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Letters |
1 Endocrine Research Laboratory St. Luke’s Medical Center Milwaukee, WI 53215
2
Department of Medicine Medical College of Wisconsin Milwaukee, WI 53226
3
Department of Allergy/Immunology Children’s Hospital of Pittsburgh Pittsburgh, PA 15213
aAddress correspondence to this author at: Endocrinology, St. Luke’s Physician’s Office Bldg., 2801 W KK River Pkwy., Suite 245, Milwaukee, WI 53215. Fax 414-649-5747; e-mail hraff{at}mcw.edu.
To the Editor:
The measurement of salivary cortisol is emerging as the simplest approach in the diagnosis of Cushing syndrome (1)(2)(3). One of the problems with previous methods is the lack of a Food and Drug Administration (FDA)-cleared method. We have evaluated an enzyme immunoassay (EIA) for salivary cortisol marketed by Salimetrics (State College, PA) and recently cleared by the FDA for in vitro diagnostic use.
We compared the new EIA with our modified RIA (2) in 147 samples. The first set of samples (n = 44) was collected at 2300 and 0700 from a group of apparently healthy adult individuals (n = 22; age range, 25–60 years; 11 females and 11 males). The second set of samples (n = 30) was collected at 2300 by patients (n = 30; age range, 12–84 years; 22 females and 8 males) to screen for Cushing syndrome. The third set of samples (n = 73) was collected between 0600 and 1000 from participants (n = 42; age range, 6–14 years; 21 females and 21 males) enrolled in a study of allergic rhinitis. The study was approved by the appropriate Institutional Review Boards, and consent was obtained. Saliva was sampled as described previously (2) with a collecting device (Salivettes with no preservative; Sarstedt).
Salivary cortisol was measured by two methods. The serum cortisol RIA [Coat-a-Count TKCO; Diagnostic Products (DPC)] was used as commonly modified for the measurement of salivary cortisol (2). The salivary cortisol EIA (product no. 1-1102; Salimetrics) was used as instructed without modification. The sample volumes were 200 and 25 µL for the modified RIA and EIA, respectively, and the incubation times were 3 h for the RIA and a total of 80 min for the EIA. The EIA calibrators ranged from 0.2 to 49.7 nmol/L and were provided in a saliva-like matrix with a nonmercury preservative.
The lower detection limit of the EIA was 0.3 nmol/L. The intraassay imprecision (CV) was 5.2% at 3.1 (SD, 0.2) nmol/L (n = 10) and 2.6% at 10.4 (0.3) nmol/L (n = 10). Interassay (total) imprecision (CV) was 11% at 2.8 (0.3) nmol/L (n = 10), 11% at 10.1 (1.1) nmol/L (n = 10), and 6.9% at 25.0 (1.7) nmol/L (n = 10).
The correlation of the salivary cortisol measured by RIA (x axis) vs EIA (y axis) yielded a slope of 0.84 (SE, 0.01) and a y-intercept of 1.2 (SE, 0.3) nmol/L (r2 = 0.96; Sy|x = 2.5 nmol/L; n = 147) with results spanning the range 0.3–130 nmol/L. The slope was significantly less than unity, and the y-intercept was significantly >0. Because the critical range for a salivary cortisol assay for screening for Cushing syndrome is 0.3–10.0 nmol/L (1)(2)(3), Fig. 1
shows only those data pairs with DPC results 
10 nmol/L. The slope was not significantly different from unity, and the y-intercept was not significantly different from 0.
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On the basis of the correlation shown in Fig. 1
, the reference interval for the Salimetrics EIA calculated from a large group of verified healthy individuals (2) at 2300 (the appropriate time for the diagnosis of Cushing syndrome) was <0.3 to 4.3 nmol/L. We then evaluated salivary cortisol at 2300 in samples from 14 patients (4 males and 10 females; age range, 20–78 years) with Cushing syndrome confirmed by subsequent measurement of additional increased salivary cortisol, increased urinary free cortisol, and/or abnormal low-dose dexamethasone suppression testing using previously described criteria (3). The mean (SD) salivary cortisol as measured by the Salimetrics EIA was 20.4 (13.4) nmol/L (range, 5.3–46.8; 95% confidence interval, 12.7–28.1 nmol/L). Salivary cortisol concentrations at 2300 in these patients with confirmed Cushing syndrome were all clearly increased.
We have previously shown that the DPC RIA yields accurate results for samples enriched with known concentrations of hydrocortisone (4). The Salimetrics EIA yielded results very close to those obtained with the DPC RIA, particularly in the critical diagnostic range of 0.3–10.0 nmol/L. This is in contrast to the results that we recently obtained with the EIA from Diagnostic Systems Laboratories (DSL), which were 
1.7-fold higher than those obtained with the DPC RIA (4). The only relevant disadvantage of the Salimetrics EIA is that the highest calibrator concentration (49.7 nmol/L) is considerably lower than those of the DSL EIA (276 nmol/L) and the DPC RIA (138 nmol/L). This will require more frequent dilution of high-concentration samples from patients with Cushing syndrome, which can have cortisol concentrations >50 nmol/L (1)(2). The advantages of the Salimetrics EIA compared with the DSL EIA are that it produces results not different from those obtained with the DPC RIA in the clinically important range and that it is FDA-cleared for in vitro diagnostic use.
We have previously demonstrated that urinary free cortisol does not correlate with salivary cortisol in healthy individuals primarily because of the episodic and circadian nature of salivary cortisol measurement (2). However, there is good correlation in patients with Cushing syndrome, primarily because of their increased cortisol concentrations throughout the circadian period (2). The standard approach to the evaluation of Cushing syndrome, therefore, has been the measurement of 24-h urinary free cortisol (3). However, a recent study has demonstrated in a large study population that several increased nighttime salivary cortisol measurements are superior to the measurement of urinary free cortisol in establishing the diagnosis of Cushing syndrome (5). Because of its accuracy, simplicity, and cost-effectiveness, we believe that measurement of nighttime salivary cortisol will become the principal method to screen for Cushing syndrome.
Acknowledgments
This study was supported in part by Aventis Pharmaceuticals.
References
The following articles in journals at HighWire Press have cited this article:
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  S. Kidambi, H. Raff, and J. W Findling Limitations of nocturnal salivary cortisol and urine free cortisol in the diagnosis of mild Cushing's syndrome Eur. J. Endocrinol., December 1, 2007; 157(6): 725 - 731. [Abstract] [Full Text] [PDF]
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 S. Kidambi, J. M. Kotchen, C. E. Grim, H. Raff, J. Mao, R. J. Singh, and T. A. Kotchen Association of Adrenal Steroids With Hypertension and the Metabolic Syndrome in Blacks Hypertension, March 1, 2007; 49(3): 704 - 711. [Abstract] [Full Text] [PDF]
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 J. W. Findling and H. Raff Cushing's Syndrome: Important Issues in Diagnosis and Management J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 3746 - 3753. [Abstract] [Full Text] [PDF]
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M. Yaneva, H. Mosnier-Pudar, M.-A. Dugue, S. Grabar, Y. Fulla, and X. Bertagna Midnight Salivary Cortisol for the Initial Diagnosis of Cushing's Syndrome of Various Causes J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3345 - 3351. [Abstract] [Full Text] [PDF]
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 M. Groschl, M. Rauh, and H.-G. Dorr Circadian Rhythm of Salivary Cortisol, 17{alpha}-Hydroxyprogesterone, and Progesterone in Healthy Children Clin. Chem., October 1, 2003; 49(10): 1688 - 1691. [Full Text] [PDF]
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M. O. van Aken, J. A. Romijn, J. A. Miltenburg, and E. G.W.M. Lentjes Automated Measurement of Salivary Cortisol Clin. Chem., August 1, 2003; 49(8): 1408 - 1409. [Full Text] [PDF]
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 H. Raff and J. W. Findling A Physiologic Approach to Diagnosis of the Cushing Syndrome Ann Intern Med, June 17, 2003; 138(12): 980 - 991. [Full Text] [PDF]
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