Reference intervals of salivary cortisol and cortisone and their diagnostic accuracy in Cushing's syndrome
Abstract
OBJECTIVE: The challenge of diagnosing Cushing's syndrome (CS) calls for high precision biochemical screening. This study aimed to establish robust reference intervals for, and compare the diagnostic accuracy of, salivary cortisol and cortisone in late-night samples and after a low-dose (1 mg) dexamethasone suppression test (DST). DESIGN AND METHODS: Saliva samples were collected at 08:00 and 23:00 h, and at 08:00 h, after a DST, from 22 patients with CS and from 155 adult reference subjects. We also collected samples at 20:00 and 22:00 h from 78 of the reference subjects. Salivary cortisol and cortisone were analysed with liquid chromatography-tandem mass spectrometry. The reference intervals were calculated as the 2.5th and 97.5th percentiles of the reference population measurements. Diagnostic accuracies of different tests were compared, based on areas under the receiver-operating characteristic curves. RESULTS: The upper reference limits of salivary cortisol and cortisone at 23:00 h were 3.6 nmol/L and 13.5 nmol/L, respectively. Using these reference limits, CS was detected with a sensitivity (95% CI) of 90% (70-99%) and specificity of 96% (91-98%) for cortisol, and a 100% (84-100%) sensitivity and 95% (90-98%) specificity for cortisone. After DST, cortisol and cortisone upper reference limits were 0.79 nmol/L and 3.5 nmol/L, respectively. CS was detected with 95% (75-100%) sensitivity and 96% (92-99%) specificity with cortisol, and 100% (83-100%) sensitivity and 94% (89-97%) specificity with cortisone. No differences in salivary cortisol or cortisone levels were found between samples collected at 22:00 and 23:00 h. CONCLUSION: Salivary cortisol and cortisone in late-night samples and after DST showed high accuracy for diagnosing CS, salivary cortisone being slightly, but significantly better.
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Salivary cortisone has been shown to correlate better with plasma free cortisol than salivary cortisol and to be less susceptible to preanalytical confounders.
“Salivary cortisone has been shown to correlate better with plasma free cortisol than salivary cortisol and to be less susceptible to preanalytical confounders (20, 23, 27, 28).”
From Salivary Cortisol and Cortisone Can Circumvent Confounding Effects of Oral Contraceptives in the Short Synacthen Test (2024) · cites this paper as [23]
No salivary samples had a cortisol:cortisone ratio greater than 1, a test for sample integrity and possible contamination.
“No salivary samples had a cortisol:cortisone ratio greater than 1, a test for sample integrity and possible contamination (23, 41).”
From Salivary Cortisol and Cortisone Can Circumvent Confounding Effects of Oral Contraceptives in the Short Synacthen Test (2024) · cites this paper as [23]
Salivary cortisone may have an advantage over salivary cortisol as a diagnostic analyte, since it is considerably more robust against confounding factors including blood contamination, topical hydrocortisone, and licorice consumption.
“Salivary cortisone may have an advantage over salivary cortisol as a diagnostic analyte, since it is considerably more robust against confounding factors including blood contamination, topical hydrocortisone, and licorice consumption (23, 27).”
From Salivary Cortisol and Cortisone Can Circumvent Confounding Effects of Oral Contraceptives in the Short Synacthen Test (2024) · cites this paper as [23]
Higher levels of cortisol overwhelm the capacity of 11β-HSD2 in the salivary glands, as shown by higher salivary cortisol:cortisone ratios with increasing concentrations.
“The present study and previous data show higher salivary cortisol:cortisone ratios with increasing concentrations, indicating that higher levels of cortisol overwhelm the capacity of 11β-HSD2 in the salivary glands (23, 47).”
From Salivary Cortisol and Cortisone Can Circumvent Confounding Effects of Oral Contraceptives in the Short Synacthen Test (2024) · cites this paper as [23]
Salivary cortisol has been shown to be less affected by CBG alterations than plasma cortisol.
“Analysis of salivary cortisol has been proposed as an alternative to analyzing plasma free cortisol and has been shown to be less affected by CBG alterations (9, 14, 20-25).”
From Salivary Cortisol and Cortisone Can Circumvent Confounding Effects of Oral Contraceptives in the Short Synacthen Test (2024) · cites this paper as [23]
In most Cushing's syndrome cases, the diagnosis was confirmed by histological examination of the ACTH- or cortisol-producing tumor.
“In a majority of the cases, the diagnosis was confirmed by histological examination of the ACTH-or cortisol producing tumor [2].”
From Salivary cortisol and cortisone in diagnosis of Cushing's syndrome: a comparison of six different analytical methods (2023) · cites this paper as [2]
The authors and others have shown that salivary cortisol and cortisone after DST have high diagnostic accuracy for Cushing's syndrome, but many methods lack an established diagnostic cut-off.
“We and others have shown salivary cortisol and cortisone after DST to have high diagnostic accuracy for CS, but many methods lack an established cut-off for diagnosis [2-5, 41, 42].”
From Salivary cortisol and cortisone in diagnosis of Cushing's syndrome: a comparison of six different analytical methods (2023) · cites this paper as [2]
A ratio cut-off around 1 is probably dependable for detecting sample contamination with most LC-MS/MS assays.
“The 97.5th percentile for the salivary cortisol:cortisone ratios was close to 1 for all three tested LC-MS/MS methods, making this likely to be a reliable cut-off for most LC-MS/MS methods to indicate sample contamination [2,13,16,44].”
From Salivary cortisol and cortisone in diagnosis of Cushing's syndrome: a comparison of six different analytical methods (2023) · cites this paper as [2]
The study's large reference population, with age and sex distributions and rates of smoking and common diseases that reflect the population, is a strength that supports external validity and clinical usefulness.
“A strength in our study is the large reference population with representative age and gender distributions, and proportions of smokers and patients with common diseases, giving a high external validity and results useful for clinical practice [2].”
From Salivary cortisol and cortisone in diagnosis of Cushing's syndrome: a comparison of six different analytical methods (2023) · cites this paper as [2]
Salivary cortisone at 11 p.m., as well as salivary cortisol and cortisone at 8 a.m. following 1-mg overnight DST, all have high diagnostic accuracy for CS.
“We have recently demonstrated that salivary cortisone at 11 p.m., as well as salivary cortisol and cortisone at 8 a.m. following 1-mg overnight DST, all have high diagnostic accuracy for CS (17).”
From The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism (2022) · cites this paper as [17]
The upper limit of normal for Sa-cortisone at 8 a.m. following DST used in this study was <5 nmol/L.
“- Sa-cortisone at 8 a.m. following DST <5 nmol/L”
From The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism (2022) · cites this paper as [17]
The upper limit of normal for Sa-cortisone at 11 p.m. used in this study was <15 nmol/L.
“- Sa-cortisone at 11 p.m. <15 nmol/L”
From The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism (2022) · cites this paper as [17]
Contamination with exogenous hydrocortisone or blood in salivary samples was determined by calculating the salivary-cortisol:cortisone ratio, and samples with a ratio over the 97.5 percentile for the whole cohort (1.0) were considered contaminated.
“Contamination with exogenous hydrocortisone, or blood, in the salivary samples was determined by calculating the salivary-cortisol:cortisone ratio as previously described (17). Samples with a ratio over the 97.5 percentile for the whole cohort (1.0) were considered to be contaminated.”
From The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism (2022) · cites this paper as [17]
The upper limit of normal for Sa-cortisol at 8 a.m. following DST used in this study was <1 nmol/L.
“- Sa-cortisol at 8 a.m. following DST <1 nmol/L”
From The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism (2022) · cites this paper as [17]
The upper limit of normal for LNSC used in this study was <3 nmol/L.
“- LNSC <3 nmol/L”
From The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism (2022) · cites this paper as [17]
Bäcklund et al. found that Sa-cortisone at 11 p.m. had a higher sensitivity than LNSC for diagnosing CS.
“Bäcklund et al. recently found that Sa-cortisone at 11 p.m. had a higher sensitivity than LNSC for diagnosing CS (17).”
From The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism (2022) · cites this paper as [17]
LNSC and Sa-cortisone at 11 p.m. had a slightly lower sensitivity than salivary samples collected after DST.
“Also, in agreement with Bäcklund et al. (17), LNSC and Sa-cortisone at 11 p.m. had a slightly lower sensitivity than salivary samples collected after DST.”
From The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism (2022) · cites this paper as [17]
Analysis of Sa-cortisol and Sa-cortisone following DST has been used in two previous studies to screen patients with suspected CS, both showing high sensitivity (95-100%) and specificity (80-95%).
“Analysis of Sa-cortisol and Sa-cortisone following DST has also been used in two previous studies to screen patients with suspected CS, both showing a high sensitivity (95-100%) and specificity (80-95%) (17, 18), in agreement with our findings.”
From The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism (2022) · cites this paper as [17]
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“It has been suggested that the measurement of salivary cortisol (F) and cortisone (E) by LC-MS/MS may improve the performance of the test because of the high analytic specificity of this structural analytic methodology [28-31, 55-60].”
BackgroundBiochemical diagnosis of Cushing’s disease: Screening and confirmatory testing
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“The saliva samples were collected at home using Salivette® Cortisol tubes (Sarstedt, Nümbrecht, Germany) after oral and written instructions on how to properly collect the saliva had been provided (17).”
BackgroundMethodResultInfluentialSelf-citationBäcklund, N., Lundstedt, S., Tornevi, A., Wihlbäck, A.-C., Olsson, T., Dahlqvist, P., & Brattsand, G. (2024) · The Journal of Clinical Endocrinology & Metabolism · doi:10.1210/clinem/dgad763 · Cited by 13 · see citing works
“Salivary cortisone has been shown to correlate better with plasma free cortisol than salivary cortisol and to be less susceptible to preanalytical confounders (20, 23, 27, 28).”
BackgroundResultSelf-citationBäcklund, N., Brattsand, G., Lundstedt, S., Aardal, E., Bartuseviciene, I., Berinder, K., Höybye, C., Burman, P., Edén Engström, B., Isaksson, A., et al. (2023) · Clinical Chemistry and Laboratory Medicine (CCLM) · doi:10.1515/cclm-2023-0141 · Cited by 9 · see citing works
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- Authors
- Bäcklund, N., Brattsand, G., Israelsson, M., Ragnarsson, O., Burman, P., Edén Engström, B., Høybye, C., Berinder, K., Wahlberg, J., Olsson, T., & Dahlqvist, P.
- Affiliations
- Umeå University (SE)
- 3Department of Internal Medicine and Clinical Nutrition, Institute of Medicine at Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
- Skåne University Hospital (SE)
- 6Department of Medical Sciences, Endocrinology and Mineral Metabolism, Uppsala University, Uppsala, Sweden
- 7Department of Molecular Medicine and Surgery, Patient Area Endocrinology and Nephrology, Inflammation and Infection Theme, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden
- Karolinska University Hospital (SE)
- Linköping University (SE)
- Corresponding author
- Bäcklund, Nils
- ORCID
- Venue
- European Journal of Endocrinology
- Publisher
- Oxford University Press (OUP)
- Published
- 2020-06
- Volume
- 182
- Issue
- 6
- Pages
- 569-582
- ISSN
- 0804-4643 (ISSN-L)
- 1479-683X
- OpenAlex
- W3013791685
- Cited by
- 46
- References
- 31
- Citations by year
- 2026: 7 · 2025: 9 · 2024: 4 · 2023: 12 · 2022: 6 · 2021: 5 · 2020: 3
- FWCI
- 4.90
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- European Journal of Endocrinology (publishedVersion)
- PubMed (publishedVersion)
- Lund University Publications (Lund University) (submittedVersion)
- Topics
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Pituitary Gland Disorders and Treatments
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adrenal Hormones and Disorders
- Health Sciences › Medicine › Surgery › Adrenal and Paraganglionic Tumors
- Keywords
- Cushing's syndrome, salivary cortisol, salivary cortisone, reference values, dexamethasone suppression test, diagnostic accuracy
- MeSH terms
- Adult, Aged, Aged, 80 and over, Circadian Rhythm, Cortisone, Cushing Syndrome, Dexamethasone, Female, Humans, Hydrocortisone, Male, Mass Screening, Middle Aged, Reference Values, ROC Curve, Saliva, Sensitivity and Specificity, Time Factors, Area Under Curve, Tandem Mass Spectrometry, Young Adult
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