Title: Late-night salivary cortisol has better performance than urinary free cortisol in the diagnosis of Cushings syndrome.

Authors: Elias PCL1, Martinez EZ2, Barone BFC1, Mermejo LM1, Castro M1, Moreira AC1.
Formatado: Ingls (EUA) Formatado: Ingls (EUA)

Affiliation: 1 Division of Endocrinology-Department of Medicine and 2Division of StatisticsDepartment of Social Medicine, School of Medicine of Ribeirao Preto - University of Sao Paulo, Ribeirao Preto, SP- Brazil.

Short title: Better performance of LNSF than UFC in the diagnosis of Cushings syndrome.

Key words: Cushings syndrome, salivary cortisol, urinary free cortisol, ROC curves

Number of words abstract = 253 words /number of words text = 3540 words Number of tables = none / figures: four

Corresponding author: Ayrton C Moreira Department of Medicine - School of Medicine of Ribeirao Preto University of Sao Paulo. Ribeirao Preto, SP, Brazil. Fax number: +55 16 3633-6695/ Telephone number: +55 16 3602-2910 e-mail: acmoreir@fmrp.usp.br This work was supported by grants from FAPESP (Fundao de Amparo a Pesquisa do Estado de Sao Paulo (07/58365-3; 10/03039-7) and CNPq (314279/2009-1).

DISCLOSURE STATEMENT: The authors have nothing to disclose.

ABSTRACT Context: The comparison of variability, reproducibility, and diagnostic performance of LNSF and UFC using concurrent and consecutive samples in Cushings syndrome (CS) is lacking. Objectives, Patients and Methods: In a prospective study, we evaluate three simultaneous and consecutive samples of late-night salivary cortisol (LNSF) by RIA and urinary free cortisol (UFC) by LM-MS/MS in Cushing disease (CD; n=43), adrenal CS patients (n=14) and obese subjects (n=18) in order to compare their diagnostic performances by ROC curves. In CS patients we also performed a modified Cushings syndrome severity index (CSI). Results: There was no difference in the coefficient of variation between LNSF and UFC among the three samples obtained for each patient with CD (3526vs3124), adrenal CS (2918vs2113) and obesity (3937 vs 4820). The area under the ROC curves for LNSF was 0.999 (95%CI 0.971-1.00) and for UFC was 0.908 (95%CI 0.789 - 0.978). The ratio between AUCs was 0.911 (95%CI 0.796 - 0.981) indicating better performance of LNSF than UFC in diagnosing CS. LNSF performed the diagnosis even in the presence of normal UFC in 18% of CS. UFC alone did not help to diagnose CS in any patient. There was no association between the severity of CSI and the degree of biochemical hypercortisolism. Conclusion: Our data, using for the first time three simultaneous and consecutive samples, thus avoiding intermittent hypercortisolism, support that LNSF is a better diagnostic tool and should replace the well established, but cumbersome, UFC as the first line test in clinical practice to diagnose CS.

INTRODUCTION

The clinical manifestations of Cushings syndrome (CS) can be variable and cyclical; nowadays CS has been suspected before patients develop classical clinical pictures. Moreover, diagnostic difficulties can arise due to the relative rarity of the disease (Aron). The diagnostic strategies to confirm endogenous hypercortisolism have
changed throughout the last decades. In the 70s, the diagnosis was established by combining basal state measurements of the daily urine-free cortisol (UFC) excretion and late evening plasma cortisol levels with low-dose (2mg/d for 48h) dexamethasone suppression test (LDDST) (Crapo 1979). Two decades later, biochemical confirmation of CS was best achieved still using LDDST, UFC and sleeping midnight plasma cortisol measurements (Newell-Price, Trainer et al. 1998). At that time, evaluation of late-night salivary cortisol (LNSF) levels was demonstrated to be as sensitive as the conventional gold standard tests for the diagnosis of CS (Raff, Raff et al. 1998; Castro, Elias et al. 1999). Finally, the last Endocrine Society clinical practice guideline of the diagnosis of CS recommends one of the following tests: at least two measurements of UFC or LNSF, or 1mg DST, or 2mg low-dose DST (Nieman, Biller et al. 2008). UFC provides an integrated assessment of cortisol secretion over 24-h period; however urine measurements may be inaccurate because of improper collection or renal insufficiency. The introduction of salivary cortisol assessment as a first line test for CS, both inpatient and outpatient settings, in many centers, has allowed the study of HPA axis response patterns without the need of repeated blood or urinary samples (Nunes, Vattaut et al. 2009; Manetti, Rossi et al. 2013). Studies have addressed the comparison of LNSF with traditional tests in large population of CS and obese subjects, the patients most frequently suspected for CS. Receiveroperating characteristic (La Brocca, Terzolo et al.) curves generated to establish the optimal threshold values for each test and their inherent diagnostic efficacy demonstrated no statistically difference in terms of sensitivity, specificity, and diagnostic accuracy, or predictive values

among midnight plasma cortisol, LNSF, and UFC, if cutoff values derived from ROC curves were applied (Putignano, Toja et al. 2003) LNSF seems to help diagnosing patients with a high index of clinical suspicion presenting mild endogenous CS with either normal or only slightly elevated UFC (Kidambi, Raff et al. 2007). In this study, the majority of patients with either normal or only slightly elevated UFC had LNSF above the upper limit of the normal; however some patients had LNSF levels in the normal range in some occasions. Similar results demonstrating patients with CS with at least one normal UFC or LNSF have been observed in other series (Friedman, Ghods et al. 2010). The limitation of these studies was the great time interval of serial measurement of either UFC or LNSF and/or non simultaneous and non consecutive collection of urine and saliva, and therefore, intermittent hypercortisolism could have led to one or the other being normal. There is one study evaluating the day-to-day variability of LNSF by three repeated measurements in healthy controls, but not in CS patients. The coefficient of variation (CV) of LNSC between- control subjects was 78% and withincontrol subjects 22%. (Viardot, Huber et al. 2005). More recently, the variability of baseline UFC was evaluated in patients with persistent/recurrent or de novo Cushings disease, which presented mean UFC 1.5 times the upper limit of the normal range, calculated from four 24-h urine samples collected over 2 weeks. The intrapatient CV across two or more UFC samples was approximately 50% and the variability increased with progressively higher UFC levels. However, patients with mild degrees of cortisol excess were not included in the study; in addition, UFC was analyzed at three different laboratories (Petersenn, Newell-Price et al. 2013). The cyclical characteristics of CS observed in some patients, the pulsatile and circadian nature of cortisol secretion may contribute to the variability of UFC and LNSF. The comparison of variability, reproducibility, and diagnostic performance of LNSF and UFC using simultaneous and consecutive samples in CS is lacking. A comprehensive understanding of these two laboratory tests may modify the diagnostic strategy of CS in clinical practice. In the present prospective study, we evaluate three concurrent and consecutive samples of LNSF and

UFC in a large group of patients with Cushings syndrome in order to compare their diagnostic performances.

SUBJECTS This study was approved by the Ethical Committee of the Ribeirao Preto Medical School University of Sao Paulo and, according to the requirements of the Declaration of Helsinki, written informed consent was obtained from all participants. Eighty patients, the majority with clinical features suspicious for hypercortisolism were studied between 2005 and 2012 at Division of Endocrinology at the University Hospital of the Ribeirao Preto Medical School, University of Sao Paulo. In addition to the clinical features of chronic hypercortisolism to diagnose endogenous hypercortisolism we adopted the 2300h salivary cortisol cutoff value of 350 ng/dL (or 9.8 nmol/L) and salivary cortisol levels after overnight 1mg DST of 150 ng/dL (or 4.2 nmol/L), as previously described (Castro, Elias et al. 1999; Castro and Moreira 2007). To evaluate the etiology of Cushings syndrome, standard tests of pituitary-adrenal function, including plasma ACTH levels, low and high dose DST, CRH test, bilateral inferior petrosal sinus sampling, and image studies were performed. The diagnosis was later confirmed by surgical approach and histopathology. Based on the adopted parameters, Cushings syndrome was diagnosed in 62 patients. The diagnosis of Cushings disease was established in 43 patients (38 female /5 male; age 31.511.7, ranging from 9 to 64 years). Fourteen patients had ACTH independent Cushings syndrome (14 female; age 35.316.4, ranging from 13 to 69 years). Five patients who presented Cushings syndrome due to ectopic ACTH secretion were excluded from the study because of disease severity; thus leading to a very small number of samples to be analyzed. Finally, eighteen patients (16 female/1 male; age 38.213.6, ranging from 18 to 71 years) were diagnosed as having obesity, associated with essential hypertension, diabetes, and idiopathic hirsutism, with no progression of clinical signs for at least six months follow up.

In all three groups we evaluated the body mass index (BMI), systolic and diastolic blood pressure, presence of glucose intolerance or diabetes. In CS patients we performed a modified Cushings syndrome severity index (CSI), as previously reported (Sonino, Boscaro et al. 2000). CSI was summarized in an overall assessment of eight clinical features: fat distribution, skin lesions, muscle weakness, mood disorder, hypertension, diabetes, hypokalemia, and sex-related disturbances, scored in a scale of zero, 1 or 2 points (absent/mild/severe forms, respectively) with a maximum of 16 points.

METHODS Study Design: Inpatient salivary cortisol samples were collected by Salivette sampling devices (Salivette; Sarstedt, Nuembrecht, Germany) at 0900h and 2300h (LNSF) on three consecutive days. At the same days, 24-h urine collections for UFC measurements were performed for each subject. Renal function was normal in all patients. The salivary and urinary samples were stored at 20C until analysis. Salivary cortisol measurements were performed by previously described RIA method on 25-l samples of saliva without prior extraction or chromatography. The antiserum specificity was 100% for cortisol and 15% for cortisone. The mean intra-assay CV was 5.5%. (Santiago, Jorge et al. 1996). All samples obtained from each subject were analyzed in duplicate in the same assay. UFC was measured by liquid chromatography associated with tandem mass spectrometry (LC-MS/MS normal range of 3-43 g/24h). The mean intra-assay CV was 4.4% (Vieira, Nakamura et al. 2005).

Statistical Analysis
Excludo:

Data are expressed as mean and standard deviation (SD). Kruskal-Wallis test was performed for multiple comparisons among different groups and the Dunn's Multiple Comparison Test was used as a post hoc test. The Wilcoxon-Mann-Whitney test was used when appropriate. The performance of CSI in reflecting severity of the disease was correlated with the values of LNSF and UFC by Spearmans method. Fishers exact test was used for categorical

Excludo: ANOVA

data such as the severity of CSI and the degree of biochemical hypercortisolism evaluated by LNSF and UFC. Statistical analyses were carried out using SAS for Windows software (SAS Institute, Cary, NC). . The level of significance was set at = 0.05. Receiver-operating characteristic (La Brocca, Terzolo et al.) curves for LNSF and UFC were obtained to discriminate between states of hypercortisolism and obesity. In addition, the comparison of the area under ROC curves (Tauchmanova, Rossi et al.) was performed by parametric approach using Bayesian estimation with non-informative prior distributions (Gelman et al, 2003), considering repeated measures. This statistical model is very similar to that presented in the section 7.2 of the book of Box and Tiao (1992), by assuming that the logarithm of LNSF and UFC follows a normal distribution and the multiple observations per individual are treated as blocks. This analysis was performed using the software OpenBugs (version 3.2.1, MRC, Cambridge, UK, 2011) based on Markov chain Monte Carlo (MCMC) estimation. The area under the curve was estimated by integration and inferences were obtained by using the MCMC method. Summaries for each parameter of interest were reported as the mean of the respective simulated chains and its 95% credible intervals (95% CI). Credible intervals are the Bayesian analogs of classical confidence intervals.

[EDSON1] Comentrio: Ayrt on,eu,commuitasinceridade, nogostoenoconfionesse GraphPadPRISM.Euoconsidero umpssimosoftwaree desaconselhoseuuso.Portanto, eunogostariadetermeunome emumartigoquedizterusado esseprograma,dadoqueseria muitoincoerentedaminhaparte norecomendloeaquiutiliz lo.Portanto,peoquemudea citaoparaoSAS. Excludo: Data analyses were carried out with the statistical package GraphPad PRISM 4.0 (GraphPad software Inc., 2003, USA). [EDSON2] Comentrio: Aqui humaconfusodeconceitos, entrenveldesignificncia, denotadopelaletragregaalfa,e ovalorp. Excludo: Significance was assumed when P < 0.05 Excludo: . Excludo: analyses Excludo: repeated measures (REF Edson) with multiple observations per individual. Excludo: The

RESULTS There were no differences in age and gender among Cushings syndrome and obese patients. The BMI (kg/m2) was no different among CD (33.47.6), adrenal CS (31.46.7), and obese (38.710.8) patients. Regarding systolic/diastolic blood pressure (mmHg) no differences were observed among CD (135.7/87.6), adrenal CS (135.6/91.0), and obese (139.4/90.6) patients (p=0.8). The majority of CD (67.4%), adrenal CS (57.1%) and obese (72.2%) patients had high blood pressure. Diabetes mellitus or glucose intolerance was observed in 67.4% and 2.3% of CD, in 28.6% and 0% of adrenal CS, and in 27.8% and 27.8% of obese patients, respectively.

The mean global CSI score was not different between CD (7.62.3, range 3 to 13) and adrenal CS (5.33.5, range 1 to 10). In addition, there was no evidence of correlation between individual CSI with the respective mean of LNSF and UFC, neither for CD (p=0.32 and p=0.48, respectively) nor adrenal CS (p=0.9 and p=0.32, respectively). In addition, we did not observe association between the severity of the CSI (8 or >8) and the degree of biochemical hypercortisolism considering LNSF and UFC below or above the respective mean values (LNSF: p=0.59 and UFC: p=0.22). All obese subjects presented salivary cortisol circadian rhythm and suppressed salivary cortisol after 1mg dexamethasone test (61.75.0 ng/dl). On the other hand, as a group, abnormal salivary cortisol circadian rhythm was observed either in CD or CS. Both groups did not suppressed salivary cortisol after 1mg DST (CD: 17801397); adrenal CS: (170612415 ng/dl). LNSF levels (ng/dl) were similar between CD (26491807) and adrenal CS (16841377) patients; both groups had higher LNSF than obese patients (268410; P<0.001). UFC levels (g/24h) were also similar between CD (329623) and adrenal CS (169206) patients; both groups had higher UFC than obese patients (21.022.6; P<0.001). Figures 1 and 2 show samples of LNSF and UFC obtained from each patient with CD and adrenal CS, respectively. There was no difference among the three LNSF (p=0.42) and UFC (p=0.87) samples for each subject with CD. No difference was also observed among the three LNSF (p=0.47) and the UFC (p=0.2) samples for each subject with adrenal CS as well as for obese patients (p=0.12). In the CD patients, the mean of LNSF intrapatient CV (calculated using three samples) was 35% (95% CI: 2744) compared with 29% (95% CI: 1839) in the adrenal CS patients and with 39% (95% CI: 2657) in obese patients. Regarding the mean of UFC intrapatient CV (calculated using three samples), CV in the CD patients was 31% (95% CI: 2439) compared with 21% (95% CI: 1328) in the adrenal CS patients and with 48% (95% CI: 3661) in obese patients. There was no difference in the CV (%) between LNSF and UFC among the three samples obtained for each patient with CD (3526vs3124), adrenal CS (2918vs2113) and obesity (3937 vs 4820).

ROC curves were created to establish the optimal threshold values for each test and its diagnostic efficiency (Figure 3). For Cushings syndrome, the sensitivity and specificity for LNSF, if cutoff value derived from ROC curves was 485 ng/dl, were 94.4% and 93.4% with a positive likelihood ratio (LR) and negative likelihood ratio (LR-) of 14.4 and 0.06, respectively. Sensitivity and specificity for UFC, with a cutoff of 45 g/24h, were 93.2% and 79.2% with LR+ and LR- of 4.5 and 0.06, respectively. The AUC by Bayesian approach for LNSF was 0.999 (95%CI 0.971-1.00) and for UFC was 0.908 (95%CI 0.789- 0.978). The ratio between AUCs was 0.911 (95%CI 0.796- 0.981) indicating a better performance of LNSF than UFC in diagnosing Cushings syndrome. After obtained the cutoff values from the ROC curves, we reanalyzed the ability of LNSF and UFC to diagnose Cushings syndrome. LNSF samples >485ng/dl diagnosed all but one (2.3%) CD patient while UFC >45g/24h failed in 6 of out 43 (14%). Regarding adrenal CS patients, LNSF failed in 3/14 (21.5%) and UFC in 6/14 (43%). A positive correlation was found between the individual mean of LNSF and of UFC in Cushings syndrome patients (r=0.64, P<0.0001; Figure 4) but not in obese patients (data not shown). The axes of a two-dimensional Cartesian system divided figure 4 into four counterclockwise quadrants based on the cutoff values of LNSF (485 ng/dl) and UFC (45/24h). Quadrant I shows that above the cutoff values either LNSF or UFC was able to diagnose Cushings syndrome in the majority of patients. Quadrant II shows that LNSF performed the diagnosis even in the presence of UFC <45g/24h in 10 patients (8 with CD, one incidentaloma, and one PPNAD). Of note, severity of CSI was not different between patients with high LNSF and UFC values (quadrant I; CSI=7.52.5) and patients with high LNSF and normal UFC values (quadrant II; CSI=7.71.6). Quadrant III shows that neither LNSF nor UFC was able to diagnose Cushings syndrome in four adrenal CS, consisting of three patients with incidentaloma and one with PPNAD. We observed no difference between CSI in these patients compared with the remaining patients (p=0.14). Finally, the quadrant IV shows that UFC alone did not help to diagnose Cushings syndrome in any patient.

Discussion
In the present study, we evaluated three simultaneous and consecutive samples of LNSF and UFC in a large group of patients with Cushings syndrome. Our data demonstrated no difference among the three absolute values of samples obtained in each patient, either for LNSF or UFC. In addition, there was no difference between LNSF and UFC coefficient of variation among the three samples. Finally, the comparison of ROC curves indicated a better performance of LNSF than UFC in diagnosing CS. Altogether these data suggest that, in clinical practice, LNSF should replace the well established, but cumbersome, UFC for diagnosing CS. Regarding clinical presentation, in the present study, the occurrence of obesity and hypertension were similar among CD, adrenal CS and obese patients while diabetes or glucose intolerance were observed more frequently in CD patients. These finding can be ascribed by the low likelihood ratio of these symptoms/signs in diagnosing CS due to overlap in clinical findings between CS and Cushingoid obesity (Aron 2010). The pretest probability in diseases with high prevalence is equal to the prevalence of the disease (Motulsky 1995; Bianchi, Alexander et al. 2009). However, it is not applicable for rare endogenous CS, which besides overlap of some clinical findings with more prevalent disorders; its epidemiological information has not been accurately known. Recently, we applied a Bayesian approach to evaluate the diagnostic probability of CS, based on clinical perception before the confirmation with biochemical tests. The pre-test probability showed a linear increase throughout the physician endocrine experience (Cipoli, Martinez et al. 2012). Clinimetrics is the domain concerned with indexes and rating scales used to measure clinical phenomena such as severity of the disease, rate of progression, and the magnitude of changes upon treatment. CSI has been described as a valid and reliable method to evaluate severity in CS patients. In the first study describing CSI to evaluate CS severity, the authors found the mean CSI of 8.52.7 in fourteen patients (Sonino, Boscaro et al.). The mean of CSI

scores observed by us is similar to the observed in that previous study with no difference in the mean global CSI score between CD (7.62.3) and adrenal CS (5.33.5) patients. In addition, there was no correlation between severity of the disease evaluated by individual CSI with the respective mean of LNSF and UFC, either for CD or adrenal CS, indicative of no association between the severity of clinical features and the degree of biochemical hypercortisolism. Our data obtained in a larger series of CS confirm previous finding evaluating only UFC and also extend this information to LNSF. Overall these data indicate that clinical score and biochemical measures do not share the same properties in diagnosing hypercortisolism. It can be ascribed to the interindividual variability and to a spectrum of glucocorticoid sensitivity and glucocorticoid tissue specificity observed in several pathological conditions and even in healthy individuals (Bamberger, Schulte et al. 1996; Chriguer, Elias et al. 2005). Moreover, the quality of CSI as a clinical discriminatory tool might be questioned since it gives the same score for each sign or symptom, independently of its prevalence (e.g., obesity, diabetes, and signs of catabolism) in general population, bringing inherent problems to this method. Recently, Elamin et al. conducted a systematic review and a meta-analysis to summarize the accuracy of biochemical tests for diagnosing CS. The UFC, LNSF and 1mg-DST had similar high accuracy, when analyzed by classical statistical methods (Elamin, Murad et al. 2008). Although the meta-analysis evaluated a very large number of CS patients, its heterogeneous nature due to the fact of isolated collection of UFC, plasma, or saliva obtained in different centers, with different patients can difficult a more precise comparison between each test. In the present study, evaluating LNSF and UFC simultaneously in three consecutive days, no difference was observed among values of the samples for LNSF and for UFC obtained from each CD, adrenal CS, and obese patients. In addition, there was no difference in the CV (%) of LNSF or UFC within the three samples obtained for each patient. Coefficient of variation of LNSF and UFC varied from 29% to 39% and 21 to 48%, respectively in CD, adrenal CS and obese patients. Previous studies evaluated the variability of LNSF in a subset of CS patients collecting LNSF in two consecutive nights. One observed variability of 35% in CS (Nunes,

Vattaut et al. 2009) while the other observed 22% in normal subjects, 32% in a suspected CS group, and 51% in CS patients, being higher in the later group (Carrasco, Garcia et al. 2012). In addition, the variability of LNSF in a subset of CS patients, in which the sample collection occurred in two nonconsecutive days, was 17% (Cardoso, Arregger et al. 2009). Concerning UFC, a recent study evaluated its variability on four nonconsecutive samples collected over a 2week period and found intrapatient coefficient of variation of 52% (Petersenn, Newell-Price et al. 2013). Our results on LNSF coefficient of variation are similar to those that used consecutive sampling collection. In the same condition, UFC coefficient of variation is also very similar. ROC curves were created to establish the optimal threshold values for each test for Cushings syndrome diagnosis. Although the sensitivity of both tests was similar, the specificity was higher for LNSF. Unlike sensitivity and specificity, likelihood ratios are independent of disease prevalence and can be used at individual level; the larger the positive likelihood ratio, the greater the likelihood of disease (Attia 2003). Indeed, our data show that LNSF had a LR14.4 compared to LR4.5 of UFC indicating a superior power of LNSF than UFC as a diagnostic test for Cushings syndrome. In addition, the Bayesian analysis of the AUCs clearly indicated better performance of LNSF than UFC in diagnosing Cushings syndrome. It is important to point out that the better LNSF performance can not be ascribed to the variability of these methods, since coefficient of variation was similar between LNSF and UFC tests. One hypothesis would be the fact that small increases in cortisol production at the circadian nadir may not be detected as an increase in UFC (Alexandraki and Grossman 2011). Another possibility is that there are intrinsic differences between methodologies. In the present study, LNSF was measured by RIA and, thus, the antibody against cortisol also cross-reacts against cortisone. As known, there is significant conversion of cortisol to cortisone in salivary glands by 11-hydroxysteroid dehydrogenase II leading to high concentration of cortisone in saliva (Smith, Maguire et al. 1996). Indeed, a recent study demonstrated lower sensitivity (74.5%) and specificity (90.1%) of LNSF in diagnosing CS using LC-MS/MS (Erickson, Singh et al. 2012). Conversely, in our study, UFC was measured by LC-MS/MS which detects cortisol and eliminates the interference of cortisol metabolites, increasing its biochemical specificity.

Therefore, what could be a limitation of salivary cortisol assayed by immunoassay-based methods, turns into an advantage for diagnosing CS. However, it is important to be aware of the different results generated by the commercially available methods and to interpret the published reference intervals appropriately (Castro and Moreira 2007; Raff 2012). Finally, the majority of CS patients were diagnosed either by LNSF or UFC. Of note, LNSF diagnosed CS in 18% of patients even in the presence of normal UFC, whereas UFC alone did not help any patient. It is important to point out that in four adrenal CS patients neither LNSF nor UFC was able to diagnose CS, in agreement to previous data demonstrating that in primary adrenal diseases the 1mg DST is preferable to LNSF or UFC (Nieman, Biller et al. 2008). Thus, in the present context, we suggest that the next task force for update recommendations for the diagnosis of CS could consider a hierarchy of screening tests. As the initial test, two samples of LNSF should be the first choice and further, the 1mg-DST. The UFC and 2mg-DST might be performed only in special situations. In conclusion, our data support that LNSF is a better diagnostic tool than UFC and should replace the well established UFC as the first line test in clinical practice to diagnose CS.

Legend of Figures

Figure 1. Individual values of three consecutive samples of late-night salivary cortisol (LNSF) and urinary free cortisol (UFC) obtained from each patient with Cushings disease. The dotted line represents the cutoff values of LNSF (485 ng/dl) and UFC (45 g/24h).

Figure 2. Individual values of three consecutive samples of late-night salivary cortisol (LNSF) and urinary free cortisol (UFC) obtained from each patient with different etiologies of adrenal Cushings syndrome: carcinoma, ACTH-independent macronodular adrenal hyperplasia (AIMAH), primary pigmented nodular adrenocortical disease (PPNAD), adenoma and incidentaloma. The dotted lines represent the cutoff values of LNSF (485 ng/dl) and UFC (45 g/24h).

Figure 3: Receiver operating characteristic curves showing the diagnostic performance of latenight salivary cortisol (LNSF continuous line) and urinary free cortisol (UFC dotted line) in diagnosis of Cushingsyndrome. Cutoff values of 485ng/dl for LNSF and 45g/24h for UFC show sensitivity/specificity of 94.4%/93.4% and 93.2%/79.2%, respectively, to predict Cushingsyndrome.

Figure 4: Correlation between mean individual values of late-night salivary cortisol (LNSF) and urinary free cortisol (UFC) in Cushings syndrome patients (n=57; r=0.54, P<0.01). Cushings disease: n=43 (circles). Adrenal Cushings syndrome: n=14 (triangle). The quadrants are numbered from I to IV.

Acknowledgements We thank Mr Jos Roberto Silva, Ms Adriana Rossi and Ms Lucimara Bueno for technical support.

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Manetti, L., G. Rossi, et al. (2013). "Usefulness of salivary cortisol in the diagnosis of hypercortisolism: comparison with serum and urinary cortisol." Eur J Endocrinol 168(3):315321. Motulsky,H.(1995).Intuitivebiostatistics.NewYork,OxfordUniversityPress. NewellPrice, J., P. Trainer, et al. (1998). "The diagnosis and differential diagnosis of Cushing's syndromeandpseudoCushing'sstates."EndocrRev.19(5):647672. Nieman, L. K., B. M. Biller, et al. (2008). "The diagnosis of Cushing's syndrome: an Endocrine Society Clinical Practice Guideline." J Clin Endocrinol Metab. 93(5): 15261540. Epub 2008Mar1511. Nunes, M. L., S. Vattaut, et al. (2009). "Latenight salivary cortisol for diagnosis of overt and subclinical Cushing's syndrome in hospitalized and ambulatory patients." J Clin EndocrinolMetab.94(2):456462.Epub2008Nov2011. Petersenn, S., J. NewellPrice, et al. (2013). "High variability in baseline urinary free cortisol valuesinpatientswithCushing'sdisease."ClinEndocrinol. Putignano,P.,P.Toja,etal.(2003)."Midnightsalivarycortisolversusurinaryfreeandmidnight serum cortisol as screening tests for Cushing's syndrome." J Clin Endocrinol Metab. 88(9):41534157. Raff, H. (2012). "Cushing's syndrome: diagnosis and surveillance using salivary cortisol." Pituitary15(1):6470. Raff, H., J. L. Raff, et al. (1998). "Latenight salivary cortisol as a screening test for Cushing's syndrome."JClinEndocrinolMetab.83(8):26812686. Santiago, L. B., S. M. Jorge, et al. (1996). "Longitudinal evaluation of the development of salivarycortisolcircadianrhythmininfancy."ClinEndocrinol(Oxf).44(2):157161. Smith,R.E.,J.A.Maguire,etal.(1996)."Localizationof11betahydroxysteroiddehydrogenase typeIIinhumanepithelialtissues."JClinEndocrinolMetab.81(9):32443248. Sonino, N., M. Boscaro, et al. (2000). "A clinical index for rating severity in Cushing's syndrome."PsychotherPsychosom.69(4):216220. Tauchmanova,L.,R.Rossi,etal.(2001)."Bonelossdeterminedbyquantitativeultrasonometry correlates inversely with disease activity in patients with endogenous glucocorticoid excessduetoadrenalmass."EurJEndocrinol.145(3):241247. Viardot, A., P. Huber, et al. (2005). "Reproducibility of nighttime salivary cortisol and its use in the diagnosis of hypercortisolism compared with urinary free cortisol and overnight dexamethasone suppression test." J Clin Endocrinol Metab. 90(10): 57305736. Epub 2005Jul5712. Vieira, J. G., O. H. Nakamura, et al. (2005). "[Measurement of free urinary cortisol and cortisone using liquid chromatography associated with tandem mass spectrometry method]."ArqBrasEndocrinolMetabol.49(2):291298.Epub2005Sep2012.

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