Midnight salivary cortisol, measured by highly sensitive electrochemiluminescence immunoassay, for the diagnosis of Cushing s syndrome

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1 Cent. Eur. J. Med. 4(1) DOI: /s y Central European Journal of Medicine Midnight salivary cortisol, measured by highly sensitive electrochemiluminescence immunoassay, Maria Yaneva, Georgi Kirilov, Sabina Zacharieva Clinical Centre of Endocrinology and Gerontology, Medical University, 1303 Sofia, Bulgaria Research Article Received 17 September 2008; Accepted 20 December 2008 Abstract: The aim of the present study is to evaluate the measurement of midnight salivary cortisol as a method of screening for Cushing s syndrome (CS). Here we tested the performance of a highly sensitive electrochemiluminescence immunoassay (ECLIA) for midnight salivary cortisol measurement in an extensive clinical study (n=104). Three groups were investigated: 30 patients with CS, 34 with obesity and 40 healthy normal weight controls. All of them collected saliva samples at 24:00 h and urine samples over the same day (24 hour period). An electrochemiluminescence immunoassay was used to measure salivary cortisol. Mean midnight salivary cortisol in healthy volunteers, obese patients and patients with CS was 8.33 ± 3.62, 8.13 ± 4.47 and ± nmol/l, respectively. No significant difference was found between midnight salivary cortisol in healthy and obese subjects (P>0.05). In contrast, salivary cortisol at midnight was significantly higher in patients with CS (P<0.001) as compared to both other groups. The cut-off point of 14.2 nmol/l yielded a sensitivity of 93.3% and a specificity of 94.2% (AUCROC=0.984 ± 0.01( ). A strong positive correlation between midnight salivary cortisol and urinary free cortisol has been found in the CS group (r=0.686, P<0.0001). Our results demonstrate that measurement of midnight salivary cortisol could be successfully used as a first-line screening method for CS. Our data approve ECLIA as a simple, reliable and timesaving method for the assessment of salivary cortisol. Automated measurement of midnight salivary cortisol by ECLIA would facilitate the routine practice in the screening for CS. Keywords: Midnight salivary cortisol Cushing s syndrome ECLIA Versita Warsaw and Springer-Verlag Berlin Heidelberg. 1. Introduction Measurement of salivary cortisol has recently become a validated method of appreciation of cortisol secretion. Several studies [1-8] have demonstrated the usefulness of midnight salivary cortisol determination for the diagnosis of Cushing s syndrome (CS). All these studies used different radioimmunoassay (RIA) techniques to measure salivary cortisol. These RIA methods require several modifications: increasing analyte volume, time of incubation, etc. They complicate the measurement, make it longer and suppose more errors in its performance. Recently, it has been reported [9] that a fully automated electrochemiluminescence immunoassay (ECLIA) measurement of salivary cortisol is a useful tool in the assessment of the activity of the hypothalamic-pituitaryadrenal axis. Since ECLIA has not been tested in a large clinical trial, we decided to use it for the determination of salivary cortisol. Thus, here we present the first results on the performance of ECLIA for midnight salivary cortisol measurement in an extensive clinical study. Midnight salivary cortisol determination is a test that could be promising for the screening of CS [1]. The authors of the Consensus Statement on the diagnosis and complications of CS [1] recommend larger studies before the validation of late-night salivary cortisol as a first-line screening test for CS. Here we show the results of midnight salivary cortisol measurement in three groups: patients with CS, obese patients and normal weight volunteers. Our data demonstrate the good diagnostic accuracy of evening salivary cortisol determination for the diagnosis of CS. Something more, we show no differences between midnight salivary cortisol levels of normal weight and obese subjects. * zacharieva@uheg.medicalnet-bg.org 59

2 Midnight salivary cortisol, measured by highly sensitive electrochemiluminescence immunoassay, 2. Material and Methods 2.1. Patient population A total of 104 subjects were studied, divided into three groups: patients with CS, patients with obesity and normal weight healthy volunteers. Patients with CS and obesity were hospitalized in the Clinical Center of Endocrinology and Gerontology, Sofia, between December 2006 and December Thirty patients had CS (5 men and 25 women; mean age ± S.E.M, 39.9 ± 12.7 yr; body mass index (BMI) 29.5 ± 7.2 kg/m²). The diagnosis of CS was based on typical clinical and biochemical features (elevated urinary free cortisol (UFC), lack of circadian cortisol rhythm and suppressibility with low dose dexamethasone). In the CS group there were 19 patients with Cushing s disease, 10 with adrenal CS and 1 with ectopic CS. The diagnosis of Cushing s disease was based on pathological confirmation after transsphenoidal surgery and/or postoperative biochemical and clinical resolution. Patients with adrenal CS were operated on and had histological confirmation. The second group (n=34) was comprised of patients that were hospitalized for evaluation of obesity. There were 10 men and 24 women (age, 41.1 ± 13.5 yr; BMI, 36.4 ± 4.8 kg/m²). Some of them had clinical features suggesting hypercortisolism: menstrual irregularities, essential hypertension, idiopathic hirsutism etc. None of the obese patients had elevated UFC; they all had a normal diurnal cortisol rhythm and displayed a sufficient suppressibility with dexamethasone (plasma cortisol at 08:00h < 50 nmol/l after 1 mg Dexamethasone), so CS had been excluded. Forty apparently healthy normal weight volunteers among the medical staff agreed to participate in the study (16 men and 24 women; age, 37.2 ± 9.2 yr; BMI, 23.3 ± 2.7 kg/m²). They formed the control group. The healthy subjects collected saliva and urine samples in ambulatory conditions. All the patients and controls provided signed informed consent to the study, which was approved by the Local Ethics Committee of the Clinical Centre of Endocrinology and Gerontology, Sofia Protocol Saliva samples were collected at 24:00 h before asleep. All patients and volunteers were instructed to have dinner and to brush their teeth before 20:00 h, to abstain from any significant physical activity after 20:00 h and to rinse the mouth at midnight before saliva collection. Participants were instructed to abstain from smoking before the investigation. Saliva samples were collected in specially designed plastic tubes, Salivettes (Sarstedt AG & Co) that contain a cotton device, placed for 2-3 min in the mouth. All studied subjects collected urine samples over the same day (24 hours period) Assays Salivary cortisol was determined by fully automated highly sensitive competitive electrochemiluminescence immunoassay using Elecsys Cortisol reagent kit (Roche). All saliva samples were run in big series in duplicate. The measuring range of the assay defined by the lower detection limit (analytical sensitivity) and the maximum of the standard curve was nmol/l. The analytical and functional sensitivity of the test determined by our laboratory did not differ significantly by those established by the manufacturer (<1 nmol/l and <8 nmol/l respectively). The dilution test showed linear results in the lowest concentration range between 1.0 and 8.5 nmol/l. Within-run precision and between-run precision were 3.2 and 4.7% respectively. The following cross-reactivities were found: corticosterone 5.8%, 11-deoxycortisol 4.1%, 17-α-hydroxyprogesterone 1.5%, prednisone 0.28% and dexamethasone 0.08%. Only 50 µl of saliva were required for the analysis. The comparison of the ECLIA cortisol assay with a commercial RIA test (Immunotech, Beckman Coulter Co, France) for the determination of cortisol in saliva gave the highly significant correlation (r =0.91). The daily excretion of free cortisol (nmol/24h) was measured in urine samples by the direct assay procedure using an RIA cortisol kit manufactured by Immunotech (Beckman Coulter Co, France). Intra-assay and interassay coefficients of variation were 5.8 and 9.2%, respectively. The analytical sensitivity of the assay was 10 nmol/tube. Extremely low cross reactivity was obtained against other naturally occurring steroids. The normal values of 24 hours urinary cortisol are between 38 and 270 nmol/24h Statistical analysis The data were analyzed using SPSS 13.0 software (SPSS, Chicago, IL, USA). The average values of each variable were compared using analysis of variance. Linear regression analysis and Wilcoxon analysis were used when appropriated. All results are expressed as mean ± standard deviation (S.D.). Significance was assumed when P<0.05. The quality of midnight salivary cortisol and UFC determination in the diagnosis of CS was tested by receiver operating characteristic (ROC) analysis and expressed as the area under the curve (AUC). 60

3 M. Yaneva, G. Kirilov, S. Zacharieva Table 1. Hormonal results of studied groups (n=104). Healthy (n=40) Obese (n=34) CS (n=30) Midnight salivary cortisol (nmol/l) 8.33 ± ± ± * Urinary free cortisol(nmol/24h) ± ± ± * Mean ± SD, * p< vs. healthy and obese subjects Figure 1. Individual data points for midnight salivary cortisol values of healthy normal weight volunteers (n=40); obese subjects (n=34); and patients with Cushing s syndrome (n=30). A broken horizontal line represents the cut-off point value (14.2 nmol/l). Figure 3. Correlation between midnight salivary cortisol (nmol/l) and urinary free cortisol (nmol/24h) in patients with Cushing s syndrome (saliva and urine samples are collected over the same day) (r=0.686, P<0.0001) nmol/l h salivary cortisol (nmol/l) healthy obese Figure 2. Area under the curve the ROC curve for midnight salivary cortisol determination ( reference line, midnight salivary cortisol determination). AUC ± standard deviation (95% confidence interval) = ± 0.1 ( ). CS urinary free cortisol (nmol/24h) 3. Results The three groups were homogenous in age and sex distribution (P>0.05). The BMI differed significantly in the three groups (P<0.0001). Hormonal results are presented in Table 1. No significant difference in midnight salivary cortisol had been observed between healthy and obese subjects (P>0.05). By contrast, salivary cortisol at 24:00 h was significantly higher in patients with CS compared to both other groups (P<0.0001) (Table 1, Figure 1). The cut-off point for midnight salivary cortisol of 14.2 nmol/l yielded a sensitivity of 93.3% and a specificity of 94.2%. The AUC derived from the ROC analysis was optimal (AUCROC=0.984 ± 0.01( ) (AUCROC ± SD (interval of confidence of 95%))) (Figure 2). UFC in obese patients and healthy normal weight volunteers was similar (P>0.05), while UFC in the CS group was significantly higher (Table 1). The cut-off point of 270 nmol/24h for UFC had a sensitivity of 81.5% and a specificity of 93.2% (AUCROC=0.975 ± 0.014( )). The AUCROC of midnight salivary cortisol and UFC did not differ significantly (P>0.05) and was close to 1, demonstrating the good performance of both tests. 61

4 Midnight salivary cortisol, measured by highly sensitive electrochemiluminescence immunoassay, We found a statistically significant positive correlation between UFC and midnight salivary cortisol in patients with CS (r=0.686, P<0.0001) (Figure 3). Salivary cortisol at 24:00h and UFC did not show a significant correlation in healthy and obese subjects. 4. Discussion Recently, Van Aken et al. [9] have reported a fully automated electrochemiluminescence immunoassay for the measurement of salivary cortisol. This method has several advantages over other radioimmunoassays and enzyme immunoassays (EIA): it is automated, samples need no pretreatment, results can be quickly obtained (within 20 minutes), and collection of a specific number of samples is not needed for efficient use. Authors suggest it as a suitable test for daily laboratory and clinical use. A detailed review of the literature shows that all big studies on the use of midnight salivary cortisol for the diagnosis of CS are based on RIA [2-8]. They all are modified RIAs for serum cortisol and need modifications of the analyte volume, time of incubation, etc. This is time consuming and suggests more errors. Moreover, performance of RIA requires a specially equipped laboratory with sophisticated methods of radioactive protection. Raff et al. [10] have reported a new EIA for salivary cortisol measurement and compared it to RIA. In another study, Raff et al. [11] tested another kit for enzyme immunoassay - EIA Salimetrics. As we see, there are different available methods of measurement of salivary cortisol and they all have their advantages and disadvantages. Since the determination of midnight salivary cortisol seems to be one of the simplest approaches in the screening for CS, clinical and laboratory efforts are focused to find the most precise, easy and cost-effective method of measurement. Until now, no clinical studies have been reported on the use of ECLIA to appreciate cortisol secretion. In this study we present the first results on ECLIA determination of midnight salivary cortisol in an extensive clinical study. Our data show significantly higher midnight salivary cortisol levels in patients with endogenous hypercortisolism compared with those of healthy normal weight volunteers (P<0.001). These data are consistent with previous studies that reveal the good sensitivity and specificity of this screening method [2-8]. The cutoff point of 14.2 nmol/l yielded a sensitivity of 93.3% (two patients with CS have values above this cut-off and nmol/l) and a specificity of 94.2% (four patients with obesity have values above 14.2 nmol/l 14.43, 14.53, 16.70, nmol/l; none of the normal weight volunteers had midnight salivary cortisol above 14.2 nmol/l) (Figure 1). The AUC derived from the ROC analysis is close to 1 (Figure 2), demonstrating the excellent quality of this test. Something more, we didn t find any significant difference between the AUCROC of midnight salivary cortisol and UFC measurment. We can conclude that both tests have the same good diagnostic performance. Our cut-off value (14.2 nmol/l) is similar to that chosen by Papanicolaou et al. [4] (15.2 nmol/l) and differs significantly from other reported cut-offs: 3.6 nmol/l [2], 5.52 nmol/l [6] and 7.7 nmol/l [3]. We can conclude that most probably the cut-off value is laboratory-dependant and not assay-dependant since Papanicolau et al. used RIA to determine salivary cortisol [4]. As mentioned in the consensus statement for the diagnosis of CS, each laboratory should establish its own values [1]. As the functional sensitivity of the test is relatively high (<8 nmol/l) we can conclude that ECLIA is better in assessing hypercortisolism than hypocortisolism. Our results show no differences between values of midnight salivary cortisol of normal weight and obese subjects (P>0.05), thus confirming data that cortisol rhythm is preserved in obese patients [6,12]. Moreover, saliva samples of healthy people were collected in ambulatory settings, while samples of obese subjects were obtained during hospitalization. As cortisol is a stress hormone, one could presume that hospitalization, as a stress factor, would elevate cortisol values. The results of the present study show that no significant differences exist between midnight salivary cortisol in healthy, ambulatory subjects and obese, hospitalized patients, suggesting that hospitalization should not be considered as a significant stress factor, causing misleading results. We found a statistically significant positive correlation between UFC and midnight salivary cortisol in patients with CS (r=0.612, P<0.005). This result confirms previously reported data from other authors [2,5,6] and suggests that midnight salivary cortisol could be used as a reliable alternative of UFC, especially in ambulatory patients. Salivary cortisol is strongly correlated with plasma free cortisol, the biologically active form of circulating cortisol [13-17]. Moreover, it is independent of the salivary flow rate [18]. Saliva sample collection is easy, non-stressful and non-invasive and does not require trained medical staff. It can be easily performed in ambulatory conditions. Samples can be stored at room temperature [19] and sent by regular mail [20,21]. Despite its advantages, salivary cortisol measurement has its limitations: in cases of dehydratation, Sjögren s syndrome, gingivitis, etc. 62

5 M. Yaneva, G. Kirilov, S. Zacharieva In conclusion, our results, obtained in a large cohort of subjects (n=104), support data that midnight salivary cortisol with its high sensitivity and specificity could be considered as a method of first-line screening for CS. Our data, obtained by ECLIA, approve it as a reliable method of assessment of salivary cortisol. Moreover, it is simple and timesaving and does not require laboratory with special radioactive protection. We suggest this automated method for a larger use in the routine clinical practice in order to facilitate screening for CS. Acknowledgements This work was supported by a grant from the Medical University of Sofia, Bulgaria. References [1] Arnaldi G., Angeli A., Atkinson A.B., Bertagna X., Cavagnini F., Chrousos G.P., et al., Diagnosis and complications of Cushing's syndrome: a consensus statement, J. Clin. Endocrinol. Metab., 2003, 88, [2] Raff H., Raff J.L., Findling J.W., Late-night salivary cortisol as a screening test for Cushing s syndrome, J. Clin. Endocrinol. Metab., 1998, 83, [3] Castro M., Elias P.C., Quidute A.R., Halah F.P., Moreira A.C., Out-patient screening for Cushing s syndrome: the sensitivity of combination of circadian rhythm and overnight dexamethasone suppression salivary cortisol tests, J. Clin. Endocrinol. Metab., 1999, 84, [4] Papanicolaou D.A., Mullen N., Kyrou I., Nieman L.K., Nighttime salivary cortisol: A useful test for the diagnosis of Cushing s syndrome, J. Clin. Endocrinol. Metab., 2002, 87, [5] Putignano P., Toja P., Dubini A., Giraldi F.P., Corsello S.M., Cavagnini F., Midnight salivary cortisol versus urinary free and midnight serum cortisol as screening tests for Cushing s syndrome, J. Clin. Endocrinol. Metab., 2003, 88, [6] Yaneva M., Mosnier-Pudar H., Dugue M.A., Grabar S., Fulla Y., Bertagna X., Midnight salivary cortisol for the initial diagnosis of Cushing's syndrome of various causes, J. Clin. Endocrinol. Metab., 2004, 89, [7] Gafni R.I., Papanicolau D.A., Nieman L.K., Night time salivary cortisol measurement as a simple, noninvasive, outpatient screening test for Cushing s syndrome in children and adolescents, J. Pediatr., 2000, 137, [8] Martinelli Jr C.E., Sader S.L., Oliveira E.B., Daneluzzi J.C., Moreira A.C., Salivary cortisol for screening of Cushing s syndrome in children, Clin. Endocrinol. (Oxf), 1999, 51, [9] Van Aken M.O., Romijn J.A., Miltenburg J.A., Lentjes E.G.W.M., Automated measurement of salivary cortisol, Clin. Chem., 2003, 49, [10] Raff H., Homar P.J., Burns E.A., Comparison of two methods for measuring salivary cortisol, Clin. Chem., 2002, 48, [11] Raff H., Homar P.J., Skoner D.P., New enzyme immunoessay for salivary cortisol, Clin. Chem., 2003, 49, [12] Pirich K, Vierhapper H., 24-hour serum concentration profile of cortisol in patients with Cushing s disease, Exp. Clin. Endocrinol., 1998, 92, [13] Laudat M.H., Cerdas S., Fournier C., Guiban D., Guilhaume B., Luton J.P., Salivary cortisol measurement: a practical approach to assess pituitary-adrenal function, J. Clin. Endocrinol. Metab., 1988, 66, , [14] Riad-Fahmy D., Read G., Walker R., Griffiths K., Steroids in saliva for assessing endocrine function, Endocr. Rev., 1982, 3, [15] Vining R.F., McGinley R.A., Maksvytis J.J., Ho K.Y., Salivary cortisol - a better measure of adrenal cortical function than serum cortisol, Ann. Clin. Biochem., 1983, 20, [16] Walker R.F., Salivary cortisol determinations in the assessment of adrenal activity, Front. Oral. Physiol., 1984, 5, [17] Vining R.F., McGinley R.A., The measurement of hormones in saliva: possibilities and pitfalls, J. Steroid. Biochem., 1987, 27, [18] Vining R.F., McGinley R.A., Transport of steroids from blood to saliva. In Immunoassays of steroids in saliva, pp Eds Read GF, Riad-Fahmy D, Walker RF & Griffiths K. Cardiff: Alpha Omega Publishing Ltd, 1984 [19] Chen Y.M., Cintron N.M., Whitson P.A., Longterm storage of salivary cortisol samples at room temperature, Clin. Chem. 1992, 38, [20] Mosnier-Pudar H., Thomopoulos P., Bertagna X., Fournier C., Guiban D., Luton J,P., Longdistance and long-term follow-up of a patient with intermittent Cushing s disease by salivary cortisol measurements, Eur. J. Clin. Endocrinol., 1995, 63

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