LC-HRMS and GC-MS Profiling of Urine Free Cortisol, Cortisone, 6Β-, and 18-Hydroxycortisol for the Evaluation of Glucocorticoid and Mineralocorticoid Disorders.

INTRODUCTION: Urine free cortisol measurements are routinely performed to evaluate hypercortisolism. Despite their analytical inaccuracy, immunoassay-based methods are frequently used. Advances in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) facilitate the incorporation of powerful diagnostic tools into clinical laboratories. In addition to its high analytical specificity and simultaneous analysis of different metabolites, accurate mass measurement allows for untargeted compound identification, which may help to identify clinically relevant metabolites or drugs. METHODS: The present study aimed to validate a simple routine LC-HRMS method to quantify cortisol, cortisone, 6ß-hydroxycortisol, and 18-hydroxycortisol simultaneously in human urine. Additionally, the study also validated a GC-MS method for the same steroids, evaluated their cross-reactivity with commercial cortisol immunoassays, and quantified the 24 h urine excretion in patients under clinical suspicion or follow-up for hypercortisolism. RESULTS: The LC-HRMS method involved liquid-liquid extraction using dichloromethane, micro-LC for chromatographic separation and detection using the accurate masses of the steroids, and simultaneous high-resolution full scan acquisition. The method presented acceptable linearity, precision, and accuracy. Significant interference from 6ß-hydroxycortisol and cortisone was demonstrated in the cortisol immunoassays, which impacted their reliability in the follow-up of patients with hypercortisolism and significant changes in these cortisol metabolites (i.e., due to drug-induced changes in CYP3A4 activity). CONCLUSION: A rapid and accurate routine LC-HRMS method was validated, which is useful for the evaluation of hypercortisolism and other disorders of glucocorticoid and mineralocorticoid metabolism.

Urinary steroid profiling using liquid chromatography-tandem mass spectrometry for the diagnosis of canine Cushing's syndrome.

Serum cortisol measurements by chemiluminescence enzyme immunoassay (CLEIA) are widely used to diagnose hypercortisolism (HC) or Cushing's syndrome in dogs. However, they are associated with problems such as the need for multiple blood collections under stressful conditions or cross-reactivity between hormones. Therefore, a less invasive and more accurate diagnostic method is required. This study aimed to develop a urinary steroid profile analysis method using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and to evaluate its clinical usefulness. Sixty-five healthy dogs and 38 dogs with suspected HC were included in the study. Using LC/MS/MS, the levels of 11 steroid hormones in the urine were determined. We established the upper limit of the reference interval for each urinary steroid-to-creatinine ratio and evaluated their diagnostic performances. The levels of the five steroid hormones were significantly higher in the 14 dogs with HC than in the 24 dogs with mimicking HC and 65 healthy dogs. The urinary corticosterone-to-creatinine ratio showed the highest diagnostic accuracy (area under the curve, 0.96). A significant correlation was seen between urinary cortisol concentrations measured by LC/MS/MS and CLEIA (rs = 0.88, P <0.001), although the CLEIA measurements were significantly higher than the LC/MS/MS measurements (P <0.001). LC/MS/MS-based urinary steroid profiles are a promising tool for diagnosing canine HC.

Personalized Noninvasive Diagnostic Algorithms Based on Urinary Free Cortisol in ACTH-dependant Cushing's Syndrome.

CONTEXT: Current guidelines for distinguishing Cushing's disease (CD) from ectopic ACTH secretion (EAS) are questionable, as they use pituitary magnetic resonance imaging (MRI) as first-line investigation for all patients. CRH testing is no longer available, and they suggest performing inferior petrosal sinus sampling (BIPPS), an invasive and rarely available investigation, in many patients. OBJECTIVE: To establish noninvasive personalized diagnostic strategies based on the probability of EAS estimated from simple baseline parameters. DESIGN: Retrospective study. SETTING: University hospitals. PATIENTS: Two hundred forty-seven CD and 36 EAS patients evaluated between 2001 and 2023 in 2 French hospitals. A single-center cohort of 105 Belgian patients served as external validation. RESULTS: Twenty-four-hour urinary free cortisol (UFC) had the highest area under the receiver operating characteristic curve for discrimination of CD from EAS (.96 [95% confidence interval (CI), .92-.99] in the primary study and .99 [95% CI, .98-1.00] in the validation cohort). The addition of clinical, imaging, and biochemical parameters did not improve EAS prediction over UFC alone, with only BIPPS showing a modest improvement (C-statistic index .99 [95% CI, .97-1.00]). Three groups were defined based on baseline UFC: < 3 (group 1), 3-10 (group 2), and > 10 × the upper limit of normal (group 3), and they were associated with 0%, 6.1%, and 66.7% prevalence of EAS, respectively. Diagnostic approaches performed in our cohort support the use of pituitary MRI alone in group 1, MRI first followed by neck-to-pelvis computed tomography scan (npCT) when negative in group 2, and npCT first followed by pituitary MRI when negative in group 3. When not combined with the CRH test, the desmopressin test has limited diagnostic value. CONCLUSION: UFC accurately predicts EAS and can serve to define personalized and noninvasive diagnostic algorithms.

A dilute and shoot method for urinary free cortisol analysis by LC-MS/MS.

24-hour urinary free cortisol (UFC) is considered as the first-line test for screening and diagnosis of Cushing's syndrome. Although 24-hour UFC assay has been extensively studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS), an accurate assay coupled with a reliable sample preparation procedure and a method-specific reference interval would be very important for reasonable diagnosis. In this study, a simple dilute and shoot method has been proposed for UFC determination by LC-MS/MS. Namely, 50 µL of urine sample was mixed with 200 µL of a 50 % methanol/water solution containing the internal standard cortisol-13C3. The mixture was centrifuged and the supernatant was used for direct analysis by LC-MS/MS. This method was validated with wide linear range from 0.625 to 500 ng/ml with coefficients of variation (CVs) ≤ 3.64 %, excellent precision (intra-day CVs ≤ 5.70 % and inter-day CVs ≤ 5.33 %) and good recovery in the range of 93.3-109 %. The preservatives were further evaluated for urine storage. It was recommended that no preservatives could be used in collection of 24-hour urine for good detecting peaks. The investigation of reference interval and diagnostic performance finally confirmed the potential usage of this LC-MS/MS assay in routing clinical testing.

Validation of a LC-MS/MS method for establishing reference intervals and assessing diagnostic performance of urinary free cortisol and cortisone.

BACKGROUND: 24-h urinary free cortisol (UFF) is recommended for screening of Cushing's syndrome (CS), a rare disease characterized by apparent cortisol and cortisone excess. We aimed to validate a simple LC-MS/MS method for accurate measurement of UFF and urinary free cortisone (UFE), establishment of reference ranges, and evaluation of performance for CS diagnosis. METHODS: Urine samples were processed using solid-phase extraction cartridges, followed by elution with methanol and acetonitrile. Analysis was performed via tandem mass spectrometry, utilizing multiple reaction monitoring and electrospray ionization source in positive ion mode. RESULTS: The assay displayed excellent linearity (r > 0.99) in the range of 0.05-100 ng/mL for cortisol and 0.25-500 ng/mL for cortisone, with lower limits of quantification (LLOQ) at 0.05 ng/mL for cortisol and 0.25 ng/mL for cortisone. The obtained results for intra-day and inter-day imprecision for both analytes were within the acceptable range of less than 10 %. The trueness values for both compounds were also within the acceptable limit of 15 %. No significant matrix effects or carry over observed in our method. The reference intervals of UFF, UFE and UFF:UFE ratio were 7.01-45.66 µg/24-h, 27.97-139.21 µg/24-h and 0.17-0.56, respectively. UFF > 56.75 µg/24-h showed 100 % specificity and 100 % sensitivity for CS diagnosis, which was superior to UFF:UFE ratio. CONCLUSIONS: We developed and validated a sensitive LC-MS/MS method to detect UFF and UFE. Our data indicate that UFF measured by the current LC-MS/MS assay exhibited high diagnostic performance for CS.

Delineating endogenous Cushing's syndrome by GC-MS urinary steroid metabotyping.

BACKGROUND: Diagnosing Cushing's syndrome (CS) is highly complex. As the diagnostic potential of urinary steroid metabolome analysis by gas chromatography-mass spectrometry (GC-MS) in combination with systems biology has not yet been fully exploited, we studied a large cohort of patients with CS. METHODS: We quantified daily urinary excretion rates of 36 steroid hormone metabolites. Applying cluster analysis, we investigated a control group and 168 patients: 44 with Cushing's disease (CD) (70% female), 18 with unilateral cortisol-producing adrenal adenoma (83% female), 13 with primary bilateral macronodular adrenal hyperplasia (PBMAH) (77% female), and 93 ruled-out CS (73% female). FINDINGS: Cluster-Analysis delineated five urinary steroid metabotypes in CS. Metabotypes 1, 2 and 3 revealing average levels of cortisol and adrenal androgen metabolites included patients with exclusion of CS or and healthy controls. Metabotype 4 reflecting moderately elevated cortisol metabolites but decreased DHEA metabolites characterized the patients with unilateral adrenal CS and PBMAH. Metabotype 5 showing strong increases both in cortisol and DHEA metabolites, as well as overloaded enzymes of cortisol inactivation, was characteristic of CD patients. 11-oxygenated androgens were elevated in all patients with CS. The biomarkers THS, F, THF/THE, and (An + Et)/(11ß-OH-An + 11ß-OH-Et) correctly classified 97% of patients with CS and 95% of those without CS. An inverse relationship between 11-deoxygenated and 11-oxygenated androgens was typical for the ACTH independent (adrenal) forms of CS with an accuracy of 95%. INTERPRETATION: GC-MS based urinary steroid metabotyping allows excellent identification of patients with endogenous CS and differentiation of its subtypes. FUNDING: The study was funded by the Else Kröner-Fresenius-Stiftung and the Eva-Luise-und-Horst-Köhler-Stiftung.

Overnight 1 mg dexamethasone suppression test and 24 h urine free cortisol-accuracy and pitfalls when screening for Cushing's syndrome.

Diagnosis of Cushing's syndrome (CS) is often delayed due to variable clinical features and its rarity. Simple and accurate screening tests are required to enhance screening for hypercortisolism. Both overnight 1 mg dexamethasone suppression test (DST) and urinary free cortisol (UFC) demonstrate high sensitivity and specificity for the diagnosis of CS. However, each test has its own distinctive features, making it preferable in specific clinical conditions. This review will discuss the pitfalls for each of those tests.

High Prevalence of Adrenal Remnant Tissue in Patients Undergoing Bilateral Adrenalectomy for Cushing's Disease.

Bilateral adrenalectomy (BLA) is a treatment option for patients with Cushing's Disease (CD) if transsphenoidal pituitary surgery fails or is not a therapeutic option. For most patients, BLA eliminates endogenous glucocorticoid and mineralocorticoid production, but for a small number of patients, endogenous secretion of adrenal hormones from adrenal tissue continues or recurs, leading to signs and symptoms of hypercortisolism. If adrenal tissue is confined to the adrenal bed, it is considered adrenal remnant tissue, while if it is outside the adrenal bed, it is considered adrenal rest tissue. We retrospectively evaluated morning serum cortisol, nighttime serum cortisol, nighttime salivary cortisol, and 24-h urine free cortisol on at least three occasions in 10 patients suspected of having endogenous cortisol production. Imaging of adrenal remnant tissue was also reviewed. Ten of 51 patients who underwent BLA during this time period had adrenal remnant/rest tissue marked by detectable endogenous glucocorticoid production; 9 of the 10 patients had signs and symptoms of hypercortisolism. Localization and treatment proved difficult. We conclude that the incidence of adrenal remnant/rest tissue in those undergoing BLA following unsuccessful pituitary surgery was 12% although there may have been a selection bias affecting this prevalence. The first indication of remnant tissue occurrence is a reduction in glucocorticoid replacement with symptoms of hypercortisolism. If this occurs, endogenous cortisol production should be tested for by cortisol measurements using a highly specific cortisol assay while the patient is taking dexamethasone or no glucocorticoid replacement. Endocrinologists need to monitor the development of both adrenal remnant tissue and Nelson's syndrome following BLA.

Diagnostic challenges and considerations of cyclical Cushing's syndrome in a 15-year-old female.

Cyclical Cushing's syndrome (CS) is a rare disorder in which cortisol secretion is cyclical and intermittent. This phenomenon makes for a challenging diagnosis, as patterns of cycling can vary widely among patients and as patients with cyclical CS do not exhibit unique clinical features compared to those without cycling. Current research suggests that cyclical CS may be present in approximately 15% of adult cases, with an even lower reported prevalence in the pediatric population. In this case study, we describe a 15-year-old female with obesity and hypertension who was then diagnosed with cyclical CS after we pursued additional screening tests of urine creatinine and 24-hour urine cortisol, dexamethasone suppression tests, bilateral inferior petrosal sinus sampling, as well as MRI. We discuss the vari-ous diagnostic modalities in the challenging diagnosis of cyclical CS as well as the importance and modalities of post-operative monitor-ing in this patient population. From this case study, we emphasize that when CS is suspected and initial screening tests are negative, clinicians should be aware of the cycling phenomenon of CS in order to consider performing additional screening tests.

Cortisol urinary metabolites in dogs with hypercortisolism, congestive heart failure, and healthy dogs: pilot investigation.

Nonadrenal diseases (NAD), including congestive heart failure (CHF), can affect the conversion of cortisone to cortisol favoring the production of cortisol's urinary downstream metabolites 5α/5ß-tetrahydrocortisol (THF) relative to tetrahydrocortisone (THE). We hypothesized that healthy dogs would have lower urinary levels of cortisol, cortisone, THF, and THE than dogs with hypercortisolism (HC) or CHF, and the latter would have higher urinary levels of THF and lower THE than dogs with HC. Four, 9, and 8 dogs with HC, CHF, and normal health, respectively, were included in a pilot prospective cross-sectional study. A single morning voided urine sample was analyzed for urinary cortisol metabolites by liquid chromatography-mass spectrometry. The percentages of conjugated urinary metabolites were significantly higher in dogs with CHF than in healthy dogs (p = 0.001), and not different in HC dogs (p = 0.07). Log-transformed urine cortisol metabolites-to-creatinine ratios in healthy dogs were significantly lower than the 2 other groups (p < 0.001). The urinary free THE:THF ratio was significantly higher (p < 0.001) than the urinary total and conjugated THE:THF ratios. Health status did not affect the total, conjugated, and free THE:THF ratios (p = 0.61). Additional studies are needed to investigate differences in cortisol metabolites between dogs with HC and NAD to accurately discriminate between the groups.

Unilateral Adrenalectomy for Primary Bilateral Macronodular Adrenal Hyperplasia: Analysis of 71 Cases.

OBJECTIVE: Primary bilateral macronodular adrenal hyperplasia (PBMAH) is characterized by benign bilateral enlarged adrenal masses, causing Cushing's syndrome (CS). The aim of the current article is to define the role of unilateral adrenalectomy (UA) in treating patients with CS related to PBMAH. METHODS: A PubMed database search was conducted to identify articles reporting UA to treat PBMAH. We also report cases of PBMAH from our medical center treated by UA. RESULTS: A total number of 71 cases of PBMAH (62 cases reported in the literature and 9 cases from our center) are presented. Most patients were women (73.2%) and most UA involved the left side (64.3%). In most cases, the resected gland was the larger one. Following UA, 94.4% of cases had remission of hypercortisolism. Recurrence rate of CS was 19.4% and hypoadrenalism occurred in 29.6%. After UA, when the size of the remained adrenal gland was equal or greater than 3.5 cm, CS persisted in 21.4% of cases, and recurrence occurred in 27.3% of cases (after 20±9.2 months). However, when the size of the remained gland was less than 3.5 cm, CS resolved in all cases and recurrence occurred in 21.2% of cases after a long period (65.6±52.1 months). High levels of urinary free cortisol (UFC) were not correlated with post-surgical CS recurrence or persistence. CONCLUSIONS: UA leads to beneficial outcomes in patients with CS related to PBMAH, also in cases with pre-surgical elevated UFC or contralateral large gland.

New insights to the potential mechanisms driving coronary flow reserve impairment in Cushing's syndrome: A pilot noninvasive study by transthoracic Doppler echocardiography.

BACKGROUND: The contribution of functional and/or structural remodeling to reduced coronary flow velocity reserve (CFVR), reflecting impaired coronary microcirculation in Cushing's syndrome (CS), has not been clearly elucidated. We aimed to identify the potential mechanisms of coronary microvascular impairment in CS. METHODS: We studied 15 CS patients (11 female, age 50 ±â€¯9 years) without clinical evidence of cardiovascular disease. Coronary flow velocity in the left anterior descending coronary artery was measured by transthoracic Doppler echocardiography, at rest, and during adenosine infusion. Average peak flow velocities, CFVR, and microvascular resistance in baseline (BMR) and hyperemic conditions (HMR) were assessed. CFVR ≤2.5 was considered a marker of microvascular disease (CMD). Diastolic function (E/e'), global longitudinal strain (GLS) and fractional pulse pressure (fPP), an index of arterial stiffness, were also assessed. RESULTS: CMD was present in 5 patients (33.3%). CMD was primarily driven by increased baseline peak flow velocity (29 ±â€¯12 versus 19.6 ±â€¯4.2 cm/s, p = .03) in the presence of decreased BMR (3.62 ±â€¯0.6 versus 5.46 ±â€¯1.4 mm Hg·s/cm, p = .03). Moreover, urinary cortisol and E/e' were higher (p = .001 and p = .001, respectively) and GLS was lower (p = .009) in patients with CMD. fPP was higher in patients with CMD (p = .01). Urinary cortisol correlated to CFVR (p = .008), E/e' (p < .0001) and GLS (p < .0001). fPP directly correlated to average peak flow velocities at rest (p = .01) and inversely to BMR (p = .03). CONCLUSIONS: Functional microvascular regulatory impairment seems to be the potential mechanism of CMD in CS. CMD seems to be related to decreased myocardial contractility and diastolic dysfunction associated with cortisol excess.

Computerized Analysis of Brain MRI Parameter Dynamics in Young Patients With Cushing Syndrome-A Case-Control Study.

BACKGROUND: Young patients with Cushing Syndrome (CS) may develop cognitive and behavioral alterations during disease course. METHODS: To investigate the effects of CS on the brain, we analyzed consecutive MRI scans of patients with (n = 29) versus without CS (n = 8). Multiple brain compartments were processed for total and gray/white matter (GM/WM) volumes and intensities, and cortical volume, thickness, and surface area. Dynamics (last/baseline scans ratio per parameter) were analyzed versus cortisol levels and CS status (persistent, resolved, and non-CS). RESULTS: Twenty-four-hour urinary free cortisol (24hUFC) measurements had inverse correlation with the intensity of subcortical GM structures and of the corpus callosum, and with the cerebral WM intensity. 24hUFC dynamics had negative correlation with volume dynamics of multiple cerebral and cerebellar structures. Patients with persistent CS had less of an increase in cortical thickness and WM intensity, and less of a decrease in WM volume compared with patients with resolution of CS. Patients with resolution of their CS had less of an increase in subcortical GM and cerebral WM volumes, but a greater increase in cortical thickness of frontal lobe versus controls. CONCLUSION: Changes in WM/GM consistency, intensity, and homogeneity in patients with CS may correlate with CS clinical consequences better than volume dynamics alone.

Gas Chromatography-Mass Spectrometry Analysis of Urinary Steroid Metabolomics for Detection of Early Signs of Adrenal Neoplasm Malignancy in Patients with Cushing's Syndrome.

The metabolomics of urinary steroids was studied by gas chromatography-mass spectrometry in 25 patients with Cushing's syndrome without malignant potential and in 12 patients with malignant potential of adrenal neoplasms (Weiss score 1-3). Patients with adrenocortical adenoma (N=24) constituted the control group. In patients with Cushing's syndrome and malignant potential, increased urinary excretion of 16-oxo-androstendiol, tetrahydro-11-deoxycortisol, and 16-hydroxypregnendiol, which had 100% specificity and sensitivity >90% for the diagnosis of malignant potential. Additionally, non-classical 5-ene-pregnenes (16-OHpregnenolone, 21-OH-pregnenolone, 3ß,16,20-pregnentriol, and 3ß,17,20-pregnentriol) were identified. The revealed changes in the metabolomics of steroids can be early signs of malignant potential in patients with Cushing's syndrome. In patients with malignant potential, three signs of reduced activity of 11ß-hydroxysteroid dehydrogenase type 2 were detected and in patients without malignant potential, one sign was found. In patients with and without malignant potential, three signs increased activity of 5ß-reductase were found.

Efficacy and safety of levoketoconazole in the treatment of endogenous Cushing's syndrome (SONICS): a phase 3, multicentre, open-label, single-arm trial.

BACKGROUND: Levoketoconazole is a ketoconazole stereoisomer in development for treatment of Cushing's syndrome and has not been assessed previously in a clinical trial in patients with Cushing's syndrome. We aimed to investigate the efficacy and safety of levoketoconazole in patients with endogenous Cushing's syndrome. METHODS: SONICS is a phase 3, multicentre, open-label, non-randomised, single-arm study in which we recruited adults (≥18 years) with confirmed Cushing's syndrome and a mean 24-h urinary free cortisol (mUFC) of at least 1·5 times the upper limit of normal from 60 hospital and community sites in 19 countries (15 countries in Europe, and Canada, Israel, Turkey, and the USA). Patients were treated with oral levoketoconazole in a 2-21 week incremental dose-titration phase starting at 150 mg twice daily (150 mg increments until mUFC normalisation, maximum 600 mg twice daily) and a 6-month maintenance phase. The primary outcome was the proportion of patients with mUFC normalisation at end of maintenance, without dose increase during the maintenance phase (in the intention-to-treat population). Prespecified adverse events of special interest were potential liver toxicity, corrected QT prolongation, and adrenal insufficiency. This trial is registered with ClinicalTrials.gov, NCT01838551. FINDINGS: Between July 30, 2014, and June 30, 2017, 201 individuals were screened and 94 patients were enrolled and received at least one dose of study medication. Of the 94 patients, 80 (85%) had pituitary Cushing's syndrome. Mean mUFC at baseline was 671·4 nmol/24 h (243·3 µg/24 h), which is 4·9 times the upper limit of normal. Of the 77 patients who advanced to the maintenance phase, 62 (81%) had mUFC normalisation by end-of-dose titration. At the end of the 6-month maintenance phase, 29 (31%) of 94 patients were responders; the least-squares mean estimate of the proportion of responders was 0·30 (95% CI 0·21-0·40; p=0·0154 vs null hypothesis of ≤0·20). The most common adverse events in the 94 patients were nausea (30 [32%]) and headache (26 [28%]). Adverse events led to study discontinuation in 12 (13%) of 94 patients. Two patients had a QT interval (Fridericia corrected) of more than 500 ms, and three patients had suspected adrenal insufficiency. Alanine aminotransferase reversibly increased to more than three times the upper limit of normal in ten (11%) patients. Four patients had serious adverse events that were considered probably or definitely related to the study drug: abnormal liver function test results (n=1), prolonged QT interval (n=2), and adrenal insufficiency (n=1). One person died from colon carcinoma unrelated to study medication. INTERPRETATION: Twice-daily oral levoketoconazole treatment led to sustained improvements in urinary free cortisol, with an acceptable safety and tolerability profile. Levoketoconazole might represent a useful therapeutic option for the medical treatment of Cushing's syndrome. FUNDING: Strongbridge Biopharma.

Long-term efficacy and safety of once-monthly pasireotide in Cushing's disease: A Phase III extension study.

OBJECTIVES: Many patients with Cushing's disease (CD) require chronic pharmacotherapy to control their hypercortisolism. We evaluated the efficacy and safety of long-acting pasireotide during a long-term extension study in patients with CD. DESIGN: Open-label extension to a 12-month Phase III study of long-acting pasireotide in CD (N = 150; NCT01374906). PATIENTS: Patients with mean urinary free cortisol (mUFC) ≤ upper limit of normal (ULN) or receiving clinical benefit at core study end could continue long-acting pasireotide during the extension. RESULTS: Eighty-one of 150 (54.0%) enrolled patients entered the extension. Median overall exposure to pasireotide at study end was 23.9 months; 39/81 (48.1%) patients completed the extension (received ≥ 12 months' treatment during the extension and could transit to a separate pasireotide safety study). mUFC was ≤ULN in 42/81 (51.9%), 13/81 (16.0%) and 43/81 (53.1%) patients at extension baseline, month (M) 36 and last assessment. Median mUFC remained within normal limits. Median late-night salivary cortisol was 2.6 × ULN at core baseline and 1.3 × ULN at M36. Clinical improvements were sustained over time. Forty-two (51.9%) patients discontinued during the extension: 25 (30.9%) before M24 and 17 (21.0%) after M24. Hyperglycaemia-related AEs occurred in 39.5% of patients. Mean fasting glucose (FPG) and glycated haemoglobin (HbA1c ) were stable during the extension, with antidiabetic medication initiated/escalated in some patients. Sixty-six (81.5%) and 71 (88.9%) patients were classified as having diabetes (HbA1c  ≥ 6.5%, FPG ≥ 7.0 mmol/L, antidiabetic medication use, or history of diabetes) at extension baseline and last assessment. CONCLUSIONS: Long-acting pasireotide provided sustained biochemical and clinical improvements, with no new safety signals emerging, supporting its use as an effective long-term therapy for CD.

Clinical utility of an ultrasensitive urinary free cortisol assay by tandem mass spectrometry.

BACKGROUND: 24 h urinary free cortisol measurement is a clinically important first-line screening test for Cushing's syndrome (CS). Tandem mass spectrometry (LC-MS/MS) assays have superior sensitivity and specificity compared to immunoassays. Our goal was to improve and validate a LC-MS/MS method to measure urinary free cortisol in both adult and pediatric patients and to characterize its clinical diagnostic performance of CS by chart review. METHODS: We improved a LC-MS/MS method previously reported for urinary free cortisol to be able to measure urinary and salivary cortisol in the same batch for increased efficiency. The sample preparation was by liquid-liquid extraction using dichloromethane followed by stepwise washing with acidic, basic and neutral solutions. The assay's analytical performance was characterized, and a retrospective patient chart review was conducted to evaluate the assay's clinical performance in diagnosing CS. RESULTS: The LC-MS/MS assay demonstrated enhanced sensitivity and was linear within an analytical measurement range of 10-10,000 ng/dL. Assay accuracy was satisfactory as determined by spike and recovery studies and highly correlated with a reference LC-MS/MS method. The assay's clinical diagnostic sensitivity and specificity in detecting CS was 96% and 91%, respectively, when compared to a urinary cortisol excretion of at least 50 µg/24 h. CONCLUSIONS: The improved LC-MS/MS method is both sensitive and specific with enhanced analytical performance and clinical diagnostic utility to screen for CS. The clinical diagnostic sensitivity and specificity were superior based on retrospective patient chart review.

Intestinal Perforation in ACTH-Dependent Cushing's Syndrome.

Previous studies have linked systemic glucocorticoid use with intestinal perforation. However, the association between intestinal perforation and endogenous hypercortisolism has not been well described, with only 14 previously published case reports. In this study, we investigated if intestinal perforation occurred more frequently in patients with ectopic ACTH syndrome and in those with a greater than 10-fold elevation of 24-hour urinary free cortisol level. Of 110 patients with ACTH-dependent Cushing's syndrome followed in two clinics in Canada, six cases with intestinal perforation were identified over 15 years. Age of patients ranged from 52 to 72, five females and one male, four with Cushing's disease and two with ectopic ACTH production, one from a pancreatic neuroendocrine tumor and one from medullary carcinoma of the thyroid. Five had diverticular perforation and one had intestinal perforation from a stercoral ulcer. All cases had their lower intestinal perforation when the cortisol production was high, and one patient had diverticular perforation 15 months prior to the diagnosis of Cushing's disease. As in previously reported cases, most had hypokalemia and abdominal pain with minimal or no peritoneal symptoms and this occurred during the active phase of Cushing's syndrome. Whereas all previously reported cases occurred in patients with 24-hour urinary free cortisol levels greater than 10-fold the upper limit of normal when measured and 11 of 14 patients had ectopic ACTH production, only one of our patients had this degree of hypercortisolism and four of our six patients had Cushing's disease. Similar to exogenous steroid use, patients with endogenous hypercortisolism also have a higher risk of intestinal, in particular diverticular, perforation and should be monitored closely for its occurrence with a low threshold for investigation and surgical intervention. Elective colonoscopy probably should be deferred until Cushing's syndrome is under control.

Performance of LC-MS/MS and immunoassay based 24-h urine free cortisol in the diagnosis of Cushing's syndrome.

24-h urine free cortisol (UFC) is an indicator of integrated cortisol secretion and established screening tool for Cushing's syndrome (CS). Doubts have been raised regarding specificity of immunoassays, and mass spectrometric techniques have been proposed as an alternative. In the present study we compared diagnostic accuracy of UFC measured with LC-MS/MS vs. immunoassay in patient with CS and patients where CS has been excluded. We examined 24-h urine samples from patients with surgically confirmed CS (n = 77; Cushing's disease (n = 44), ectopic CS (n = 5), adrenal CS (n = 28)) and patients in whom Cushing's syndrome was excluded (n = 97) by long-term follow up. UFC was first measured by automated chemiluminescence immunoassays (ADVIA Centaur, Siemens; LIAISON, DiaSorin). Aliquots of all samples were also analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Statistics: Passing-Bablok Regression, Receiver operating characteristic (ROC) analysis with Youden's index calculation. UFC of CS patients were higher with both immunoassays compared to LC-MS/MS (913 +/- 235 vs. 303 +/- 155 µg/24 h (ADVIA) and 898 +/-216 vs. 399 +/- 196 µg/24 h (LIAISON)). Similarly, UFC were higher with immunoassays than with LC-MS/MS in the control group (223 +/- 10 vs. 23 +/- 2 µg/24 h (ADVIA) and 105 +/- 6 vs. 27 +/- 4 ug/24 h for (LIAISON)). Passing-Bablok regression showed good correlation between LC-MS/MS and ADVIA as well as between LCMS/MS and LIAISON (r = 0.96 and r = 0.99, p < 000.1) but less correlation in controls (r = 0.83 and r = 0.74, respectively, p < 000.1). ROC calculation revealed the highest ROC AUC (0.89) for the LIAISON immunoassay, followed by LC-MS/MS (0.82) and the ADVIA (0.80). In direct comparison, AUCs from LC-MS/MS and immunoassays in the same patient were not statistically different (p < 0,001). Best cut-off concentration to identify patients with CS was 234 µg/24 h (LIAISON), 51 µg/24 h for LC-MS/MS and 330 µg/24 h (ADVIA Centaur). In summary, UFC values were measured substantially higher by both immunoassays compared to LC-MS/MS. This is most likely due to cross-reactivity from interfering glucocorticoid metabolites. Nevertheless, all three methods correlated well. ROC analysis revealed the highest AUC for one of the immunoassays, although differences between the three methods were not significant. Direct comparison with LC-MS/MS indicates that high diagnostic accuracy can be obtained with suitable immunoassays.