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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., (© 2015 John Wiley & Sons Ltd.)

Objective: The performance of late-night salivary cortisol (LNSC) to accurately screen for postoperative recurrence of Cushing's disease (CD) at an early stage is unknown. The aim of this study was to compare the accuracy of multiple sampling strategies to suggest the optimal number of LNSC samples needed for diagnosing post-surgical recurrences of CD at an early stage., Design: Retrospective analysis in a single centre., Patients and Measurements: Thirty-six patients in surgical remission of CD had successive measurements of LNSC, defined as 'sequences', using a locally modified RIA assay as part of long-term follow-up (69·2 ± 10·6 months). Patients underwent an extensive biochemical evaluation within 3 months before or after a sequence of saliva sampling and were classified as being in remission or in early-stage recurrence. The accuracy of three diagnostic strategies combining two, three or four LNSC results from a sequence was estimated using areas under the ROC curves (AUC), sensitivity, specificity and predictive values., Results: Forty-four sequences of LNSC measurements were available. Fifty-two percent of sequences were performed during early-stage recurrence. The intrasequence variability of LNSC was higher during recurrence than during remission (medians of SDs: 2·1 vs 0·5 nm; P < 0·0001). AUCs from ROC curves ranged from 0·93 to 0·96 depending on the strategy. For 90% sensitivities, the best specificities (92·9% and 90·9%) were achieved by strategies taking into account three or four measurements summarized either by their mean or their maximum value., Conclusions: Increase in LNSC concentration is an early abnormality during post-surgical recurrence of CD. However, due to a major within-patient variability of LNSC from 1 day to another, a screening strategy using three or four samples collected on successive days may be recommended to detect early-stage recurrence of CD with a high accuracy., (© 2014 John Wiley & Sons Ltd.)

Objectives: Obesity with abdominal body fat distribution (A-BFD) and hypothalamic-pituitary-adrenal (HPA) axis activity are somehow linked, but the exact interactions still need clarification. Obese subjects display normal circulating plasma cortisol concentrations with normal circadian rhythms. However, when the HPA axis is pharmacologically challenged, body fat distribution matters and then A-BFD obese women differ from those with subcutaneous body fat distribution (P-BFD). We hypothesized that lower dose provocative and suppressive tests than those used to diagnose hypercortisolism of tumour origin or adrenal insufficiency would shed some light on the characteristics of the HPA axis activity in relation with body fat distribution., Patients and Methods: Fifty premenopausal obese women were grouped according to their body fat mass distribution. Their plasma cortisol responses to (i) two low doses of dexamethasone (0.25 and 0.5 mg) with (ii) low dose of the ACTH analogue tetracosactrin (1 microg) were assessed. Salivary cortisol was also determined during the ACTH test., Results: A-BFD differed from P-BFD women in terms of HPA axis responsiveness. They had comparatively: (i) increased nocturnal cortisol excretion (9.38 +/- 2.2 vs. 6.82 +/- 0.91 nmol/micromol creatinine, A-BFD vs. P-BFD, respectively, P = 0.03); (ii) increased salivary cortisol response to ACTH stimulation (1 microg) [salivary cortisol peak: 33.4 (14.1-129) vs. 28.5 (13.2-42.8) nmol/l; salivary AUC: 825 (235-44738) vs. 537 (69-1420) nmol/min/l; A-BFD vs. P-BFD, P = 0.04 for both]; and (iii) increased pituitary sensitivity to dexamethasone testing [postdexamethasone (0.25 mg) plasma cortisol levels: 163 (26-472) vs. 318 (26-652) nmol/l and postdexamethasone (0.5 mg) plasma cortisol levels: 26 (26-79) vs. 33 (26-402) nmol/l; A-BFD vs. P-BFD, P = 0.01 for both)., Conclusions: These data demonstrate differences in the HPA axis activity and sensitivity to glucocorticoids between obese women differing in their body fat distribution, with both enhanced negative and positive feedback in those with abdominal obesity. Several mechanisms may explain these differences: central vs. peripheral hypotheses. Thus, abdominal obesity does not appear to be linked solely to one pathophysiological hypothesis.

Objective: The dexamethasone-CRH test (combination of dexamethasone-induced suppression of HPA axis function and subsequent stimulation with oCRH) (Dex-CRH test) has been proposed to fully distinguish ACTH-dependent Cushing's disease (CD) from pseudo-Cushing's states (PCS), i.e. tumoural vs. functional hypercortisolism. A plasma cortisol concentration greater than 38 nmol/l 15 min after CRH injection has been demonstrated to identify all cases of CD and to exclude all cases of PCS. Although obviously not a PCS from a clinical point of view, anorexia nervosa (AN) is associated with CRH-driven hyperactivity of the HPA axis. This study reports the response of AN patients, a model of functional biological hypercortisolism, to the Dex-CRH test., Patients and Methods: Nineteen women affected with anorexia nervosa and 6 healthy sex-matched controls were studied., Results: Three of 19 AN patients had an abnormal 24-h urinary free cortisol excretion (UFC), whereas 1 of 19 AN had increased overnight UFC. AN subjects had inadequately suppressed plasma cortisol after low-dose dexamethasone suppression test (LDDST) (cortisol 192.8 +/- 63.4 vs. < 27 nmol/l, AN vs. controls, respectively). Seven of 19 AN patients had plasma cortisol levels above 50 nmol/l after LDDST. None of the AN patients had CRH-induced increases in plasma ACTH or cortisol (basal cortisol 192. 8 +/- 63.4 and peak cortisol 181.7 +/- 59.9 nmol/l). Despite unresponsivenessto CRH and because of the lack of suppression after dexamethasone, using the single plasma cortisol threshold value of 38 nmol/l obtained at 15 min during the Dex-CRH test would have been misclassified in half of our AN population (9 of 19)., Conclusion: Since anorexia nervosa represents a model of functional hypercortisolism that shares similar pathophysiological mechanisms to the other causes of pseudo-Cushing's states, we suggest testing all causes of pseudo-Cushing's states using the dexamethasone-CRH approach to (i) describe the actual responses of clinically relevant pseudo-Cushing's states and (ii) to improve our knowledge of the pathophysiological discrepancies between the various causes of pseudo-Cushing's states. Lastly, the evaluation of dexamethasone metabolism (absorption, volume of distribution, clearance) may help to gain more insight into the diagnostic value of the dexamethasone-CRH test.

Objective: Leptin concentrations in humans are known to decrease in response to fasting. The aim of this work was to investigate whether leptin levels might also be modified by exercise-induced negative energy balance., Subjects: Eight male runners reported in the morning from 0800 to 1200 h for (i) one resting session (sitting) and (ii) one exercise-and-rest session (2 h run and 2 h rest)., Measurements: Plasma leptin, free fatty acids (FFA), glycerol, cortisol and salivary cortisol were assayed in both sessions at 1200 h., Results: After exercise-and-rest the leptin concentrations were lower than after rest (1.7 +/- 0.1 vs 2.5 +/- 0.2 micrograms/l, P < 0.05), i.e. a mean decrease of 30.3 +/- 4.5% (range 9.5-45.8). Plasma FFA, glycerol and cortisol concentrations increased: FFA 0.78 +/- 0.08 vs 0.18 +/- 0.04 mmol/l, glycerol 0.13 +/- 0.01 vs 0.04 +/- 0.01 mmol/l, and cortisol 428 +/- 36 vs 279 +/- 27 nmol/l. A negative correlation was found between plasma FFA and leptin levels (r = -0.5, P < 0.05) and between plasma glycerol and leptin levels (r = -0.05, P < 0.05). No correlation was found between leptin and cortisol levels., Conclusions: In normal subjects with low body fat, a strenuous exercise-and-rest lowers leptin levels by a mean of 30%. A role of lipolysis possibly via increased plasma free fatty acids and glycerol levels is suggested. Cortisol does not seem to be involved.

Objective: The present study was conducted in order to describe human hypothalamo-pituitary adrenal (HPA) axis adaptation in a model of repeated physical stress (endurance training) that causes a moderate increase in cortisol levels., Subjects: We performed the same stimulation tests (adrenal stimulation with ACTH or pituitary stimulation with combined CRH/LVP) in a population of 8 endurance-trained athletes in two distinct situations: resting (baseline cortisol values) and 2 h after the end of strenuous exercise (increased cortisol values) to evaluate the HPA axis sensitivity to endogenous sustained increases in cortisol concentrations., Measurements: During these tests, saliva and plasma cortisol (Fs and Fp, respectively) were assessed and compared., Result: Cortisol values in both plasma and saliva at the end of 2 h of exercise were significantly higher than in rested controls: Fs 11.5 +/- 1.3 vs 6.5 +/- 0.8 nmol.l-1 and Fp 428 +/- 36 vs 279 +/- 27 nmol.l-1 (post exercise vs post rest sessions, respectively, P < 0.001 for both). After either hormone test (CRH/LVP or ACTH), cortisol levels in plasma and saliva increased similarly when rest was compared to post exercise. Saliva variations (delta %) under exogenous hormone stimulation were dramatically greater than plasma variations. For example, under ACTH stimulation, the relative increments in cortisol were on control day: delta Fs 980 +/- 139 vs delta Fp 218 +/- 43% (saliva vs plasma, respectively, P < 0.05) and on exercise day: delta Fs 605 +/- 89 vs delta Fp 102 +/- 14% (saliva vs plasma, respectively, P < 0.05)., Conclusions: In endurance-trained athletes, displaying a moderate but sustained endogenous cortisol increase: (1) ACTH responses following pituitary stimulation are not blunted, (2) cortisol responses following maximal adrenal stimulation are not blunted. Our results favour the hypothesis of a decreased pituitary sensitivity to cortisol negative feedback whereas the hypothesis of a major decreased adrenal sensitivity to ACTH was discarded. The greater ability of saliva assays to detect a cortisol increase strongly supports its use in the study of HPA physiology, whether under basal or dynamic conditions.

Objective: The collection of urine over 24 h to measure free cortisol (UFC) is used to diagnose Cushing's syndrome. However, a complete collection of urine is not easy to achieve and the sampling is frequently inaccurate, so a 24 h collection for the determination of UFC excretion is used as a confirmatory rather than a screening test for Cushing's syndrome. Our objective was to evaluate a more convenient urine collection for screening patients suspected of Cushing's syndrome., Design: We have studied the possibility of using night-time (from 2000 h to 0800 h) UFC excretion as a screening test for Cushing's syndrome ('overnight UFC')., Patients: Thirty patients with proven Cushing's syndrome were studied before treatment (21 cases of Cushing's disease, two cases of ectopic ACTH secretion, seven of adrenal adenoma). The results were compared to those from 150 control obese subjects., Measurements: Overnight UFC, and creatininuria (UC), were evaluated at least once in the patients and in all subjects. The 24 h-UFC and UC were determined at least once in the patients and in 56 control subjects., Results: The overnight UFC/UC ratio clearly separated all but one patient of the two groups: 194 +/- 386 vs 5.7 +/- 3.4 nmol/mumol (P < 0.0005) (ranges 16.2-2024 vs 0.6-17.4, Cushing's syndrome vs controls, respectively). The only patient with Cushing's syndrome who had an overnight UFC/UC ratio that overlapped with that of controls suffered from renal failure. The 24 h UFC/UC ratio of the patients differed from the ratio of controls: 184 +/- 365 vs 8.4 +/- 4.1 nmol/mumol (P < 0.0005) (ranges 14-1639 vs 1.5-21.2, respectively). Four patients with Cushing's syndrome had 24 h UFC/UC ratios lower than the upper control ratio., Conclusion: The overnight urinary sampling is a simple procedure compared to the 24 h urine collection performed in out-patients. For a sensitivity set at 100%, the specificities were 97% for the overnight UFC/UC ratio and 87% for the 24 h-UFC/UC ratio. This therefore appears to provide a good screening method for Cushing's syndrome.

Acromegaly favours the development of extrapituitary neoplasms presumably promoted through increased GH and IGF-I levels. We describe here two patients which acromegaly and benign and malignant melanocytic tumours of the eye choroid. The first patient had a 15 year history of progressive acromegaly despite trans-sphenoidal surgery, radiation and bromocriptine therapy when she presented with melanoma of the eye and a contralateral benign melanocytic tumour. The second patient was referred with typical acromegaly. The initial fundal examination revealed a right sided benign melanocytic tumour of the choroid. Subsequent follow up of the naevi has not shown any change in the tumours after one and three years respectively. The occurrence of melanoma has not been reported in acromegaly. Increased growth of benign naevi might be expected as GH and IGF-I receptors have been described on melanocytes. As naevi may degenerate into melanomas in normal subjects and radiation therapy could trigger their transformation, we suggest that prior to irradiation, acromegalic subjects should undergo a careful ocular examination in order to assess the presence of ocular naevi.

We report the cases of four patients with occult, and one patient with overt, ectopic ACTH syndrome. Cushing's syndrome was cured by removal of the ACTH secreting tumour, op'DDD, ketoconazole or bilateral adrenalectomy. Six to 14 months after remission of hypercortisolism, follow-up computed tomography or magnetic resonance imaging of the thorax revealed in all five patients an anterior mediastinal mass 3-5 cm in length that suggested a thymic carcinoma. Exploratory thoracotomy was performed in four cases and allowed the removal of an enlarged, but normal, thymus. Histological examination revealed no tumour but showed benign thymic hyperplasia with negative immunostaining for ACTH. Thymic enlargement spontaneously disappeared 11 months after its demonstration in the patient who did not undergo thoracotomy. The mechanism of this abnormality is thought to be thymic depletion resulting from high plasma cortisol concentrations followed by 'rebound' thymic hyperplasia when cortisol levels fall. Physicians involved in the management of patients with Cushing's syndrome must be aware of this entity in order to avert a diagnostic thoracotomy.

Ketoconazole, an imidazole derivative which inhibits adrenal steroidogenesis, has been used with success for the metabolic control of Cushing's disease. Few data are available about the use of ketoconazole in the management of the ectopic ACTH syndrome. We have used ketoconazole in eight patients: four patients with Cushing's disease, two patients with overt and two with occult ectopic ACTH syndrome. Among patients with Cushing's disease, reversible hypoadrenalism occurred once. All had full clinical and biochemical regression of the disease for more than 6 months with 400-1200 mg ketoconazole per day. Patients with ectopic ACTH syndrome received 1200 mg ketoconazole per day for at least 2 months. Partial biochemical regression was observed in two and a secondary escape to adrenal blockade in two others. These findings further indicate that ketoconazole is a valuable tool for the metabolic control of Cushing's disease. On the contrary, in ectopic ACTH syndrome, this aim can be impossible to reach with ketoconazole although the reasons for its ineffectiveness remain to be determined.