Simultaneous assay of cortisol and dexamethasone improved diagnostic accuracy of the dexamethasone suppression test
Abstract
Objectives: The overnight dexamethasone (DXM) suppression test (DST) has high sensitivity, but moderate specificity, for diagnosing hypercortisolism. We have evaluated if simultaneous measurement of S-DXM may correct for variable DXM bioavailability and increase the diagnostic performance of DST, and if saliva (sa) is a feasible adjunct or alternative to serum. Design and methods: Prospective study of DST was carried out in patients with suspected Cushing’s syndrome (CS) (n = 49), incidentaloma (n = 152) and healthy controls (n = 101). Cortisol, cortisone and DXM were assayed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Results: Three hundred and two subjects underwent DST; S-cortisol was ≥50 nmol/L in 83 patients, of whom 11 had CS and 27 had autonomous cortisol secretion. The lower 2.5 percentile of S-DXM in subjects with negative DST (n = 208) was 3.3 nmol/L, which was selected as the DXM cut-off level. Nine patients had the combination of low S-DXM and positive DST. Of these, three had been misdiagnosed as having autonomous cortisol secretion. DST results were highly reproducible and confirmed in a replication cohort (n = 58). Patients with overt CS had significantly elevated post-DST sa-cortisol and sa-cortisone levels compared with controls; 23 of 25 with autonomous cortisol secretion had elevated sa-cortisone and 14 had elevated sa-cortisol. Conclusions: Simultaneous measurement of serum DXM and cortisol reduced false-positive DSTs by 20% and improved the specificity. S-DXM >3.3 nmol/L is sufficient for the suppression of cortisol <50 nmol/L. Measurement of glucocorticoids in saliva is a non-invasive and easy procedure and post-DST sa-cortisone was found particularly useful in the diagnosis of CS.
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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 [3]
The current study established upper reference limits for salivary cortisol after DST of 0.7-1.0 nmol/L for LC-MS/MS methods and 2.4-5.4 nmol/L for immunoassays.
“The current study established the URLs for salivary cortisol after DST to 0.7-1.0nmol/L for the LC-MS/MS methods and 2.4-5.4 nmol/L for the IAs, which is in line with previous smaller studies [3,4,43].”
From Salivary cortisol and cortisone in diagnosis of Cushing's syndrome: a comparison of six different analytical methods (2023) · cites this paper as [3]
Salivary cortisone upper reference limits for late-night samples (13.5-16.6 nmol/L) and after DST (3.0-3.5 nmol/L) compare well with other studies.
“Salivary cortisone URLs for late-night samples (13.5-16.6 nmol/L) and after DST (3.0-3.5 nmol/L) compare well with other studies [3,4].”
From Salivary cortisol and cortisone in diagnosis of Cushing's syndrome: a comparison of six different analytical methods (2023) · cites this paper as [3]
In a recent study from Norway, salivary cortisone at 8 a.m. following 1-mg overnight DST was found to be useful in patients with hypercortisolism, both CS and ACS.
“Also, in a recent study from Norway, salivary cortisone at 8 a.m. following 1-mg overnight DST was found to be useful in patients with hypercortisolism, both CS and ACS (18).”
From The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism (2022) · cites this paper as [18]
Sa-cortisol and Sa-cortisone at 8 a.m. following DST has only been studied once before in the context of screening for ACS.
“Sa-cortisol and Sa-cortisone at 8 a.m. following DST has to our knowledge only been studied once before in the context of screening for ACS (18).”
From The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism (2022) · cites this paper as [18]
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 [18]
In that study, 13 of 25 patients with ACS had elevated post-DST Sa-cortisol and 23 of 25 had elevated Sa-cortisone, with 90% and 82% specificity, respectively.
“In that study, 13 of 25 patients with ACS had elevated post-DST Sa-cortisol and 23 of 25 had elevated Sa-cortisone, with 90% and 82% specificity, respectively.”
From The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism (2022) · cites this paper as [18]
P-dexamethasone was not measured in our study.
“Finally, P-dexamethasone, analysed together with P-cortisol in association with DST, in order to decrease the number of false positive results (18), was not measured in our study.”
From The diagnostic value of salivary cortisol and salivary cortisone in patients with suspected hypercortisolism (2022) · cites this paper as [18]
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“For all of these reasons, the interest in the simultaneous quantification of F and Dex circulating concentrations is constantly growing, as it represents a valuable analytical tool for obtaining more reliable test results [18,19].”
MethodSerum cortisol assay performance following the 1 mg overnight dexamethasone suppression test
Atkins, J. S., Hawley, J. M., Owen, L. J., Clayton, J. C., Scargill, J., & Keevil, B. G. (2023) · Annals of Clinical Biochemistry International Journal of Laboratory Medicine · doi:10.1177/00045632231179560 · Cited by 15 · see citing works
“17,18 As such, dexamethasone was analysed on all samples using a previously published LC-MS/MS method 17 and those with dexamethasone ≤ 3.3 nmol/L were excluded ( n = 18).”
MethodInfluentialde Graaf, A. J., Mulder, A. L., & Krabbe, J. G. (2019) · Annals of Clinical Biochemistry: International Journal of Laboratory Medicine · doi:10.1177/0004563219870834 · Cited by 14 · see citing works
Berndt, V., Dahlqvist, P., de Verdier, J., Ryberg, H., & Ragnarsson, O. (2022) · Frontiers in Endocrinology · doi:10.3389/fendo.2022.1028804 · Cited by 15 · see citing works
“following DST has to our knowledge only been studied once before in the context of screening for ACS (18).”
BackgroundMethodResultInfluentialUtility of Salivary Cortisol and Cortisone in the Diagnostics of Adrenal Insufficiency
Hellan, K. K., Lyngstad, M., Methlie, P., Løvås, K., Husebye, E. S., & Ueland, G. Å. (2024) · The Journal of Clinical Endocrinology & Metabolism · doi:10.1210/clinem/dgae486 · Cited by 12 · see citing works
Self-citationPetersenn, S. (2022) · Pituitary · doi:10.1007/s11102-022-01249-5 · Cited by 12 · see citing works
“Of note, post-DST cortisol was not associated with serum creatinine, but inversely correlated with BMI [5].”
BackgroundInfluentialCortisol: Analytical and clinical determinants
Vega‐Beyhart, A., Castro, M. A., Hanzu, F. A., & Casals, G. (2022) · Advances in clinical chemistry · doi:10.1016/bs.acc.2022.11.005 · Cited by 12 · see citing works
Olsen, H., & OLSEN, M. E. (2023) · Frontiers in Endocrinology · doi:10.3389/fendo.2022.1055298 · Cited by 11 · see citing works
“(7) Interestingly, a study by Schorr et al.”
BackgroundPuvaneswaralingam, S., Kjellbom, A., Lindgren, O., Löndahl, M., & Olsen, H. (2020) · Clinical Endocrinology · doi:10.1111/cen.14357 · Cited by 11 · see citing works
High prevalence of smoking in patients with adrenal incidentalomas: causality or case selection?
Olsen, H., Kjellbom, A., Löndahl, M., & Lindgren, O. (2020) · European Journal of Endocrinology · doi:10.1530/eje-20-0033 · Cited by 10 · see citing works
Bäcklund, 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
The impact of mild autonomous cortisol secretion and proposed interventions
Iriarte-Durán, M. B., Donato, S., Herrera-Martínez, A. D., Vega, A., Casinello, J. M. J., Marazuela, M., & Castro, M. A. (2025) · Expert Review of Endocrinology & Metabolism · doi:10.1080/17446651.2025.2480704 · Cited by 9 · see citing works
“The authors conclude that the serum DM level of >3.3 nmol/L is sufficient to suppress serum cortisol to 50 nmol/L (1.8 µg/dL) [37].”
InfluentialGarg, R., Memon, S. S., Lila, A. R., Sarathi, V., Patil, P., Jamale, T., Bose, S., Karlekar, M. P., Patil, V. A., Shah, N. S., et al. (2024) · Journal of the Endocrine Society · doi:10.1210/jendso/bvae002 · Cited by 9 · see citing works
“…during the ONDST have been reported in healthy volunteers (Ueland et al 2.8 [1.2-7.3] ng/mL, Ceccato et al 4.9 [3.1-6.3] ng/mL, Genere et al 3.4 [2.5-4.4] ng/mL, Farinelli et al 3.69 [1.4-7.9] ng/mL) with normal renal function, which were similar to our study (4.48 [2.35-8.06] ng/mL) [19, 23-25].”
ResultInfluentialUeland, G. Å., Methlie, P., Heie, A., Stokland, A. M., Dahle, A. L., Sævik, Å. B., Løvås, K., & Husebye, E. S. (2023) · European Journal of Endocrinology · doi:10.1093/ejendo/lvad076 · Cited by 8 · see citing works
Self-citationCeccato, F., Terzolo, M., Gatto, F., Cannavò, S., Bianchi, A., Isidori, A. M., Colao, A. A. L., Ferone, D., Aimaretti, G., Giraldi, F. P., et al. (2025) · Journal of Endocrinological Investigation · doi:10.1007/s40618-025-02758-3 · Cited by 10 · see citing works
LC-MS/MS the First 20 years: A Personal View
Keevil, B. G. (2021) · Annals of Clinical Biochemistry International Journal of Laboratory Medicine · doi:10.1177/00045632211040059 · Cited by 6 · see citing works
Who and how to screen for endogenous hypercortisolism in patients with mood disorders
Ferrante, E., Simeoli, C., Mantovani, G., & Pivonello, R. (2024) · Journal of Endocrinological Investigation · doi:10.1007/s40618-024-02457-5 · Cited by 8 · see citing works
“Specifically, drugs such as carbamazepine and primidone accelerate DMX metabolism by induction of CYP 3A4, therefore reducing the plasma DMX concentrations, and causing false positive results [44–47].”
Castro, M. A., Gutiérrez, M. C., Ramírez, P. A. P., Torre, E. L. M., de Miguel, P., Paja, M., Mangas, M. Á., Lamas, C., & Hanzu, F. A. (2025) · Endocrine · doi:10.1007/s12020-025-04408-3 · Cited by 7 · see citing works
Paopongpaiboon, P., Santisitthanon, P., Houngngam, N., Snabboon, T., & Boonchaya‐anant, P. (2024) · Clinical Endocrinology · doi:10.1111/cen.15020 · Cited by 6 · see citing works
“7,8 The strength of this study is the enrolment of a large number of healthy participants with a wide range of BMI levels including participants with obesity class 1 and 2 according to Asian BMI ranges.”
BackgroundCushing Disease: Diagnosis and Treatment
Tatsi, C., & Stratakis, C. A. (2019) · Contemporary Endocrinology · doi:10.1007/978-3-030-11339-1_5 · Cited by 5 · see citing works
Castro, M. A., Gómez, A. C., Rosillo, M., Rodríguez, A., Madrid, I., Corrales, E. P., & Cano, A. M. G. (2025) · Clinical Endocrinology · doi:10.1111/cen.70065 · Cited by 4 · see citing works
“Furthermore, the recommended threshold in the serum dexamethasone level proposed as an enough level to achieve a proper hypothalamic pituitary adrenal axis (HPAA) suppression is variable across these studies [4, 6, 8].”
InfluentialCasteràs, A., Fidilio, E., Comas, M., Zabalegui, A., Flores, V., Giralt, M., Troyano, N. D., Ferrer, R., Vilallonga, R., Ciudin, A., et al. (2025) · European Journal of Endocrinology · doi:10.1093/ejendo/lvaf053 · Cited by 4 · see citing works
Salivary cortisol and cortisone are stable after long-term storage
Imamovic, M., Bäcklund, N., Lundstedt, S., Brattsand, G., Aardal, E., & Dahlqvist, P. M. (2024) · Scandinavian Journal of Clinical and Laboratory Investigation · doi:10.1080/00365513.2024.2403005 · Cited by 4 · see citing works
Improving the Dexamethasone Suppression Test
Keevil, B. G. (2021) · Clinical Chemistry · doi:10.1093/clinchem/hvab076 · Cited by 4 · see citing works
“also found that the use of dexamethasone measurement reduced the false positive rate for the DST by a similar magnitude; however, they did not assess the effect of varying the cortisol cut-off on diagnostic sensitivity and specificity (12).”
MethodKlahr, J. D., Carroll, T. B., Nerenz, R. D., & Raff, H. (2025) · AACE Endocrinology and Diabetes · doi:10.1016/j.aed.2025.07.008 · Cited by 3 · see citing works
“17,22 We found a comparable 9% false positive rate in the Post-Re fl ex-oDST group demonstrating preservation of test performance with Re fl ex implementation.”
Analysis of steroid profiles by mass spectrometry: A new tool for exploring adrenal tumors?
Cambos, S., Chanson, P., & Tabarin, A. (2020) · Annales d Endocrinologie · doi:10.1016/j.ando.2020.12.001 · Cited by 3 · see citing works
Castro, M. A., Paja, M., Boillos, M. G., Corrales, E. P., Ramírez, P. A. P., Rojas‐Marcos, P. M., Cano, A. M. G., Ruiz-Sánchez, J. G., Vicente, A., Hoyos, E. G., et al. (2024) · European Journal of Endocrinology · doi:10.1093/ejendo/lvae143 · Cited by 3 · see citing works
Hamidi, O., St-Jean, M., Lacroix, A., & Bancos, I. (2026) · The Journal of Clinical Endocrinology & Metabolism · doi:10.1210/clinem/dgag095 · Cited by 5 · see citing works
Current challenges in Cushing's syndrome testing: blood, saliva, urine, or hair?
Ferrière, A., & Tabarin, A. (2025) · Current Opinion in Endocrinology Diabetes and Obesity · doi:10.1097/med.0000000000000923 · Cited by 2 · see citing works
Balderrama-Soto, A., Rivera-Hernández, A. D. J., Reza-Albarrán, A. A., la Vega, A. N.-D., Vergara-López, A., Monteros-Sánchez, A. L. E. D. L., Navas-Minero, C. E., Abreu-Rosario, C., Cuevas-Ramos, D., Espinosa-Cárdenas, E., et al. (2023) · Revista Mexicana de Endocrinología Metabolismo y Nutrición · doi:10.24875/rme.m23000025 · Cited by 2 · see citing works
Bilateral Adrenal Nodules Presenting With Mild Autonomous Cortisol Secretion
Sandooja, R., Hamidi, O., Zhang, C. D., Dogra, P., Athimulam, S., Rahimi, L., Villavicencio, C. A., Chacko, S. R., Young, W. F., & Bancos, I. (2025) · Endocrine Practice · doi:10.1016/j.eprac.2025.06.019 · Cited by 2 · see citing works
Polycystic Ovarian Syndrome: A Diagnosis of Exclusion
Aubuchon, M. (2022) · Springer eBooks · doi:10.1007/978-3-030-92589-5_3 · Cited by 2 · see citing works
Improved salivary cortisol rhythm with dual-release hydrocortisone
Ceccato, F., Selmin, E., Sabbadin, C., Costa, M. D., Antonelli, G., Plebani, M., Barbot, M., Betterle, C., Boscaro, M., & Scaroni, C. M. (2018) · Endocrine Abstracts · doi:10.1530/endoabs.56.p56 · Cited by 2 · see citing works
Management of adrenal incidentalomas: who, why and how?
Araujo-Castro, M., Iriarte-Durán, M. B., Ramírez, P. A. P., & Donato, S. (2025) · Current Opinion in Endocrinology Diabetes and Obesity · doi:10.1097/med.0000000000000917 · Cited by 1 · see citing works
Performance of afternoon (16:00 h) serum cortisol for the diagnosis of Cushing’s syndrome
Dupuis, H., Merlen, E., Elices-Diez, J., Balayé, P., Cortet, C., Jannin, A., Cao, C. D., Douillard, C., SOULEZ, B., Ramdane, N., et al. (2025) · Clinical Chemistry and Laboratory Medicine (CCLM) · doi:10.1515/cclm-2025-0133 · Cited by 2 · see citing works
Gao, Y., Mu, D., Ma, Y.-C., Li, Y., Li, M., Hu, Y.-Y., Qiu, L., Yu, S., & Cheng, X.-Q. (2025) · Endocrine Practice · doi:10.1016/j.eprac.2025.12.017
Parasiliti‐Caprino, M., Ponzetto, F., Bianco, A. D., Barba, L., Febbraro, E., Sola, C., Leoni, L., Bima, C., Mengozzi, G., Aimaretti, G., et al. (2026) · European Journal of Endocrinology · doi:10.1093/ejendo/lvag132 · Cited by 1 · see citing works
Wehbeh, L., Alwahab, U. A., Mikhael, A., Dobri, G. A., Yogi-Morren, D., Kennedy, L., & Hamrahian, A. H. (2023) · Endocrine · doi:10.1007/s12020-023-03327-5 · Cited by 1 · see citing works
Challenges and controversies in adrenal surgery: A practical approach
Beninato, T. M., Duh, Q., Long, K. L., Kiernan, C. M., Miller, B. S., Patel, S. G., Randle, R. W., Wachtel, H., Zanocco, K. A., Zern, N. K., et al. (2023) · Current Problems in Surgery · doi:10.1016/j.cpsurg.2023.101374 · Cited by 1 · see citing works
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Deksametazon Süpresyon Testlerinin Hiperkortizolizm Ayırıcı Tanısında Kullanımı
Bayraktaroğlu, T., Tekin, S., & Topaloğlu, Ö. (2021) · Turkish Journal of Diabetes and Obesity · doi:10.25048/tudod.889268 · Cited by 1 · see citing works
“Likit - tandem mass spektrometri yöntemiyle ölçülen serum deksametazon seviyesinin 3,3-3,6 nmol/L seviyesinde olması endojen kortizol üretiminin baskılanması için yeterlidir (19).”
Evaluation of a Salivary Cortisol Elisa Kit in a Critical Obstetrical Unit
Zerón, H. M., Reyes-Mendoza, L. E., Tovar, C. F. L., & Osorio, E. A. A. (2016) · Research Journal of Obstetrics and Gynecology · doi:10.3923/rjog.2017.11.16
“However, some techniques used to quantify salivary hormones are very expensive 3 .”
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Confirmatory tests in steroid endocrinology
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Honour, J. W. (2023) · Elsevier eBooks · doi:10.1016/b978-0-12-818124-9.00019-x
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“The test is straightforward, requiring administration of 1 mg dexamethasone orally at midnight followed by a morning F measurement; it exhibits a sensitivity of up to 95% and a specificity between 80% and 95% [ 9–11].”
All about dexamethasone testing in Cushing's syndrome
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Mild Autonomous Cortisol Secretion
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New tools for the diagnosis of hypercortisolism
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The utility of salivary 11-dehydrodexamethasone as a marker of dexamethasone absorption
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Research Progress in Diagnostic Methods of Cushing’s Syndrome
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Bueling, Z., Douri, D. R., Berg, A. H., Garrity, C. E., Olson, B. M., Riehl, B., Westby, C. M., Swan, J. T., Ghozy, S. A., Rabadi, I. A., et al. (2026) · Endocrine Practice · doi:10.1016/j.eprac.2026.09.121
MACS: mild by definition, challenging by nature
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- Authors
- Ueland, G. Å., Methlie, P., Kellmann, R., Bjørgaas, M., Åsvold, B. O., Thorstensen, K., Kelp, O., Thordarson, H. B., Mellgren, G., Løvås, K., & Husebye, E. S.
- Affiliations
- Haukeland University Hospital (NO)
- University of Bergen (NO)
- St Olav's University Hospital (NO)
- Norwegian University of Science and Technology (NO)
- Akershus University Hospital (NO)
- 2Department of Medicine
- Corresponding author
- Ueland, Grethe Å
- ORCID
- Venue
- European Journal of Endocrinology
- Publisher
- Oxford University Press (OUP)
- Published
- 2017-06
- Volume
- 176
- Issue
- 6
- Pages
- 705-713
- ISSN
- 0804-4643 (ISSN-L)
- 1479-683X
- OpenAlex
- W2602237045
- Cited by
- 94
- References
- 39
- Citations by year
- 2026: 12 · 2025: 15 · 2024: 7 · 2023: 13 · 2022: 10 · 2021: 13 · 2020: 10 · 2019: 5 · 2018: 6 · 2017: 2 · 2016: 1
- FWCI
- 4.97
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- European Journal of Endocrinology (publishedVersion)
- PubMed (publishedVersion)
- 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
- dexamethasone suppression test, Cushing's syndrome, hypercortisolism, cortisol, cortisone, autonomous cortisol secretion, incidentaloma, diagnostic accuracy, false positives
- MeSH terms
- Adenoma, Adolescent, Adrenal Gland Neoplasms, Adult, Aged, Aged, 80 and over, Chromatography, Liquid, Cortisone, Cushing Syndrome, Dexamethasone, Female, Humans, Hydrocortisone, Male, Middle Aged, Prospective Studies, Saliva, Case-Control Studies, Incidental Findings, Tandem Mass Spectrometry, Young Adult
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