prohibited when 5HIAA has to be assayed. Acetaminophen may interfere with electrochemical detection depending on high-pressure liquid chromatography (HPLC) parameters. No interference is known with mass spectrometric assays but with the one described for metanephrines determination. Some drugs interfere however with serotonin and catecholamines secretion and/or metabolism (monoamine oxidase inhibitors, serotonin or dopamine recapture inhibitors, etc.). Conclusion Revisited recommendations are provided for the diet, the drugs and the preservatives before HPLC coupled with electrochemical and mass spectrometry assays.

International audience; Alterations in endocrine functions and low-grade systemic inflammation represent fundamental characteristics of obesity. These biological systems have been repeatedly linked to fatigue symptoms. The aim of the study was to assess the relationship between fatigue dimensions and metabolic/inflammatory markers in a sample of non-diabetic obese children. The possibility that inflammation-induced alterations in tryptophan metabolism relates to specific dimensions of fatigue was also investigated in a subsample of patients. The study was conducted in 41 obese children, median aged 12 [9-15] years, recruited in a pediatric tertiary center. Three dimensions of fatigue (e.g., general fatigue, sleep/rest, cognitive fatigue) were assessed using the Pediatric Quality of Life Inventory Multidimentional Fatigue Scale. In addition, a principal component analysis was performed to identify fatigue dimensions that were specific to the population under study. This analysis extracted five relevant dimensions corresponding respectively to concentration, energy, self-perceived cognitive efficiency, 'sleep/rest and motivation/anhedonia. Blood samples were collected for the measurement of inflammatory and metabolic markers, including, high sensitivity C-reactive protein (hs-CRP), insulin, uricemia and glycaemia. Tryptophan, kynurenine and neopterin levels were also determined in a subsample of 17 patients. In the whole population under study, cognitive fatigue and reduced motivation/anhedonia were associated with BMI, independently of sex and age. The dimension of reduced motivation/anhedonia was associated with insulin resistance and inflammatory biomarkers. The association with insulin resistance persisted when the extent of fat mass (BMI-SDS) was taken into account. No association was found between tryptophan metabolism and specific dimensions of fatigue, but kynurenine and the kynurenine/tryptophan ratio correlated with insulin and HOMA-IR. These data indicate that insulin resistance in non diabetic obese children is associated with both cognitive fatigue and reduced motivation/anhedonia and with alterations in tryptophan metabolism. Further investigations are needed to determine whether inflammation-induced alterations in tryptophan metabolism is directly or indirectly implicated in insulin resistance and related fatigue.

Le diagnostic biologique d’hyperplasie congenitale des surrenales (HCS) repose sur l’augmentation, souvent apres test au synacthene, de la concentration serique de 17-hydroxyprogesterone (17OHP) pour les deficits en 21-hydroxylase et du 11-desoxycortisol (11DC) pour les deficits en 11-βhydroxylase. L’interpretation du resultat fait intervenir le 95e percentile des valeurs de reference obtenues dans une population controle. Ces valeurs sont historiquement etablies par immunodosage. Nous proposons de les determiner par spectrometrie de masse (LCMS). Les resultats des tests au synacthene de 102 femmes ont ete repris retrospectivement : pour 92 d’entre elles (21 [14 ;62]ans, mediane [5e ;95e] percentiles) les diagnostics de HCS et d’insuffisance surrenalienne ont ete ecartes. Pour 10 autres (31[17 ;46]ans) l’HCS est confirmee ou fortement suspectee. Les valeurs du cortisol serique (irF) ont ete obtenues par immunodosage, celles de la ms17OHP et ms11DC et des autres steroides par LCMS. A T0 min et T60 min, les concentrations de ms11DC sont de 0,6 [0,2 ;3,4] & 2,2 [0,7 ;6,9] pour la population controle vs 1.0 [0,3 ;3,2] & 3,4 [0,8 ;8,6] nmol/L pour HCS (p > 0,05). Celles de ms17OHP sont de 1,9 [0,4 ;6,3] & 3,5 [1,7 ;9,9] vs 24,1 [14,1 ;38,3] & 95,1 [48,9 ;190,2] nmol/L (p

Introduction La plus grande precocite des diagnostics de diabete chez l’enfant nous amene a questionner le retentissement de la carence en insuline sur le developpement cerebral et les performances cognitives. Dans un modele de ratons diabetiques, nous avons fait l’hypothese que les troubles mnesiques et les anomalies du developpement cerebral etaient en lien avec une alteration de la regulation de l’axe corticotrope entrainant une elevation de l’impregnation glucocorticoide responsable d’une toxicite sur les neurones hippocampiques. Methode Le diabete a ete induit par streptozotocine a 21 jours de vie. Nous avons constitue 3 groupes de ratons : controles, diabetiques non-traites par insuline, diabetiques traites par insuline (implant sous-cutane). A 2 semaines d’induction du diabete, nous avons etudie les performances mnesiques, le volume et la structure hippocampique en IRM et l’activite corticotrope. La neurogenese hippocampique et l’activite de la 11β-HSD1 hippocampique ont ete etudiees au sacrifice. Resultats Dans notre modele, le diabete induit par la streptozotocine chez le raton s’accompagne d’un defaut de performance de memoire hippocampo-dependante, d’alterations de l’organisation structurale de l’hippocampe et d’un defaut de retrocontrole negatif de l’axe corticotrope qui ne sont pas totalement corriges par l’apport d’insuline, bien que cet apport garantisse une croissance adequate et une nette amelioration de l’equilibre glycemique. L’apport d’insuline est en mesure de controler l’activite 11β-HSD1 hippocampique mesuree au moment du sacrifice. Discussion Fort de ces resultats, nous souhaitons approfondir le role joue par la 11β-HSD1 hippocampique dans la genese de ces alterations. (Travail presente en communication orale a la SFD 2018).

International audience; Objective Total urinary cortisol metabolites represent cortisol production and metabolism. We hypothesized that to assay metabolites could add some information to the one provided by a sole cortisol assay. Design and patients We set up an inexpensive multiplex mass spectrometry assay to quantify cortisol metabolites. We investigated 43 patients with benign secreting (AT+) or silent (AT−) adrenal tumours compared to 48 lean (Nl) or 143 obese (Ob) subjects, and to 26 patients with a Cushing's disease (CD). The initial investigation included immunoreactive quantification of urinary free cortisol (UFC). Results Cortisol metabolites were overexcreted in CD but not in Ob subjects. Nl and Ob were thus pooled in a control population (Ctl). Cortisol, tetrahydrocortisol (THF) and tetrahydrocortisone (THE) excretions were significantly increased in AT compared to Ctl subjects, whereas immunoreactive UFC was similar. A logistic regression retaining cortisol, THF, and α- and β-cortolone as significant analytes allowed the construction of a receiver-operating characteristics (ROC) curve significantly better than the curve generated by cortisol alone (area under the curve (AUC) 0·927 vs 0·729, respectively; P < 0·0001). More importantly, although there was no significant difference between Ctl vs AT− subjects for cortisol metabolites, a logistic regression retaining cortisol, allo-THF, and α- and β-cortolone as significant analytes generated a ROC curve performing significantly better than cortisol alone (AUC 0·910 vs 0·635, respectively; P < 0·0001). Conclusion Cortisol metabolite excretion is modified in AT, including AT−, patients even without modification of UFC. Clinical usefulness of these biomarkers has to be investigated in prospective studies following up patients with AT.

Objectifs Les recommandations preconisent de realiser le dosage du cortisol libre dans les urines de 24 heures (CLU) pour depister le syndrome de Cushing (SC). La chromatographie liquide haute performance couplee a la spectrometrie de masse (LC-MS) est la technique de reference maintenant recommandee car elle presente une meilleure specificite que les immunodosages. En l’absence de trousse de dosage adaptee a la matrice urinaire, les centres specialises equipes de LC-MS doivent developper leur propre methodologie. Ce travail vise a comparer les resultats de CLU obtenus par LC-MS dans 4 centres differents selon 4 methodologies differentes. Patients et methodes Les echantillons urinaires de 78 temoins sains (T) et 20 patients atteints d’un SC ont ete anonymises, aliquotes, congeles et envoyes vers chaque centre. Les dosages y ont ete realises par 4 techniques et appareillages differents, apres extractions liquide–liquide ou liquide–solide. Resultats Les coefficients de variation intra- et inter-series de chaque centre sont 0,90. La repartition des valeurs de CLU T et SC par rapport au 95e percentile est identique quel que soit le centre. Neanmoins, il existe un biais allant jusqu’a 80 % pour certaines urines dans des concentrations 100 μg/24 h. Discussion Meme pour une technique aussi specifique que la LC-MS, notre travail souligne l’importance des comparaisons entre differents centres afin de mettre en evidence les points critiques dans le processus de traitement de l’echantillon et d’eviter les biais analytiques.

Objectif Une exposition chronique aux glucocorticoides (GC) est responsable d’effets deleteres en particulier sur le systeme nerveux central. Ceci est en partie lie a une augmentation de leur biodisponibilite intracellulaire via l’action opposee des 11βhydroxysteroides–deshydrogenasses (HSD) de type 1 et 2. HSD1 convertit les GC inactifs (cortisone [C] ou de hydrocortisone [DHC]) en GC actif (cortisol ou corticosterone) et inversement pour HSD2. Ce travail vise a developper une methode de quantification de l’activite de ces enzymes dans des lignees cellulaires hippocampique (HT22) et gliale (BV2). Methodes L’expression des ARNm des enzymes est etudiee par pPCR (condition basale ou apres dexamethasone [Dex]). L’activite enzymatique est etudiee par la quantification en spectrometrie de masse de la conversion de C ou de DHC ajoute dans le milieu de culture. Resultats Pour les HT22, il n’existe ni expression de HSD1 (basale ou sous Dex), ni conversion de DHC en C. L’expression de HSD2 est faible mais augmente significativement sous Dex. L’activite HSD2 est moderee et augmente peu mais significativement sous Dex. Pour les BV2, HSD1 est exprimee et sa transcription augmentee significativement sous Dex ; l’activite enzymatique HSD1, tres importante sans traitement, est faiblement mais significativement augmentee par la Dex. HSD2 est exprimee sans modification significative par la Dex alors que son activite, faible en condition basale, est significativement augmentee par la Dex. Discussion Au vue des discordances observees entre expression des ARNm et activite enzymatique, notre travail souligne l’utilite d’evaluer l’activite des 2 enzymes afin d’eviter des interpretations inappropriees.

Steroid hormone measurement, first developed with radioimmunoassay, is now becoming easier with the use of automated platforms of immunoassay. However, some hormones remain uneasily detectable because of their low blood concentration, their structural homology or the presence of interferences. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be considered as an alternative to immunoassays. This approach allows the simultaneous determination of several parameters thanks to its selectivity led by the detector mass spectrometer and the separate dimension of chromatography liquid. In addition, recourse to UHPLC (ultra high performance liquid chromatography) allows improving selectivity and sensitivity while limiting the samples volumes. The "ready-to-use" kits are now available and added to the "homemade" techniques developed by laboratories, thus giving opportunity for measurement of a wide steroid panel with only one sample. Finally, mass spectrometry methods, including a prior extraction step, allow the use of varied biological fluids (blood, urine, saliva…). Also, several clinical indications could gain from mass spectrometry, especially when hormone levels are low, when several steroids have to be identified, when the sample volume is low. However, this technology represents an important financial investment and in-depth staff training. In addition, some steroids are not easily quantifiable by mass spectrometry. It is likely by immunoassay and mass spectrometry, well-matched technologies, that we could answer the best to clinical questions about steroids.

Background Serum cortisol is routinely quantified by immunoassays. In intensive care units serum free cortisol (FC) determination has been described as a better indicator of survival than total cortisol (TC). To estimate FC different methods are available including saliva sampling. We compared five methods to estimate FC, before and after an ACTH stimulating test in patients suspected of adrenal insufficiency. Method Serum and saliva was collected from 130 patients from the Endocrine Department of a university hospital before and after tetracosactide injection for TC determination. FC was estimated: after serum ultrafiltration, quadratic (Coolens') or cubic (Dorin's) equations, using TC/cortisol-binding globulin concentrations ratio or using cortisol concentration determination in saliva. Results FC concentrations obtained by different techniques were significantly correlated and Passing-Bablok regressions showed no deviation from linearity between salFC and filtFC or quadFC. Using the routine assumption that the patients were correctly diagnosed using a post-tetracosactide TC threshold of 550 nmol/L the FC methods generating the best ROC curves were salFC and filtFC or cubFC 30 min after tetracosactide injection. Conclusions FC concentrations obtained by different techniques are significantly but not similarly correlated with TC. As, salFC and filtFC are more convenient to perform than methods involving CBG assays and are better correlated to TC during tetracosactide tests they may be preferred as FC surrogate assays.

Objective To extend knowledge about the clinical performances of a new chemiluminescent immunoassay (CLIA) for aldosterone set up in available analysers. Design and patients We compared the results of a RIA assay to those of a CLIA assay in 198 serum and 80 urine samples from patients in endocrine and hypertension departments. Furthermore, for serum samples the concordance of results for postural tests was analysed. Results RIA and CLIA aldosterone serum concentration was linearly correlated with a slope of 0.988 and an intercept of 70.4 pmol/L. The variations of aldosterone serum concentration obtained with the two assays during postural tests were very consistent. There was no significant difference of aldosterone concentrations after thawing with the CLIA assay. RIA and CLIA aldosterone urine concentrations were linearly correlated with a slope of 0.787 and an intercept of − 2.64 nmol/L. Omitting the preservative boric acid from urine samples did not modify aldosterone concentration at least up to 48 h after collection. Conclusion The RIA and CLIA assays were well correlated for the most useful serum samples. It is well suited to circumvent isotopic assays with the throughput of available analysers.

Les syndromes de Cushing severes beneficient d’un traitement par metopirone et/ou ketoconazole avec un blocage de la steroidogenese imposant une surveillance de l’apparition d’une insuffisance surrenalienne. Toutefois, ce blocage est responsable de l’apparition de steroides, normalement peu abondants, responsables de reactions croisees dans les immunodosages de cortisol (serique ou urinaire). Nous rapportons l’exemple d’un corticosurrenalome traites par metopirone + ketoconazole (& mitotane). L’evolution de l’excretion urinaire de cortisol libre a ete favorable : 566 ± 252 a 80 ± 40 μg/j (LC-MSMS, N Ces resultats sont nettement en faveur du suivi par spectrometrie de masse du cortisol lors de l’initiation d’un traitement par inhibiteurs de la steroidogenese surrenalienne. Des valeurs specifiques de cortisolemies ou des dosages de cortisolurie seraient souhaitables pour les patients sous mitotane. Sans mitotane, les valeurs normales pourraient etre utilisees.

ObjectivesWe set up plasma normetanephrine (pNMA) and metanephrine (pMA) assays as they demonstrated their usefulness for diagnosing phaeochromocytomas. Our scope is to describe some practical laboratory aspects and the clinical relevance of these assays in our endocrinological or cardiological departments.MethodsWe retrospectively reviewed the results of MA from a population of in- and outpatients over a 7-year period. Subjects (n=2536) from endocrinological or cardiological departments were investigated (66 phaeochromocytomas). Urinary NMA (uNMA) and pNMA, and urinary MA (uMA) and pMA were assayed by HPLC with electrochemical detection.ResultspNMA and pMA assays are now more frequently requested than uNMA and uMA. This changed our laboratory work load with improved delivery, sensitivity and reliability of plasma assays as well as reduced apparatus maintenance time.The pNMA and pMA upper reference limits (URLs) of subjects with no phaeochromocytoma were 1040 and 430 pmol/l respectively. Sensitivity and specificity based on receiver operating characteristic curves optimal points were 83 and 93% for pNMA at 972 pmol/l and 67 and 98% for pMA at 638 pmol/l. Sensitivity and specificity of paired tests of pMA (positive test: at least one analyte above its URLs) were 100 and 91% respectively.ConclusionThe very low concentration of analytes requires a sustained very good apparatus analytical sensitivity. This can be obtained in an up-to-date laboratory. In terms of clinical performances, assays in plasma or urine are equivalent. Depending on local preferences, populations, strategies or departments, requests for one or the other assay may sustain the need for specifically defined reference ranges.

Chantier qualité GA; International audience; Vitamin A metabolite retinoic acid (RA) plays a major role in the aging adult brain plasticity. Conversely, chronic excess of glucocorticoids (GC) elicits some deleterious effects in the hippocampus. We questioned here the involvement of RA and GC in the expression of target proteins in hippocampal neurons. We investigated proteins involved either in the signaling pathways [RA receptor (RAR) and glucocorticoid receptor (GR)] or in neuron differentiation and plasticity [tissue transglutaminase 2 (tTG) and brain-derived neurotrophic factor (BDNF)] in a hippocampal cell line, HT22. We applied RA and/or dexamethasone (Dex) as activators of the pathways and investigated mRNA and protein expression of their receptors and of tTG and BDNF as well as tTG activity and BDNF secretion. Our results confirm the involvement of RA- and GC-dependent pathways and their interaction in our neuronal cell model. First, both pathways regulate the transcription and expression of own and reciprocal receptors: RA and Dex increased RAR and decreased GR expressions. Second, Dex reduces the expression of tTG when associated with RA despite stimulating its expression when used alone. Importantly, when they are combined, RA counteracts the deleterious effect of glucocorticoids on BDNF regulation and thus may improve neuronal plasticity under stress conditions. In conclusion, GC and RA both interact through regulations of the two receptors, RAR and GR. Furthermore, they both act, synergistically or oppositely, on other target proteins critical for neuronal plasticity, tTG and BDNF.

Objectif Avec des sensibilites et specificites equivalentes pour le diagnostic des pheochromocytomes, le dosage des metanephrines plasmatiques presente des avantages logistiques alors que le prelevement urinaire represente l’accumulation de leurs filtrations sur 24 h. Nous avons etudie retrospectivement les concentrations de normetanephrine plasmatiques et urinaires (NMAp/NMAu) et de metanephrine (MAp/MAu) contemporaines (intervalle ≤ 3j) afin d’etudier la concordance des resultats. Methodes Pour chaque couple NMA/MA urinaires ou plasmatiques, nous avons calcule une probabilite d’existence d’un pheochromocytome en utilisant les coefficients de regression logistique decrits precedemment (Brossaud, 2013). Nous avons considere comme discordance significative les probabilites se situant, pour urine et plasma, de part et d’autre d’un seuil arbitraire de 5 %. Resultats Les dosages plasmatiques (N = 3229) et urinaires (N = 2156) ont ete etudies (HPLC, detection electrochimique, 1381F/1221 M, âge 52 [0,1–90]ans, mediane [min-max]) ; 1004 dosages NMA/MA plasmatique et urinaires etaient synchrones. Leurs concentrations etaient : NMAp & MAp 525 [189–21 693] & 250 [200–12 423]pmol/L (references [250–1040] & [250–430]pmol/L), NMAu & MAu 1663 [196–72 846] & 488 [26–86 491]nmol/j (references [510–4030] & [150–1020]nmol/j). Trente-trois paires urine/plasma, soit 3 %, etaient discordantes (31 patients). Faux negatifs : chez 2 patients les dosages plasmatiques predisaient une probabilite Discussion Des divergences entre concentrations de metanephrines urinaires et plasmatiques sont rares. Cette etude ne plaide pas en faveur de doubles prelevements plasmatique et urinaire.

Objectif Avec de hautes sensibilites et specificites cliniques pour le diagnostic des pheochromocytomes, le dosage des metanephrines plasmatiques presente des avantages logistiques par rapport aux dosages des metanephrines urinaires des 24 h. La variabilite des concentrations ponctuelles, pour un meme individu, est toutefois un element a considerer vis-a-vis d’un seuil decisionnel. Methodes Nous avons etudie retrospectivement les resultats de dosages plasmatiques de normetanephrine (NMAp) et de metanephrine (MAp) prescrits, au CHU de Bordeaux, a intervalle bref (≤ 3j) afin d’evaluer leur concordance. Resultats Entre 2012–2015, 3141 dosages ont ete realises (HPLC, detection electrochimique) chez 2065 sujets (1125F/640 M, 52 [0,1 − 90] ans, mediane [min-max]). Les valeurs de NMAp et MAp etaient 514 [189–38 719] & 250 [200–56 790] pmol/L (valeurs de ref [250–1040] & [250–430] pmol/L). Ont ete retrouvees, 464 paires de dosages a intervalle bref (30 % des dosages, 81 % des paires provenait d’endocrinologie, 13 % de cardiologie). La difference des paires etait : NMAp 151 [0–11 759] & MAp 0 [0–4571] pmol/L. Le coefficient de concordance entre paires etait 0,995 pour NMAp et pour MAp. Nous avons recherche les paires de dosages discordantes par rapport a la valeur de reference haute (VRH) : 1,5*VRH. 4 paires etaient discordantes sur la NMAp, 5 sur la MAp soit Discussion La grande concordance entre les concentrations de NMAp et MAp retrouvees dans des prelevements rapproches de moins de 3j plaide pour limiter les prelevements repetes aux cas ambigus.

Objectif Decrire l’activite de l’axe corticotrope (AC) et le niveau d’anxiete des enfants diabetiques de type 1 en les comparant a des enfants temoins. Patients et methodes Des enfants diabetiques (DT1) et des fratries d’enfants diabetiques (CT) prepuberes ont ete inclus. Le questionnaire State-Trait Anxiety Inventory (STAI) a ete propose a l’inclusion. Les metabolites des glucocorticoides/creatinine (GCs/creat) dans les urines de la nuit et le cortisol salivaire au reveil ont ete mesures a domicile pendant 5 jours consecutifs sans episode connu d’hypoglycemie nocturne. Les resultats correspondent a la moyenne des dosages sur 5 jours. Resultats Quarante-neuf DT1 (âge moyen 9,0 ± 1,7 ans) et 26 Ct (9,3 ± 1,4 ans) ont ete inclus. Les scores STAI n’etaient pas differents entre DT1 et Ct. La somme des GCs/creat etait diminuee chez les DT1 vs Ct (552 ± 170 vs 673 ± 170 μg/mmol, p Conclusion Il existe des modifications subtiles dans l’activite de l’AC des enfants DT1, particulierement lors de la synthese nocturnes des GCs et independantes d’episodes connus d’hypoglycemies.

Objectif Les fonctions du recepteur des glucocorticoides (GR) peuvent etre modifiees de maniere importante par phosphorylation grâce a des kinases cytoplasmiques. L’acide retinoique (AR), metabolite de la vitamine A, est capable de moduler l’activite de ces kinases. Nous avions precedemment montre que l’AR module les effets des glucocorticoides sur les neurones. L’objectif etait ici de chercher a savoir si l’action de l’AR passait par une modification de la phosphorylation du GR. Methodes Des cellules de lignee cellulaire hippocampique HT22 ont ete cultivees 4j, avec ou sans dexamethasone (Dex) ou AR (10 −6 M). Des western-blots ont ete realises : GR phosphorylee (pGR pSer220GR), cyclin-dependent kinase 5 (CDK5), ses co-facteurs p35 et p25, brain derived neurotrophic factor (BDNF), la tissue transglutaminase (tTG) ainsi que des transfections de l’element de reponse du GR (GRE) couple au gene de la luciferase. Ces elements ont ete testes avec ou sans inhibiteur de CDK5 (roscovitine) ou siRNA de CDK5. Resultats L’AR augmente l’expression nucleaire de pGR par rapport au traitement par Dex uniquement. Le traitement par l’AR n’a pas d’effet sur l’expression de CDK5 mais augmente l’expression du ratio p35/p25. La roscovitine et le siRNA suppriment l’augmentation de l’expression nucleaire de pSer220GR par le RA. Enfin, la roscovitine diminue l’activite du GR apres transfection avec le GRE-luciferase ainsi que l’expression de 2 proteines cibles, la tTG et le BDNF. Conclusion Cette etude montre que l’AR module la voie des glucocorticoides par augmentation de la phosphorylation du GR en position Ser220 via la modulation de l’activite CDK5.

International audience

International audience; Background: After modification of our routine cortisol assay, we questioned the reference ranges for basal and stimulated cortisol plasma concentration in children. Methods: We retrospectively addressed the relevance of using the manufacturer's normal reference range for basal cortisol and investigated its response to glucagon-betaxolol testing. Results: Basal morning cortisol was 260 (98-604) nmol/L [manufacturer's normal range (185-624) nmol/L: 26% subjects had "low" basal cortisol]. Upon testing cortisol increased to 502 (117-856) nmol/L. If a recently described 100% specificity threshold (403 nmol/L) is used it would amount to 31% adrenal insufficient children in apparently unaffected children. Basal and stimulated cortisol obtained with our prior radioimmunoassay (RIA) in a sub-group of subjects were lower: 411 (187-1061) and 770 (329-1542) nmol/L. Using the 403 nmol/L threshold with the radioimmunoassay would result in only 5% adrenal insufficient children. Conclusions : This shows again that laboratories have to advertise the need to establish reference values for given populations, both for basal or stimulated hormone levels. Failure to apply this rule will elicit false-positive and more critically, false-negative results.

International audience; BACKGROUND: Erythropoietin (EPO) is an oxygenregulated hormone promoting the differentiation of erythroid progenitor cells. Apart fromhypoxia, few data is available about release by secretagogues including hormones. AIM: To investigate EPO serum concentration in subjects with endocrine diseases. MATERIAL AND METHODS: A retrospective study evaluating serumEPO concentrations in serumleftovers fromsubjects with various endocrine disorders. RESULTS: EPO is not noticeably influenced by thyroid hormone or cortisol concentrations and the relationship with hemoglobin concentration is preserved. In acromegalic patients, the latter is lost but EPO is neither statistically influenced by GH/IGF-I. This may reflect a dual action of GH and/or IGF-I on erythroid progenitors proliferation as well as on EPO synthesis. CONCLUSION: EPO is not noticeably modified by endocrine disorders although GH and or IGF-I may alter EPO relationship with blood hemoglobin concentration.

International audience

Primary hyperaldosteronism diagnosis is helped by assaying renin concentrations either by immunodetection (active renin) or by enzymatic reaction followed by angiotensin I immunoassay (plasma renin activity). We investigated the impact of pre-analytical and analytical conditions on a PRA assay.PRA was assayed using a commercial kit. Firstly, a retrospective analysis of PRA results from 1yr was performed. Secondly, the impact of pre-analytical temperature conveyance/storage of samples was tested (4 and 25 degrees C). Thirdly, 2 durations of enzymatic reaction 1.5 and 18h were tested.Retrospectively, 9.5% samples displayed elevated "blanks": calculating PRA with or without "blank" subtraction elicited a difference of10% in 1/3 samples. There was a significant decrease of PRA values in samples left at 25 degrees C vs 4 degrees C (50% "blank" increase). Finally, the longer the enzymatic reaction (1.5 vs 18h), the higher the production of angiotensin I and the better the PRA assay sensitivity.We propose a 2 step procedure: a brief 1.5h enzymatic reaction time followed by a prolonged reaction (18h) only if PRA is0.2ng mL(-1) h(-1). We would recommend performing "blank" samples for the brief enzymatic reaction time. Finally, conveyance/storage temperature does modify PRA results albeit in our hands this had little clinical impact.

Background Different pathophysiological situations such as congenital adrenal hyperplasia, adrenocortical carcinoma, metyrapone treatment, etc. elicit specificity problems with serum cortisol assay. Methods We assayed cortisol using 2 kits and performed cross reaction studies as well as multiple regression analysis using 2 other steroids: 11-desoxycortisol and 17-OH progesterone. Results Analysis showed the existence of an analytical bias. Importantly, significantly different biases were demonstrated in newborns or patients taking metyrapone. Multiple regression analysis and cross reaction studies showed that 11-desoxycortisol level significantly influenced cortisol determination. Moreover, despite using the normal ranges provided by manufacturers discrepant results occurred such as 17% discordance in the diagnosis of hypocorticism in infants. Conclusion We wish to raise awareness about the consequences of the (lack of) specificity of cortisol assays with regard to the evaluation of hypocorticism in infants or when “unusual” steroids may be increased.