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Endocrine Research Laboratory, St. Luke’s Medical Center, Milwaukee, WI 53215
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Department of Medicine, Medical College of Wisconsin, Milwaukee, WI 53226
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Clement J. Zablocki, VA Medical Center, Milwaukee, WI 53295
aAddress correspondence to this author at: Endocrinology and Diabetes, St. Luke’s Physician’s Office Building, 2801 W KK River Pkwy., Suite 245, Milwaukee, WI 53215. Fax 414-649-5747; e-mail hraff{at}mcw.edu.
To the Editor:
The concentration of cortisol in the saliva reflects the activity of the hypothalamic-pituitary-adrenal axis (1), and an increased late-night salivary cortisol has >95% sensitivity and specificity for Cushing syndrome (1)(2).
We compared a commonly used modification of a RIA for serum cortisol with a new enzyme immunoassay (EIA) specifically designed to measure salivary cortisol in 352 saliva samples. The first samples (n = 195) were sent to our laboratory to screen for Cushing syndrome; all were collected at 2300 from 90 patients (age range, 17–77 years; 74 women and 16 men). The second set of samples (n = 104) was obtained at 2300 and 0700 from healthy, elderly patients (n = 52; age range, 67–82 years; 21 women and 31 men) who were part of a large, longitudinal study (3). The third set of samples (n = 53) was obtained at 2300 and 0700 from a group of apparently healthy individuals (n = 27; age range, 12–63 years; 16 women and 11 men). The study was approved by the appropriate Institutional Review Boards, and consent was obtained. Saliva was sampled as described previously (2)(4) 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) was used as commonly modified for the measurement of salivary cortisol (2)(4). The salivary cortisol EIA (product no. 10-67100; Diagnostic Systems Laboratories) 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 45 min for the EIA. The EIA calibrators ranged from 2.8 to 276 nmol/L and were provided as cortisol in a solution of bovine serum albumin.
The lower detection limit of the EIA was 0.4 nmol/L. The intraassay imprecision (CV) was 9.5% at 6.3 (SD, 0.6) nmol/L (n = 12), 4.4% at 10.6 (SD, 0.5) nmol/L (n = 9), and 5.6% at 53.7 (SD, 3.0) nmol/L (n = 12). Interassay (total) imprecision (CV) was 9.7% at 4.3 (SD, 0.4) nmol/L (n = 13), 11% at 7.4 (SD, 0.8) nmol/L (n = 13), and 7.8% at 59.6 (SD, 4.7) nmol/L (n = 13).
The regression lines for the data from the three groups of individuals were not different from each other, so the combined data are shown in Fig. 1
. The slope of the regression for RIA salivary cortisol values <11 nmol/L was not significantly different from the slope for the complete data set (Fig. 1
, inset). Because the EIA showed consistently higher salivary cortisol concentrations, we assayed the RIA calibrators in the EIA, and vice versa. The EIA gave values for the RIA calibrators that were the same as the RIA stated values, and vice versa. When we added the 27.6 nmol/L EIA calibrator to saliva, the RIA returned a value of 21.2 nmol/L. When we added the 13.8 nmol/L RIA calibrator to saliva, the EIA returned a value of 15.8 nmol/L.
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To determine which method was correct, cortisol (Hydrocortisone; cat. no. H 4001; Sigma Chemical) was accurately weighed to create a stock solution of 2.76 mmol/L and then diluted. Cortisol (23 nmol/L) or vehicle was added to saliva and assayed 10 times. The difference between saliva with cortisol added and saliva with vehicle added was 22.4 (SD, 0.7) nmol/L in the RIA and 27.3 (SD, 0.6) nmol/L in the EIA. Therefore, the RIA was much closer to the expected result (23 nmol/L) than the EIA, and the EIA overestimated the concentration of cortisol in the saliva.
The most important finding is that the EIA returned consistently higher salivary cortisol values than the RIA. The RIA gave results much closer to the expected value of an independently created cortisol stock solution diluted in saliva. Because salivary cortisol measurement is increasing in popularity, it is important to be aware of the different results generated by these two commercially available methods and to interpret the published reference intervals appropriately.
Acknowledgments
This study was supported in part by GCRC Grant M01 RR00058-38 and a grant from the Aid Association for Lutherans.
References
The following articles in journals at HighWire Press have cited this article:
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  W. M. Pratt and D. Davidson Role of the HPA Axis and the A118G Polymorphism of the {micro}-Opioid Receptor in Stress-Induced Drinking Behavior Alcohol Alcohol., July 1, 2009; 44(4): 358 - 365. [Abstract] [Full Text] [PDF]
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 A. C Heijboer, F. Martens, and M. A Blankenstein Measuring salivary cortisol with the Architect i2000 random access analyser Ann Clin Biochem, May 1, 2009; 46(3): 261 - 262. [Full Text] [PDF]
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 S. K. Baid, N. Sinaii, M. Wade, D. Rubino, and L. K. Nieman Radioimmunoassay and Tandem Mass Spectrometry Measurement of Bedtime Salivary Cortisol Levels: A Comparison of Assays to Establish Hypercortisolism J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3102 - 3107. [Abstract] [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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C. de Weerth, G. Graat, J. K. Buitelaar, and J. H.H. Thijssen Measurement of Cortisol in Small Quantities of Saliva Clin. Chem., April 1, 2003; 49(4): 658 - 660. [Full Text] [PDF]
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H. Raff, P. J. Homar, and D. P. Skoner New Enzyme Immunoassay for Salivary Cortisol Clin. Chem., January 1, 2003; 49(1): 203 - 204. [Full Text] [PDF]
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D. A. Papanicolaou, N. Mullen, I. Kyrou, and L. K. Nieman Nighttime Salivary Cortisol: A Useful Test for the Diagnosis of Cushing's Syndrome J. Clin. Endocrinol. Metab., October 1, 2002; 87(10): 4515 - 4521. [Abstract] [Full Text] [PDF]
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, referred clinical samples (n = 195); •, healthy elderly individuals (104 samples); 
, non-elderly healthy individuals (53 samples). - - -, line of identity. Regression statistics: slope = 1.7 (SE, 0.0); y-intercept = 0.2 (SE, 0.3) nmol/L; r2 = 0.94; F(1,350) = 5150.5; P <0.001; Sy|x = 5.2 nmol/L (n = 352). Inset, regression of the points with salivary cortisol RIA values <11 nmol/L. Slope = 1.9 (SE, 0.1); y-intercept = -0.3 (SE, 0.2) nmol/L; r2 = 0.85 (n = 280).