CLINICAL STUDY Performance of salivary cortisol in the diagnosis of Cushing’s syndrome, adrenal incidentaloma, and adrenal insufficiency Filippo Ceccato 1 , Mattia Barbot 1 , Marialuisa Zilio 1 , Sergio Ferasin 1 , Gianluca Occhi 1 , Andrea Daniele 1 , Sara Mazzocut 1 , Maurizio Iacobone 2 , Corrado Betterle 1 , Franco Mantero 1 and Carla Scaroni 1 1 Endocrinology Unit, Department of Medicine DIMED and 2 Minimally Invasive Endocrine Surgery Unit, Department of Surgery, University-Hospital of Padova, Via Ospedale, 105, 35128 Padova, Italy (Correspondence should be addressed to C Scaroni; Email: carla.scaroni@unipd.it) Abstract Objective: Salivary cortisol has recently been suggested for studies on the hypothalamic–pituitary– adrenal (HPA) axis. The lackof circadian rhythm is a marker of Cushing’s syndrome (CS), and some authors have reported that low salivary cortisol levels may be a marker of adrenal insufficiency. The aim of our study was to define the role of salivary cortisol in specific diagnostic settings of HPA axis disease. Subjects and methods: We analyzed morning salivary cortisol (MSC) and late-night salivary cortisol (LNSC) levels in 406 subjects: 52 patients with Cushing’s disease (CD), 13 with ectopic CS, 17 with adrenal CS, 27 with CD in remission (a mean follow-up of 66G39 months), 45 with adrenal incidentaloma, 73 assessed as having CS and then ruled out for endogenous hypercortisolism, 75 with adrenal insufficiency, and 104 healthy subjects. Results: A LNSC value above 5.24 ng/ml differentiated CS patients from controls with high sensitivity (96.3%) and specificity (97.1%); we found higher LNSC levels in ectopic CS patients than in CD patients. We found no difference in MSC and LNSC levels between patients with CD in remission and healthy subjects. Both MSC and LNSC levels were higher in patients with adrenal incidentaloma than in healthy controls. A MSC value below 2.65 ng/ml distinguished patients with adrenal insufficiency from controls with high sensitivity (97.1%) and specificity (93.3%). Conclusions: Salivary cortisol is a useful tool to assess endogenous cortisol excess or adrenal insufficiency and to evaluate stable CD in remission. European Journal of Endocrinology 169 31–36 Introduction Salivary cortisol is increasingly being used to assess hyper- or hypocortisolism (1, 2). The measurement of late-night salivary cortisol (LNSC) levels in combination with the 1 mg dexamethasone suppression test (DST) and that of urinary free cortisol (UFC) levels have been proposed as the first-line laboratory tests in the diagnosis of Cushing’s syndrome (CS) (3). The lack of a circadian rhythm is a peculiar marker of CS (4), so the LNSC test seems to be the best choice to screen for CS because of its noninvasive, stress-free, easy collection in outpatients (1). There is a marked variation between studies in the performance of the LNSC test, reflecting differences in laboratory assays, sample collection, severity of CS, and control groups (2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). It is also important to have reference data not only for healthy volunteers but also for a control group of obese, hypertensive, diabetic, and depressed subjects or other clinical features of CS that are common in the general population (3, 15). Patients with adrenal incidentaloma are another interesting control group, where mild hypothalamic–pituitary– adrenal (HPA) axis activation is found in a high percentage (16, 17). Although the diagnosis of primary adrenal insuffi- ciency is simple, that of the secondary form is a challenge, and dynamic tests are needed to confirm clinical suspicions (18). Healthy people show physio- logically high levels of salivary cortisol when they wake up, and some authors have reported that low salivary cortisol levels may thus be a marker of adrenal insufficiency (2, 6, 19). None of these studies have looked for differences between primary and secondary adrenal insufficiency; in the latter, some residual HPA axis activity may be confounding. The aims of our study were to determine the performance of salivary cortisol in specific diagnostic settings: i) as a first test to rule out CS; ii) in a large number of newly diagnosed CS patients of different European Journal of Endocrinology (2013) 169 31–36 ISSN 0804-4643 q 2013 European Society of Endocrinology DOI: 10.1530/EJE-13-0159 Online version via www.eje-online.org