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Briefly, the assay requires 50 µL of saliva for duplicate measurements. Salivary cortisol was measured in duplicate by EIA (Salimetrics 1-3102, State College, PA) as described and validated previously. Saliva samples were obtained using the Sarstedt Plain Cotton Salivette. Subjects obtained saliva samples at their normal bedtime and upon their normal awakening time.
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Subjects were instructed to refrain from smoking for at least 2 hours before sampling.
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The only other exclusion was the use of any form of exogenous corticosteroids including topical, inhaled, nasal, and/or oral. All of the subjects were daytime workers (no nightshift workers), and all reported normal sleep-wake patterns. The only information obtained from the subjects was age and sex (35 females/19 males). Because samples were de-identified, the institutional review board determined that written informed consent was not required. Luke’s Medical Center under a protocol approved by the Aurora Institutional Review Board. Healthy SubjectsĪdult subjects (N = 54) were recruited from employees at the Aurora Research Institute and Aurora St. We also sought to validate a modified LC-MS/MS method using only 50 µL of saliva as we often receive clinical samples with <100 µL of saliva, and compare its performance to our standard EIA. In particular, our focus is whether there is a requirement to stay awake until 2300 to 2400 hours as is now routinely done, or whether subjects and patients can sample at their normal bedtime. The purpose of this study is to explore salivary cortisol and cortisone measurements in healthy subjects with a wide age range and compare those results to the standard, US Food and Drug Administration-cleared salivary cortisol enzyme immunoassay (EIA) currently in use. This has led to the possibility that, under certain circumstances, the measurement of salivary cortisone by liquid chromatography-tandem mass spectrometry (LC-MS/MS) may be useful in addition to salivary cortisol, or perhaps even superior in the evaluation of the HPA axis. Furthermore, the measurement of salivary cortisol and cortisone has been used in the evaluation of the dynamics of the hypothalamic-pituitary-adrenal (HPA) axis in humans including the awakening response. Because the salivary gland expresses 11- β-hydroxysteroid dehydrogenase, salivary cortisone is significantly greater than salivary cortisol, and the assessment of salivary cortisone may be useful in the diagnosis of Cushing syndrome, as an index of cortisol exposure, and in the evaluation of the stress response in humans. The majority of prior studies used immunoassays for the measurement of salivary cortisol with a sensitivity and specificity for Cushing syndrome of greater than 90%. Measurement of late-night salivary cortisol is now one of the mainstays of the diagnosis of Cushing syndrome. Late-night salivary cortisol, awakening, immunoassay, mass spectrometry, sex We conclude that (i) salivary cortisol and cortisone can be reliably measured by LC-MS/MS in small volumes of saliva and (ii) that patients can be evaluated using saliva sampled at their normal bedtime, rather than being required to stay awake until 2300 to 2400 hours. There were no significant effects of age or sex on any of the salivary steroid measurements. Furthermore, the upper limit of normal of salivary cortisol by EIA for bedtime samples was lower than the previously published upper limit of normal with sampling required at 2300 to 2400 hours. We found that the salivary cortisol and cortisone results by LC-MS/MS correlated well with salivary cortisol measured with the US Food and Drug Administration-cleared EIA. We allowed patients to sample at their normal bedtime (2025 to 2400 hours) to answer a common question as to whether sampling at the normal bedtime is equivalent to the standard required sampling at 2300 to 2400 hours. We measured salivary cortisol by enzyme immunoassay (EIA) and salivary cortisol and cortisone by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in only 50 µL of saliva sampled from 54 healthy subjects (aged 20 to 64 years). Because the salivary glands express 11- β-hydroxysteroid dehydrogenase, the measurement of salivary cortisone may improve the performance of salivary corticosteroid measurements. Furthermore, the measurement of salivary cortisol is useful in assessing the cortisol awakening response. The measurement of late-night salivary cortisol is a mainstay in the diagnosis of Cushing syndrome.