ALTHOUGH CUSHING S SYNDROME occurs rarely, it

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1 /02/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 87(10): Printed in U.S.A. Copyright 2002 by The Endocrine Society doi: /jc Nighttime Salivary Cortisol: A Useful Test for the Diagnosis of Cushing s Syndrome DIMITRIS A. PAPANICOLAOU, NANCY MULLEN, IOANNIS KYROU, AND LYNNETTE K. NIEMAN Department of Internal Medicine (D.A.P.), Emory University, Atlanta, Georgia 30322; and Department of Nursing (N.M.), Warren Grant Magnuson Clinical Center, and Pediatric and Reproductive Endocrinology Branch (I.K., L.K.N.), National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland ALTHOUGH CUSHING S SYNDROME occurs rarely, it is often suspected because of common clinical features such as obesity, hypertension, hirsutism, menstrual disorders, fractures, impotence, psychiatric disturbances, failure of growth, and weakness (1). Thus, because it may be difficult to identify those patients with endogenous hypercortisolism on the basis of clinical features, screening tests must be used to confirm the diagnosis. Unfortunately, each of the currently available biochemical tests for the diagnosis of Cushing s syndrome has limitations. Glucocorticoid excretion has been the gold standard that reflects directly the underlying endogenous hypercortisolism of Cushing s syndrome. However, urine measurements may be inaccurate because of improper collection technique. Furthermore, some individuals without Cushing s syndrome have hypercortisoluria that is caused, presumably, by functional stimulation of the CRH neuron, leading to overactivation of the entire hypothalamic-pituitary-adrenal axis (2). This type of hypercortisolism, termed a pseudo- Cushing state, has been associated with certain psychiatric disorders (depression, anxiety disorder, obsessive-compulsive disorder), morbid obesity, poorly controlled diabetes mellitus, and alcoholism (3 6) but also may be seen without any associated condition (6). The 1-mg overnight dexamethasone suppression test (DST) (7) is simple and inexpensive, but it has a false positive rate of up to 30%, especially in chronic illness, obesity, and psychiatric disorders (8, 9) and even in healthy individuals Abbreviations: AM, Morning salivary cortisol; CV, coefficient of variation; DST, dexamethasone suppression test; IP, inpatient; 17OHCS, 17-hydroxycorticosteroid; OP, outpatient; PM, evening salivary cortisol; UFC, urinary-free cortisol. Clinical features such as weight gain, depression, hypertension, and menstrual irregularities, although common in the general population, may raise the possibility of Cushing s syndrome. Up to 30% of urine cortisol and dexamethasone suppression screening tests may return an incorrect result, suggesting that better tests are needed. This study evaluated the utility of nighttime salivary cortisol measurement as a screening test for Cushing s syndrome. We evaluated 139 inpatients and 4 outpatients with possible Cushing s syndrome, 16 inpatients and 7 outpatients with other nonadrenal disorders, and 34 healthy outpatients. Using cut points that excluded all subjects without Cushing s syndrome, we compared the sensitivity for the detection of Cushing s syndrome of nighttime salivary cortisol levels (2330 and 2400 h for inpatients and bedtime for outpatients), simultaneous inpatient serum cortisol levels, and urine glucocorticoid excretion. An assayspecific inpatient 2400-h salivary cortisol or an outpatient bedtime salivary cortisol greater than 550 ng/dl (15.2 nmol/ liter) identified 93% of patients with Cushing s syndrome (confidence interval, 89 98%) and excluded all individuals without the disorder. Salivary cortisol measurements worked as well as plasma measurements and better than urine glucocorticoid excretion. We concluded that bedtime salivary cortisol measurement is a practical and accurate screening test for the diagnosis of Cushing s syndrome. (J Clin Endocrinol Metab 87: , 2002) (10). Thus, the low specificity of both urinary-free cortisol (UFC) and the 1-mg DST leads to further testing in many individuals without Cushing s syndrome. The 2-mg 2-d DST is cumbersome and also has a relatively low diagnostic accuracy (71%) (1, 6) if urine end points are used. However, its specificity and diagnostic accuracy improves dramatically if plasma cortisol is used instead (6, 11). The dexamethasone-crh test also has a high diagnostic accuracy for the identification of Cushing s syndrome (98%) (6, 12). However, like the 2-d DST, this test is relatively impractical, and it is also more expensive because of the costs of CRH and test personnel and the requirements for placement of an iv line and perfect timing of the CRH injection in relation to the last dose of dexamethasone. The metabolic clearance of dexamethasone may confound test results in patients receiving substances (e.g. alcohol, rifampin, phenytoin, phenobarbital) that induce the cytochrome P450-related enzymes, which enhance dexamethasone clearance, and in patients with renal or hepatic failure, which retard dexamethasone clearance (13, 14). In such cases, any of the dexamethasone tests can give either false positive or false negative results. We previously exploited the preservation of the physiologic circadian rhythmicity of cortisol secretion (15) in pseudo-cushing states, compared with Cushing s syndrome, showing that serum cortisol measurement at 2400 h can distinguish the two conditions with a diagnostic accuracy of 95% (16). However, obtaining a blood sample at 2400 h is impractical and costly. Because current screening tests for Cushing s syndrome have the disadvantages of a high false positive rate, cost, or inconvenience, many investigators have sought to develop better tests. Because salivary cortisol reflects the unbound, 4515

2 4516 J Clin Endocrinol Metab, October 2002, 87(10): Papanicolaou et al. Salivary Cortisol in Cushing s Syndrome biologically active form of serum cortisol, we hypothesized that it might work as well as serum cortisol measurement for the detection of Cushing s syndrome. This strategy, if effective, would have the potential advantage of convenient selfcollection at home, eliminating the stress and cost of hospitalization and iv line placement. We have demonstrated that evening salivary cortisol works well to identify Cushing s syndrome in children (17). Three other groups have shown that 2400-h salivary cortisol has a sensitivity of 92 93% for discrimination of Cushing s syndrome, using comparison groups of up to 40 individuals with obesity or suspected Cushing s syndrome (18 20). The present study compared the diagnostic utility of UFC, evening serum cortisol, and salivary cortisol in 156 inpatients (IPs) evaluated for Cushing s syndrome. To develop an outpatient (OP) test, we compared these results with those obtained using bedtime specimens collected at home by normal individuals and patients with either nonadrenal disorders or possible Cushing s syndrome. Subjects Subjects and Methods A total of 156 adult patients referred with clinical findings suggestive of Cushing s syndrome and elevated 24-h UFC were admitted for evaluation of hypercortisolism at the NIH Clinical Center between June 1994 and February The patients were followed up until a diagnosis of Cushing s syndrome or pseudo-cushing states was made. The diagnosis of Cushing s syndrome was established by tissue pathology or hypocortisolism after adrenalectomy or removal of a tumor producing ACTH or by resolution of clinical and laboratory abnormalities after surgical therapy. The diagnosis of pseudo-cushing states was based on negative tests for Cushing s syndrome, including a dexamethasone-crh stimulation test (6), a loperamide suppression test (17), and/or a 1-mg overnight DST (7), and/or resolution of hypercortisolism over up to 18 months follow-up. Thirteen patients had one or more of these tests; the remainder was followed up with UFC. Some of the patients who underwent dexamethasone-crh testing were reported previously (6, 12). Patients with suspected pseudo-cushing states also underwent a psychiatric evaluation. For comparison purposes, we studied 16 IPs admitted for evaluations not involving the adrenal gland. To evaluate the potential use of salivary cortisol measurement in an OP setting, 34 healthy volunteers, 7 patients with nonadrenal conditions, and 6 patients referred for the evaluation of Cushing s syndrome provided saliva samples collected at home. One hundred twenty-two (121 IPs and 1 OP) patients had Cushing s syndrome (Cushing s disease, 98; ectopic ACTH production, 12; primary adrenal disease, 12) and 21 (18 IPs and 3 OPs) had pseudo-cushing states. Eighty-five patients with Cushing s disease had histological confirmation of the diagnosis. The remaining 13, in whom no definitive ACTH-staining tumor was found during transsphenoidal surgery, had undetectable plasma cortisol concentrations and 24-h UFC excretion after surgery. In four patients with presumed ectopic ACTH production (based on inferior petrosal sinus sampling results), the source of ACTH production has not been found; ACTH immunohistochemistry was positive in tumor tissue removed from the other seven patients with ectopic ACTH secretion. Obesity (18 of 21) and psychiatric disorders (12 of 21) were the most common diagnoses in the pseudo-cushing group. Five patients were diagnosed with depression, four with anxiety disorder, two with bipolar disorder, and one with obsessive-compulsive and personality disorder. Other diagnoses included chronic alcoholism (one), poorly controlled diabetes mellitus (two), chronic pain (two), polycystic ovarian syndrome in association with sleep disorder (one), and morbid obesity (body mass index, 30 kg/m 2 ) (two). The remaining patient had no specific diagnosis. Twenty-three patients (16 IPs and 7 OPs) with other disorders served as control subjects. The control patients had obesity (two), McArdle s disease (one), prolactinoma (two), acromegaly (one), meningioma (one), depression (two), cured Cushing s syndrome currently eucortisolemic (three), inappropriate secretion of TSH (one), aldosterone-producing tumor (one), polycystic ovarian syndrome (one), Carney complex without adrenal involvement (two), unaffected family members of patients with Carney complex (four), and multiple endocrine neoplasia-1 without adrenal abnormalities (two). None of these patients had signs or symptoms suggestive or adrenal insufficiency or hypercortisolism, and none had a history of recent glucocorticoid treatment. Nineteen of the 219 subjects studied were excluded from analysis. Fifteen without a final diagnosis included three with possible recurrence of Cushing s disease, who received no treatment; three with Carney complex and uncertain adrenal status; and nine with inconclusive diagnostic data. Other exclusions included two patients with acute anxiety and marked pain during blood drawing, one with factitious hypercortisolism, and one receiving glucocorticoid therapy. Protocol All patients were studied under protocols approved by the National Institute of Child Health and Human Development Investigational Review Board after giving informed written consent. IPs collected urine for 24 h, starting at 0600 h, for measurement of cortisol, 17-hydroxycorticosteroids (17OHCS) and creatinine. On the same day, an indwelling vein catheter was placed at 2200 h and blood was drawn for serum cortisol determination at 2330 and 2400 h and 0730 and 0800 h the next morning. At the same times, the patients expectorated into 5-ml plastic tubes, identical to those used to store plasma aliquots in an earlier study of the utility of plasma cortisol for the diagnosis of Cushing s syndrome (21). The saliva samples were refrigerated at 4 C for a maximum of 10 h and were eventually stored at 20 C. Because food ingestion and physical stress may increase circulating cortisol levels, patients were requested to abstain from physical activity and food from 2100 h until the conclusion of sampling. Patients traveling from a time zone different from that of Bethesda, Maryland, were studied at least 3 d after arrival, corresponding to the maximal number of time zones traversed. Customarily, patients on this unit are awakened between 0600 and 0800 h for blood withdrawal or vital signs and go to sleep between 2300 and 2400 h. OPs collected saliva in the same way as the IPs, at bedtime and upon awakening and observing the same dietary and activity restrictions. After collection, samples were stored in the home freezer until transported to the Clinical Center on ice by the subject. Assays Serum cortisol was measured by fluorescence polarization immunoassay (Abbott Laboratories, Abbott Park, IL) with an intra- and interassay coefficient of variation (CV) of 2.1% and 4.1%, respectively. UFC was measured by RIA (SmithKline Bioscience Laboratories, King of Prussia, PA) and urine 17OHCS were measured by the Porter-Silber method (intra- and interassay CV of these assays were 6% and 11.5%, respectively). HPLC measurements of urine cortisol were not routinely available during the study. Salivary cortisol was measured by RIA (Covance Laboratories, Inc., Vienna, VA), using a modification of an unextracted RIA for serum cortisol (22). Briefly, after centrifugation for 10 min at 2500 rpm, 50 l saliva were pipetted into assay tubes and [1, 2, 6, 7-3 H] cortisol and rabbit antisera against cortisol acetate-3 CMO: BSA were added. Following a 3-h incubation at room temperature and charcoal separation, free counts were measured. Cortisol standards ranged from pg/tube. Control samples in each assay included charcoal-stripped saliva, with and without the addition of cortisol to a final concentration of 300, 1000, and 2000 ng/dl. The detection limit ranged from ng/dl ( nmol/liter) and intra- and interassay CV were 3.4% and 12.8%, respectively. All samples from each patient were batched and run in the same assay. Samples were assayed as they were received. Twenty-eight samples were run a second time in the same assay and at the same time in a second assay, the Coat-a-Count assay (Diagnostic Products, Los Angeles, CA) as modified by other authors for saliva (18). The detection limit for this assay when performed at Covance Laboratories, Inc. was 100 ng/dl (2.76 nmol/liter).

3 Papanicolaou et al. Salivary Cortisol in Cushing s Syndrome J Clin Endocrinol Metab, October 2002, 87(10): Statistical analysis IP serum and salivary cortisol at four individual time points, the mean salivary cortisol of the morning (AM) and the evening (PM) time points, the ratio between these means (PM/AM), and excretion of cortisol and 17OHCS per gram of creatinine were evaluated. For OPs, the bedtime and wakening salivary cortisol values were examined. Results are expressed as mean sem. Undetectable salivary cortisol results in the Covance Laboratories, Inc. assay were assigned a value of 210 ng/dl (5.8 nmol/liter), the highest detection limit in various assay runs. Undetectable salivary cortisol results in the Coat-a-Count assay were assigned a value of 100 ng/dl (2.76 nmol/liter), the detection limit of the assay run. Data were analyzed with the Mann-Whitney U test. Using the cut-off criterion that gave 100% specificity for the diagnosis of Cushing s syndrome, we calculated the sensitivity and diagnostic accuracy of each parameter for the diagnosis of Cushing s syndrome, both for all patients and for those with mild to moderate hypercortisolism, defined as all patients with pseudo-cushing states and only those patients with Cushing s syndrome whose UFC was within the range seen in the patients with pseudo-cushing states (i.e. up to 242 g/d, 668 nmol/d). The 95% confidence intervals were calculated (23). We also compared the accuracy of salivary cortisol measurements with that of serum cortisol and UFC for the diagnosis of Cushing s syndrome. Salivary cortisol measurements from IPs and OPs without Cushing s syndrome were compared using an unpaired t test. A paired t test and regression analysis were used to compare the Covance Laboratories, Inc. and Coat-a-Count salivary cortisol assay results. P values of less than 0.05 were considered significant. Results Subjects Seventy-four percent of Cushing s syndrome, 86% of pseudo-cushing states, 50% of the control patients, and none of the healthy volunteers were obese (body mass index, 27 kg/m 2 ) (Table 1). Biochemical data Inpatients Compared with either the pseudo-cushing state or the control patient group, Cushing s syndrome patients had significantly greater urine cortisol and 17OHCS excretion and ratio between the average evening and morning salivary cortisol concentrations (Table 2). Cushing s syndrome patients also had significantly higher mean salivary cortisol values at all time points, compared with pseudo-cushing TABLE 1. Clinical characteristics of the subjects states and control patients (P for both, Fig. 1). There was no statistically significant difference between the mean salivary cortisol concentrations in pseudo-cushing states and control patient groups at any time point. OPs Bedtime ( h) salivary cortisol values in normal volunteers were ng/dl (range, ) ( nmol/liter; range, ) and morning ( h) values were ng/dl (range, ) ( nmol/liter; range, ). The seven control patients had bedtime ( h) salivary cortisol values of ng/dl (range, ) ( nmol/liter; range, ) and morning ( h) values of ng/dl (range, ) ( ; range, ). All three OPs with pseudo-cushing states had undetectable bedtime ( h) salivary cortisol values; their morning ( h) values were ng/dl (range, ) ( ; range, nmol/liter). The OP with Cushing s syndrome had a bedtime salivary cortisol concentration of 630 ng/dl (17 nmol/liter). In the non-cushing s syndrome patients, there was no statistical difference between the bedtime salivary cortisol values (OPs) and either the 2330-h or the 2400-h values (IPs). Criteria for the diagnosis of Cushing s syndrome Cut points for the diagnosis of Cushing s syndrome at 100% specificity were determined for each of the diagnostic parameters (Table 3). The highest salivary cortisol in patients without Cushing s syndrome, either at bedtime (from 2100 to 0200 h) in OPs or at 2330 or 2400 h in IPs was 550 ng/dl (15.2 nmol/liter) (Fig. 2). At 100% specificity, at 2400 h (for IPs) or bedtime (for OPs) salivary cortisol value more than 550 ng/dl (15.2 nmol/liter) correctly identified Cushing s syndrome with 93% sensitivity (confidence interval, 89 98%). Using this cut point, the sensitivity of salivary cortisol at 2330 h was similar to that obtained at 2400 h; 100% specificity was maintained, and the sensitivity decreased to 91%. The diagnostic value of these nighttime salivary cortisol measurements was similar to that of the serum cortisol measure- Diagnosis Age (yr) (range) Gender (M/F) (%F) BMI (kg/m 2 ) (range) Cushing s syndrome 36 1 (18 67) 29/90 (76) ( ) Pseudo-Cushing syndrome 38 2 (24 52) 5/16 (76) ( ) Control patients 37 2 (18 57) 8/15 (65) ( ) Normal volunteers 41 2 (22 73) 10/24 (71) ( ) a M, Male; F, female. a P vs. all other groups. TABLE 2. Indices of hypercortisolism in IPs Diagnosis 24-h UFC ( g/d) 17OHCS (mg/gcr/d) PM/AM Cushing s syndrome ( ) a 15 1(3 139) a ( ) a Pseudo-Cushing syndrome (21 242) b 6 1(3 12) b ( ) Control patients 51 5 (24 80) 3 0(2 5) ( ) 17OHCS/gCr, 24-h excretion of 17OHCS per gram of excreted creatinine. To convert UFC to SI units, multiply by Ranges are in parentheses. a P vs. pseudo-cushing syndrome and control patients. b P vs. control patients.

4 4518 J Clin Endocrinol Metab, October 2002, 87(10): Papanicolaou et al. Salivary Cortisol in Cushing s Syndrome FIG. 1. Morning and evening salivary cortisol levels in IPs with Cushing s syndrome (E), pseudo-cushing states (F), and patient controls (f). To convert salivary cortisol nanograms per deciliter to nanomole per liter, multiply by *, P vs. pseudo-cushing states (PCS) and controls (CTRL). TABLE 3. Sensitivity [% (confidence interval)] of biochemical parameters for the diagnosis of Cushing s syndrome, using a criterion that achieved 100% specificity Parameter Criterion All IPs (n 121) Patients with UFC 242 g/d (n 50) 24-h UFC 242 g/d 56 (47 65) 0 17OHCS/Cr 11.8 mg/gcr/d 53 (43 62) 33 (20 47) F 2330 h 10.1 g/dl 90 (84 95) 81 (70 92) F 2400 h 8.8 g/dl 97 (93 100) 89 (81 98) sf 2330 h 510 ng/dl 91 (86 96) a 86 (76 96) sf 2400 h 550 ng/dl 93 (89 98) 84 (73 94) 17OHCS/Cr, 17OHCS per gram of urinary creatinine; F, serum cortisol; sf, salivary cortisol. To convert UFC to SI units, multiply by 2.76; to convert plasma cortisol to SI units, multiply by 27.6; to convert sf to SI units, multiply by a P 0.57 vs. midnight cortisol. ments at the same times, as shown in Table 3 (P 0.33 and P 0.61, salivary vs. serum cortisol values at 2330 h and 2400 h, respectively). This is not surprising because a strong correlation existed between the 2400-h salivary and plasma values (r 0.67). At 100% specificity, all morning serum and salivary cortisol end points and the PM/AM salivary cortisol value had a sensitivity of 41% or less (data not shown). Glucocorticoid excretion discriminated Cushing s syndrome slightly better, with a sensitivity of 56% for UFC and 53% for 17OHCS (Table 3). The distinction between Cushing s syndrome and pseudo- Cushing states is most difficult in the setting of mild to moderate hypercortisolism. To evaluate the utility of nighttime salivary cortisol measurements in this setting, we analyzed the data of all patients with UFC in the pseudo- Cushing range [i.e. 242 g/d (668 nmol/d)]. In this subset of patients, the sensitivity of nighttime salivary cortisol measurements remained superior to all other measures (Table 3). Seven patients with Cushing s syndrome had normal UFC [ 90 g/d (248 nmol/d)] at the time of evaluation. A 2400-h salivary cortisol of more than 550 ng/dl (15.2 nmol/liter) correctly identified four of these patients, and a 2330 salivary of more than 510 ng/dl (14.1 nmol/liter) correctly identified six patients (Fig. 3). Comparison of the Covance Laboratories, Inc. in-house and Coat-a-Count salivary cortisol assay results for the same samples showed that the Covance Laboratories, Inc. assay consistently reported higher values. Fifteen Coat-a-Count results at or below the detection limit of the assay (100 ng/dl) corresponded with in-house results between 110 ng/dl (detection limit, n 3) and 350 ng/ml. Results in the Coat-a- Count assay greater than 100 ng/dl corresponded to values in the Covance Laboratories, Inc. in-house assay that were, on average, twice as high (mean se, vs ng/dl, P 0.001). Discussion This study demonstrated that a bedtime salivary cortisol measurement can exclude the diagnosis of Cushing s syndrome while identifying more than 90% of patients with the disorder. Our envisioned use of the salivary cortisol measurement as a screening test for Cushing s syndrome included bedtime sampling and an assay-specific cut point of 550 ng/dl (15.2 nmol/liter), which carried 100% specificity at a wide range of time points in this study. When compared at 100% specificity, the diagnostic sensitivity of a bedtime salivary cortisol measurement was equivalent to that of a 2400-h cortisol measurement and superior to all other parameters. Although this study demonstrated that 2400 h and bedtime salivary cortisol measurements are of equal value in non-cushing s patients, further studies in Cushing s patients in an OP setting will be necessary to prove the usefulness of a bedtime sampling strategy for the diagnosis of Cushing s syndrome. Salivary cortisol measurement offers several advantages: It reflects the unbound fraction of circulating cortisol, and thus it is not affected by alterations in cortisol-binding globulin (as, for example, during treatment with oral contraceptives) (24). Salivary cortisol concentration is not influenced by saliva flow rate (24), making it a reliable end point. The advantages of collection at home at bedtime are obvious: The cost of an office or hospital visit is eliminated, and potential increases in cortisol levels because of anxiety or an unfamiliar environment is mitigated. However, an unsupervised OP environment may allow for other stressors. Thus, we emphasize that the patients abstain from food and physical activity for 3 h before they collect the saliva because these activities or other severe emotional or physical disturbance may elevate cortisol levels (24) and give false results. Although nighttime salivary cortisol measurement was superior to any other parameter in detecting patients with mild Cushing s syndrome, eight patients with Cushing s syndrome (7%) had 2400-h salivary cortisol within the pseudo-cushing range. All had mild hypercortisolism [UFC 250 g/d (690 nmol/d)], and three had normal UFC at the time of evaluation. This is not surprising. In certain patients with Cushing s syndrome, the diurnal rhythmicity is preserved; furthermore, Cushing s syndrome can be periodic,

5 Papanicolaou et al. Salivary Cortisol in Cushing s Syndrome J Clin Endocrinol Metab, October 2002, 87(10): FIG. 2. Nighttime salivary cortisol values in healthy volunteers ( ), patient controls (Œ), pseudo-cushing states patients (F), and patients with Cushing s syndrome (E). Samples were collected at 2400 h from IPs and at bedtime ( h) from OPs. The horizontal line represents the highest salivary cortisol value (550 ng/dl, 15.2 nmol/liter) for non-cushing s syndrome subjects. To convert salivary cortisol nanograms per deciliter to nanomoles per liter, multiply by with a normal biochemical profile during the quiescent phase (25, 26). Thus, even though salivary cortisol measurements are very accurate for the diagnosis of Cushing s syndrome, single measurements may be of limited value in cases of periodic Cushing s syndrome. Because Cushing s syndrome is generally a progressive disease, prolonged follow-up and possibly other tests, such as Dexamethasone Suppression CRH stimulation test (6), may be necessary to make the diagnosis in such cases. Salivary cortisol values have been advocated for the evaluation of hypercortisolism as an end point in the 1-mg DST (20, 24, 27 29) and to identify intermittent hypercortisolism on an OP basis (30, 31). Several investigators have shown that mean 2400-h salivary cortisol concentrations are higher in Cushing s syndrome than in normal individuals (24, 27, 28, 31, 32), and three studies (18 20) have shown that obese patients or individuals suspected of having Cushing s syndrome can be distinguished from patients with the disorder. All of these studies demonstrate a sensitivity of more than 90% for the detection of Cushing s syndrome, with high specificity. Although the present study confirms and extends those findings, it highlights important issues in the use of salivary cortisol as a screening test for Cushing s syndrome. Despite the similarity in diagnostic performance, the current salivary cortisol data differ from previous studies, mainly in terms of the range of responses seen in individuals without Cushing s syndrome. The possible reasons for this difference represent issues that should be considered by investigators and clinicians when using the test to screen for Cushing s syndrome. Although it is possible that the subjects without Cushing s syndrome were different in the various studies and the differences represent a true finding, it is also possible that the assays or cut points for their interpretation differed or that the collection method influenced the results. In this study, the cut point of 550 ng/dl represented the highest evening sal-

6 4520 J Clin Endocrinol Metab, October 2002, 87(10): Papanicolaou et al. Salivary Cortisol in Cushing s Syndrome FIG. 3. Diagnostic utility of urine cortisol and 2400-h salivary cortisol measurement in IPs with mild hypercortisolism (UFC 250 g/d). E, Patients with Cushing s syndrome; F, patients with pseudo-cushing states and controls. The horizontal line represents the diagnostic cut point for salivary cortisol (550 ng/dl); the vertical line indicates the upper limit of normal urine cortisol (90 g/d). To convert salivary cortisol nanograms per deciliter to nanomoles per liter, multiply by ; to convert UFC micrograms per day to nanomoles per day, multiply by ivary cortisol result in a patient without Cushing s syndrome. Others have set cut points of ng/dl based on use of the mean plus two sd or use of the value at the 90th or 97.5th percentile of the normal subjects (18 20). Use of these strategies in the current study would yield cut points of ng/dl, still higher than other studies. All recent studies of salivary cortisol use RIA, but these assays differ. For example, the modified Coat-a-Count assay used by Raff et al. (18) has a detection limit of 14 ng/dl (0.4 nmol/liter). By contrast, RIAs used in the present study and by Castro et al. (19) are less sensitive, with detection limits of 80 and 62 ng/dl, respectively. Furthermore, the Coat-a- Count assay returned values about half as great as the Covance Laboratories, Inc. assay, when performed at Covance Laboratories, Inc. Raff et al. (33) recently found a similar disparity when comparing their Coat-a-Count assay with an enzyme immunoassay. In that study, the enzyme immunoassay results were nearly 2-fold those of the Coat-a-Count assay. Further analysis using a stock solution of cortisol standard diluted in saliva suggested that the Coat-a-Count assay returned results closest to the expected value. The technique for collection of saliva either direct expectoration or use of the Salivette collection device also might lead to different results. It is possible that the cotton wad in the Salivette may trap cortisol, leading to a lower result than what would be found if the saliva had been collected directly. However, the similarity of salivary cortisol cut points proposed for interpretation of the overnight DST in studies using Salivette (103 ng/dl) (29) and direct collection ( ng/dl) (20, 27) suggests that collection technique may not be an important factor. Although we required IPs to refrain from exercise and eating, the stress of venipuncture or hospital admission might result in a higher salivary cortisol value and account for the differences between studies. Although IPs had a catheter placed 90 min before the blood and saliva sampling, we have observed that the stress of venipuncture is transient, with any increase in plasma cortisol returning to baseline within 30 min. The similarity between evening salivary cortisol values in OPs and IPs with nonadrenal disease also suggests that hospitalization per se did not account for the higher values in this study. This study supports previous endorsements of the nighttime salivary cortisol as a simple, convenient, accurate, and cost-effective screening test for Cushing s syndrome. However, it is important to recognize that the nighttime salivary cortisol cut point used to interpret the results varies among studies. These criteria are based on results from a relatively small number of patients with obesity, nonadrenal disorders, or pseudo-cushing states and may be influenced by periodic hypercortisolism and assay variability. Thus, optimally salivary cortisol assays should be chosen and interpreted based on assay-specific normative data in combination with results from patients suspected but proven not to have Cushing s syndrome. We also suggest that commercial laboratories consider largescale validation of these assays to make them more widely available to practicing clinicians.

7 Papanicolaou et al. Salivary Cortisol in Cushing s Syndrome J Clin Endocrinol Metab, October 2002, 87(10): Acknowledgments Received April 4, Accepted July 16, Address all correspondence and requests for reprints to: Lynnette K. Nieman, M.D., Pediatric and Reproductive Endocrinology Branch, National Institutes of Health, Building 10, Room 9D42 MSC 1583, 10 Center Drive, Bethesda, Maryland niemanl@nih.gov. References 1. Orth DN 1995 Cushing s syndrome. N Engl J Med 332: Gold PW, Loriaux DL, Roy A, Kling MA, Calabrese JR, Kellner CH, Post R, Pickar D, Gallucci W, Avgerinos P, Schulte H, Paul S, Nieman LK, Oldfield EH, Cutler GB, Chrousos GP 1986 Responses to corticotropin-releasing hormone in the hypercortisolism of depression and Cushing s disease. Pathophysiologic and diagnostic implications. N Engl J Med 314: Rees LH, Besser GM, Jeffcoate WJ, Goldie DJ, Marks V 1977 Alcohol-induced pseudo-cushing s syndrome. 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