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Performance of afternoon (16:00 h) serum cortisol for the diagnosis of Cushing’s syndrome

  • Hippolyte Dupuis EMAIL logo , Emilie Merlen , Julien Elices-Diez , Pierre Balayé , Christine Cortet , Arnaud Jannin , Christine Do Cao , Claire Douillard , Benoit Soulez , Nassima Ramdane , Benoit Soudan , Marie-Christine Vantyghem and Stéphanie Espiard
Published/Copyright: July 11, 2025
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM)

Abstract

Introduction

Late-night salivary cortisol (LNSC), which assesses the loss of the circadian rhythm of cortisol, is one of the first-line tests performed to diagnose Cushing’s syndrome (CS). Unfortunately, access to LNSC is still limited in some institutions. Alternatively, midnight serum cortisol can be measured, often combined with additional cortisol sampling over 24 h. This study investigates the performance of afternoon serum cortisol (F16h) for the positive diagnosis of hypercortisolism.

Methods

Retrospective study including consecutive patients evaluated for suspicion of CS by at least two tests among urine-free cortisol (UFC), overnight 1-mg dexamethasone suppression test (DST), and midnight serum cortisol (F00h). Patients assessed for adrenal incidentalomas or Cushing’s disease recurrence were excluded.

Results

Of the 589 included patients (41.3 % women, mean age 50.7 ± 16.3 years), 49 (8.3 %) were diagnosed with CS. F16h demonstrated significant correlations with DST, UFC, and F00h (r=0.24, p<0.001, r=0.41, p<0.001 and r=0.42, p<0.001, respectively). The optimal cut-off of the F16h was 218 nmol/L, achieving 83.7 % sensitivity, 67.4 % specificity, and a 97.8 % negative predictive value. The area under the ROC curve (AUC) for the F16h did not differ from UFC (0.83 vs. 0.79, p=0.3), yet its sensitivity was higher using the optimal diagnostic threshold. The AUC for the F16h was significantly lower than that for F00h and DST.

Conclusions

Given the limited diagnostic accuracy of both F16h and UFC, particularly in mild to moderate CS, efforts should prioritize expanding access to reliable LNSC assays for circadian rhythm assessment. Meanwhile, F16h may help rule out CS when LNSC is unavailable.

Keywords: Cushing’s syndrome; hypercortisolism; serum cortisol; urine free cortisol; dexamethasone suppression test
Corresponding author: Hippolyte Dupuis, Department of Endocrinology, Diabetology and Metabolism, Hospital Huriez, University Hospital of Lille, F-59000 Lille, France, E-mail:

  1. Research ethics: Lille hospital data warehouse (INCLUDE project) has been approved by the French National Data Protection Authority (CNIL) from 2019 (authorization number: 1754053). INCLUDE data warehouse ensures that researchers at Lille University Hospital can process end-to-end data in a secure, GDPR-compliant environment.

  2. Informed consent: Not applicable.

  3. Author contributions: HD and SE wrote the manuscript. HD and SE analyzed the data. NR performed statistical analyses. EM, JED, PB, CC, AJ, CDC, CD, BS, NR, BS and MCV reviewed the manuscript and provided comments. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  4. Use of Large Language Models, AI and Machine Learning Tools: None declared.

  5. Conflict of interest: The authors declare that they have no conflict of interest in this manuscript.

  6. Research funding: None declared.

  7. Data availability: The data that support the findings of this study are available from Lille University Hospital but restrictions apply to the availability of these data due to patient privacy regulations, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Lille University Hospital.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2025-0133).

Received: 2025-02-05
Accepted: 2025-07-03
Published Online: 2025-07-11

© 2025 Walter de Gruyter GmbH, Berlin/Boston

https://doi.org/10.1515/cclm-2025-0133
Keywords for this article
Cushing’s syndrome; hypercortisolism; serum cortisol; urine free cortisol; dexamethasone suppression test
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