Salivary Cortisol and Cortisone After Low-Dose Corticotropin Stimulation in the Diagnosis of Adrenal Insufficiency

Ingrid Yin Fung Mak; Benjamin Yick Toa Au Yeung; Ying Wai Ng; Cheung Hei Choi; Heidi Yan Ping Iu; Chi Chung Shek; Sau Cheung Tiu


J Endo Soc. 2017;1(2):96-108. 

In This Article

Abstract and Introduction


Context: Basal and poststimulation salivary cortisol and cortisone levels can be useful in the diagnosis of adrenal insufficiency. However, little is known about the optimal cutoffs and performance characteristics of these tests.

Objective: To derive the cutoff values and study the performance characteristics of salivary cortisol and salivary cortisone in the diagnosis of adrenal insufficiency.

Design and Setting: Prospective study in a regional hospital in Hong Kong from January 2014 to September 2015.

Participants: Fifty-six Chinese healthy volunteers and 171 patients suspected of having adrenal insufficiency.

Main Outcome Measures: All participants underwent low-dose short Synacthen test (LDSST) with intravenous injection of 1 μg of tetracosactide (Synacthen 1–24). Serum cortisol, salivary cortisol and cortisone levels were measured at baseline and 30 and 60 minutes afterward.

Results: Using the reference cutoff (mean − 2 standard deviations of post-LDSST peak serum cortisol) derived from healthy volunteers as the gold standard, receiver operating characteristic analysis of patients' data revealed that both post-LDSST peak salivary cortisol and cortisone performed better than basal tests. The most optimal cutoff values for serum cortisol as measured by immunoassay and for salivary cortisol and salivary cortisone as measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS) were 376, 8.6, and 33.5 nmol/L, respectively, for post-LDSST peak values, and 170, 1.7, and 12.5 nmol/L, respectively, for basal values.

Conclusions: We established method-specific reference cutoffs for serum cortisol, salivary cortisol, and salivary cortisone during LDSST. Both post-LDSST peak salivary cortisol and cortisone performed well as diagnostic tests for adrenal insufficiency. Their measurements by LC-MS/MS can alleviate problems associated with serum cortisol immunoassays.


An accurate diagnosis of adrenal insufficiency (AI) is important because the condition is potentially life-threatening if missed; however, overdiagnosis may lead to unnecessary replacement of glucocorticoids, leading to decreased quality of life and increased mortality.[1] Various tests, including the insulin tolerance test, the glucagon stimulation test, and the standard (250 μg) and low-dose (1 μg) short Synacthen (tetracosactide) tests (SSTs), have been used to diagnose AI. The SSTs are the most widely used, owing to their convenience, safety, and reasonably good correlation with the insulin tolerance test.[2–5] However, the reference cutoff levels for the SSTs differed in different studies, ranging from 418 to 574 nmol/L, depending on the nature of the study population, the gold standard used to define AI, the dosage of ACTH and time of sampling, the type of statistical analysis used, and the type of cortisol assay used.[4,6–9] The fluorimetric cortisol assays adopted in older studies also measured corticosterone, thus overestimating the cortisol levels by 20% to 30%.[10,11] Even among modern immunoassays, different commonly used assays can yield different results.[8,9,12]

The levels of the binding proteins, cortisol-binding globulin (CBG) and albumin, may also contribute to the variation in cutoff levels and the performance characteristics of the post-SST serum total cortisol.[1,7,13] Serum free cortisol has been shown to provide a more accurate measurement of the circulating glucocorticoid status than serum total cortisol.[13–15] However, the direct measurement of serum free cortisol requires laborious laboratory techniques that are not commonly available, and the accuracy of calculated free cortisol[13,14] is not widely accepted. Salivary cortisol may serve as a better alternative, as it is noninvasive and reflects the level of serum free cortisol.[16–19] Measurement of salivary steroids with liquid chromatography–tandem mass spectrometry (LC-MS/MS) enhances the sensitivity by 10- to 100-fold,[19–21] and it improves the specificity by eliminating cross-reactivity among various metabolites,[16,19,20] thus enabling concomitant measurement of cortisone in addition to cortisol.

The potential utility of salivary cortisone was proposed by Perogamvros et al.[19] in 2009. Salivary cortisone is formed predominantly from serum free cortisol, by the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) type 2, as the latter passes through the salivary gland.[22] Diffusion of serum free cortisone into the saliva contributes to a lesser extent.[23] Total and free cortisol levels are higher than total and free cortisone levels in the serum,[22–24] yet the ratio is reversed in the saliva, with salivary cortisone to cortisol ratio of 4:1 to 10:1,[19,24] thus allowing more accurate measurement of cortisone. Both salivary cortisone and cortisol correlate strongly with serum cortisol; however, at low serum total cortisol levels of < 74 nmol/L, when salivary cortisol is undetectable, salivary cortisone can still be detected.[24] In AI patients on hydrocortisone replacement, salivary cortisone can also be used for monitoring, as it is not contaminated by oral hydrocortisone intake.[18,23,24]

The aims of this study were to derive the cutoff values and study the performance characteristics of salivary cortisol and salivary cortisone, as measured by LC-MS/MS, in the diagnosis of AI.