Defining normative cortisol responses to the insulin tolerance test: A comparison of Elecsys®Cort I and II assays

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The study aim was to define the normative cortisol response to the ITT with the first- and second-generation Roche Elecsys ® Cortisol immunoassays (Elecsys ® Cort I and II). Methods : Ninety participants underwent an ITT after an overnight fast. Blood samples were analysed by both Elecsys ® Cort I and II. The normative cortisol cut-off limit to the ITT was defined as the 2.5 th percentile of the peak cortisol level for each assay. The percentage of healthy participants with peak cortisol below the cut-off limits defined for the ACTH test, 500 nmol/L for Elecsys ® Cort I and 420 nmol/L for Elecsys ® Cort II, respectively, was calculated as the false-positive rate. Results : The mean peak cortisol was 608 ± 102 nmol/L with Elecsys ® Cort I and 501 ± 89 nmol/L with Elecsys ® Cort II. The 2.5 th percentile was 404 nmol/L with Elecsys ® Cort I and 320 nmol/L with Elecsys ® Cort II; 5 th percentiles were 451 nmol/L and 348 nmol/L, respectively. False-positive rates were 15% with Elecsys ® Cort I and 18% with Elecsys ® Cort II. Conclusion : Elecsys ® Cort II demonstrated a lower normative cortisol response to the ITT compared with Elecsys ® Cort I, and the commonly used ACTH test thresholds. These data underline the need for assay- and test-specific cut-off limits to confirm or reject adrenal insufficiency. Cortisol insulin tolerance test cut-off immunoassay adrenal insufficiency and HPA-axis Figures Figure 1 Figure 2 Figure 3 Introduction The insulin tolerance test (ITT) is considered the gold standard for assessing the function of the hypothalamic-pituitary-adrenal axis (HPA-axis), with assessment of cortisol response to hypoglycemia as a driver of central stimulation of the HPA-axis. It is therefore superior in detecting central adrenal insufficiency and in cases of hypothalamic-pituitary dysfunction or impairment, for example, following radiotherapy and shortly after pituitary damage [1]. After the introduction of second-generation cortisol immunoassays, cortisol cut-off limits for adrenal insufficiency were determined for the corticotropin (ACTH) stimulation test, whereas similar assessments have not been performed for the ITT yet. Previous research indicates that the diagnostic cortisol cut-off limit for adrenal insufficiency depends on the type of stimulation test [2, 3], and the immunoassay used for the analysis of serum cortisol [4, 5]. Studies have evaluated different contemporary second-generation cortisol immunoassays, which use more specific antibodies than previous assays. They have suggested cortisol cut-off values for the ACTH test in the range of 350-460 nmol/L [6–12], which is approximately 20-30% lower than the previously recommended cut-off values of 500-550 nmol/L [11–13]. The switch to new and more specific assays, such as Elecsys ® Cortisol II, thereby has significant clinical implications, as it entails the need for an adjustment of the cut-off limits defining adrenal insufficiency, which guide the clinicians’ decision whether to treat or not. A similar change in the cortisol cut-off remains to be defined for the ITT. This study aimed to define the normative cortisol response to the ITT measured with thefirst- and second-generation Roche Elecsys ® Cortisol immunoassays (Elecsys ® Cort I and II), respectively. Methods Participants : The participants were recruited by newspaper advertisements or web-based recruiting http://www.forsogsperson.dk [14]. All the participants gave their written informed consent before enrollment. A total of 90 participants were included in the study (Table 1). The inclusion criterion was age >18 years. The exclusion criteria were malignant disease, pituitary or adrenal disease, epilepsy, ischaemic heart disease, or cerebrovascular disease, ongoing treatment with glucocorticoids, spironolactone, or opioids, steroid injection within 3 months before the test, pregnancy, breastfeeding, and alcohol or drug abuse. Sample collection and handling :All 90 participants underwent an ITT starting between 8:00 and 10:00 AM after an overnight fast. An intravenous (i.v.) line was established, followed by i.v. administration of 0.05– 0.15 U/kg insulin (Actrapid ® ; Novo Nordisk). Blood was drawn from the i.v. line 15 minutes before insulin administration, immediately before administration (t = 0 min) and 15, 30, 45, 60, 75, and 90 minutes after administration of insulin. During the test, 87 out of 90 participants achieved adequate hypoglycaemia, with a nadir blood glucose level below 2.2 mmol/L, accompanied by clinical symptoms, confirming appropriate testing. Blood samples were centrifuged, and serum was stored at -80°C until analysis of cortisol. All samples from each participant were measured in the same assay series. Laboratory analysis :Cortisol was quantified by the immunoassay Elecsys ® Cort I and II on Cobas 8000 e-modules (Roche GmbH, Germany), which uses electro-chemiluminescent detection and has interassay coefficients of variation (CVs) of 2.1%, 1.6%, and 1.8% at concentrations of 161, 532, and 837 nmol/L, respectively[15]. Statistics : Statistical analyses were performed using R Version 4.3.1 (R Foundation for Statistical Computing, Austria) and MicrosoftExcel Version 16.75.2 (Microsoft Corporation, USA). The agreement between the two immunoassays for measuring cortisol was assessed using Passing-Bablok regression and Bland-Altman plot with limits of agreement (LOA) defined as mean difference (bias) ± 2 SD (standard deviation). The cut-off limit for a normal adrenal function was calculated both as the 2.5 th percentile of the peak s-cortisol concentration and as the 5 th percentiles for the peak s-cortisol concentration. Differences in peak s-cortisol concentrations measured by the two assays were assessed using a paired t-test, while differences between sexes for each assay were evaluated using unpaired t-tests. The associations of age, sex, and body composition with cortisol concentrations during the ITT were examined using univariate and multivariate regression analyses. A difference was deemed statistically significant when P <0.05. Moreover, the percentage of false-positive cases relative to the recommended cortisol cut-off based on the ACTH test values was calculated. Results Immunoassay comparison : The mean peak cortisol was 608 ± 102 nmol/L measured by Elecsys ® Cort I and 501 ± 89 nmol/L by Elecsys ® Cort II, respectively (Table 1). Histograms of the distribution of the peak cortisol concentrations of the participants analysed by both immunoassays illustrated that Elecsys ® Cort II systematically measured lower cortisol concentrations compared to Elecsys ® Cort I (Fig. 1). Passing-Bablok regression confirmed that Elecsys ® Cort II measured cortisol concentrations 11% (95% CI: 4-19%) lower compared with Elecsys ® Cort I (Fig. 2A). The regression equation indicated both a fixed difference (intercept: −37 nmol/L) and a proportional difference (slope: 0.89). Comparison of the variability between the two immunoassays using Bland-Altman plots demonstrated a mean cortisol difference (bias) of 107 nmol/L (95% CI: 35-178 nmol/L), with higher values measured by Elecsys ® Cort I (Fig. 2B). The variability between the two immunoassays was 143 nmol/L. Cortisol cut-off limits : The 2.5 th percentile of peak cortisol was 404 nmol/L with Elecsys ® Cort I and 320 nmol/L with Elecsys ® Cort II (Table 2). The 5 th percentiles of peak cortisol with Elecsys ® Cort I was 451 nmol/L, while it was 348 nmol/L with Elecsys ® Cort II. Both the 2.5 th and 5 th percentiles were significantly lower when measured by Elecsys ® Cort II (P < 0.001) (Table 2). The gender specific 2.5 th percentile cortisol for men (Elecsys ® Cort I 390 nmol/L; Elecsys ® Cort II 313 nmol/L) and women (Elecsys I 474 nmol/L; Elecsys II 406 nmol/L) were also significantly different, with mean differences of 76 nmol/L (95% CI: 74–78) for Elecsys Cortisol I and 88 nmol/L (95% CI: 87–90) for Elecsys Cortisol II, respectively (Table 2). The percentage of patients who failed the ITT based on assay-dependent cortisol cut-off limits derived from the ACTH-test was 15% with a peak cortisol below 500 nmol/L with Elecsys ® Cort I (Fig. 3), while 18% had a peak cortisol below 420 nmol/L with Elecsys ® Cort II, respectively. Factors influencing the insulin tolerance test results: Neither sex, age nor body composition measurements (BMI, weight, height, waist, and hip) were significantly associated with peak s-cortisol in univariate and multivariate regression analyses (data not shown). Discussion This study established test- and assay-specific 2.5 th and 5 th percentiles for peak cortisol measured during the ITT with thefirst- and second-generation Roche Elecsys ® Cortisol immunoassays (Elecsys ® Cortisol I and II) to allow comparison with findings from previous research. The 2.5 th and 5 th percentiles of peak cortisol were 320 nmol/L and 348 nmol/L, respectively with Elecsys ® Cort II, which systematically reported 11% (95% CI: 4-19%) lower cortisol concentrations compared with Elecsys ® Cort I. Applying the cortisol cut-off limits recommended for diagnosing adrenal insufficiency determined with the ACTH test to the ITT led to misdiagnosis of nearly 20% of the healthy participants. These findings address a critical gap in the current knowledge regarding the diagnosis of adrenal insufficiency. The explanation for lower cortisol concentration measured by Elecsys ® Cort II is the change from using polyclonal antibodies in Elecsys ® Cort I to more specific monoclonal antibodies in Elecsys ® Cort II, which has reduced the variability and enhanced the specificity of the immunoassay as cortisol now binds to a single site on the target antigen reducing the possibility of cross-reactivity with other substances. [16]. Accordingly, studies have shown that cortisol concentrations and cut-off limits are lower when measured with monoclonal antibody-based immunoassays compared with polyclonal immunoassays and radioimmunoassays [2, 5, 6, 9]. The increased specificity has made the second-generation immunoassays move closer to the gold standard method for steroid measurements, liquid chromatography–tandem mass spectrometry (LC-MS/MS) [5, 9, 12]. The finding of lower cortisol concentrations and cut-off limits with the second-generation assay compared to the first-generation assay was consistent with earlier research in this field [5, 6, 10–12]. Previous studies, however, have assessed assay performances for the ACTH stimulation test, not the ITT. The present data suggest a cut-off limit for the ITT from healthy controls that is significantly lower than the one suggested for the ACTH stimulation tests. Previous studies of cortisol cut-off limits defining adrenal insufficiency for the ITT were performed two decades ago by less specific methods and reported cut-off limits ranging from 500-600 nmol/L, which was very similar to those determined for the ACTH test [17–21]. The way of defining cut-off points has varied between studies. Some defined the cortisol cut-off limit as the 5 th percentile from healthy controls [2, 20, 22]. Cho et al. argued for using the 5 th percentile due to the significant variation in peak cortisol levels in response to the ITT [2]. They determined a cortisol cut-off limit for the ITT of 15 μg/dL (414 nmol/L) which was lower compared with cut-off limits determined for the low-dose and high-dose ACTH tests [2]. Similarly, Simsek et al. defined a cortisol cut-off for the ITT of <300 nmol/L based on concordant results from low-dose ACTH and glucagon stimulation tests [3]. They also found that the cut-off limit in response to ITT was significantly lower than those obtained for both low-dose ACTH and glucagon stimulation tests, in accordance with the present findings. Such data have resulted in the suggestion of implementation of stimulation test-specific cortisol cut-off limits [2, 23] to improve the diagnostic accuracy. The lower cortisol cut-off limits observed with the ITT compared to the ACTH test might be explained by the ITT’s mechanism of action. The ITT generates a natural stress response in the body due to hypoglycaemia-induced endogenous cortisol release, while the ACTH stimulation test generates a supra-physiological response to the injection of a high dose of synthetic ACTH, acting directly on the adrenal glands and exceeding the body’s natural production. Mean peak cortisol was not significantly different between sexes, which is in concordance with previous findings [3, 24]. Our data indicated a lower cut-off point in men as compared to women, although gender differences are not usually considered [5, 12]. The reason for the difference remains unknown, and validation in a larger study is required. Failure to pass a cut-off limit for diagnosing adrenal insufficiency based on healthy controls should not stand alone in the diagnosis of adrenal insufficiency. While such cut-offs provide useful reference data, their clinical utility depends on validation in patient populations with varying pre-test probabilities, allowing the determination of threshold with acceptable predictive values. A recent study comparing the overnight metyrapone test with the ACTH stimulation test demonstrated that in patients with low pre-test probability, a normal response to ACTH test may suffice. However, in patients with higher likelihood of central adrenal insufficiency, confirmation by a reference standard test such as the overnight metyrapone test or ITT remains necessary [25]. Thus, in accordance with clinical experience, patient outcomes play a crucial role for optimizing the diagnosis [24]. Further research is needed to establish evidence-based cut-offs for the ITT, ideally by correlating test results with clinical outcomes in terms of hydrocortisone need, Addison crises etc. The main limitation of this study is the relatively small number of healthy controls, which reflects the labour-intensive and time-consuming nature of the ITT. Additionally, data from patients with a high pre-test probability of HPA axis deficiency are needed to validate an appropriate cut-off that balances sensitivity and specificity. Nevertheless, our findings provide substantial evidence to support the reevaluation of the existing cortisol cut-off limit when changing assays due to new technology and the evolution and differences in clinical practice. These findings support the transition towards test- and assay-specific guidelines in endocrine testing protocols, particularly in settings where accurate diagnosis of adrenal insufficiency carries critical clinical implications. Conclusion Using different assays and different stimulation tests requires different cortisol cut-off limits to define adrenal insufficiency. Based on healthy controls, we defined 2.5 th and 5 th percentile normative cortisol response measured with first- and second-generation Roche Elecsys ® Cortisol immunoassays for the ITT, which were considerably lower than commonly applied cut-off limits determined for the ACTH test measured with the same assays to confirm or reject adrenal insufficiency. Our data emphasise the importance of the clinician's understanding of cut-offs depending on the technique used in the laboratory, as well as the type of stimulation test. This is particularly essential in patients with low a priory likelihood of adrenal insufficiency, because the medical decision-making is more challenging due to the risk of error, when the biochemical outcome lies in the proximity of a chosen cut-off limit. The risk is a false positive outcome according to Bayes' Theorem [26], thereby compromising and concealing an accurate, clinically meaningful diagnosis. Statements and Declarations The study was approved by the local ethical committee (J.nr. H-B-2008–122) and the Danish Data Protection Agency (J.nr. 30–0330). The authors declare no conflict of interest. Acknowledgments: The authors thank laboratory technician Casper Kok for his valuable assistance. The study was funded by unrestricted grants from: The A.P. Moller Foundation for the Advancement of Medical Science, Arvid Nilsson’s Foundation, Christenson-Ceson’s Foundation, Axel-Muusfeldt’s Foundation and Else and Mogens Wedell-Wedellsborg’s Foundation. MK’s research salary was sponsored by Skibsreder Per Henriksen, R. & Hustrus Fund. UFR’s research salary was sponsored by a grant from Kirsten and Freddy Johansen’s Fund. SWB’s research salary was sponsored by the Novo Nordisk Foundation: NNF20OC0063280. 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All participants Men Women P- value Gender n = 87 57 (66%) 30 (34%) Age (years) 35 (19−66) 35 (19−65) 34 (20−66) 0.9 Weight (kg) 78 (43−137) 81 (62−119) 64 (43−137) 0.002 Height (cm) 176 ± 10 181 ± 7 167 ± 6 <0.001 BMI (kg/m 2 ) 24 (17−47) 24 (19−35) 23 (17−47) 0.8 Waist (cm) 87 ± 13 89 (74−118) 78 (62−126) 0.001 Hip (cm) 102.7 ± 11 101 ± 8 103 ± 15 0.7 Peak S-cortisol (nmol/L), Elecsys ® Cortisol I 608 ± 102 601 ± 103 622 ± 97 0.4 Peak S-cortisol (nmol/L), Elecsys ® Cortisol II 501 ± 89 490 ± 89 523 ± 85 0.1 Blood glucose, nadir 1.6 ± 0.4 1.7 ± 0.4 1.5 ± 0.4 0.03 *Data is presented as mean ± SD or median (range). One outlier was removed before analysis. Table 2. Assay specific cortisol cut-off limits based on normative cortisol responses to the insulin tolerance test. Elecsys Cortisol I Elecsys Cortisol II Mean difference (95% CI) P-value All participants (N=87) 2.5 th 404 nmol/L 320 nmol/L 89 (86-91) nmol/L <0.001 5 th 451 nmol/L 348 nmol/L 95 (93-98) nmol/L <0.001 Men (N=57) Women (N=30) Mean difference (95% CI) P-value Elecsys ® Cortisol I 2.5 th 390 nmol/L 474 nmol/L 76 (74-78) nmol/L <0.001 5 th 425 nmol/L 488 nmol/L 56 (54-59) nmol/L <0.001 Elecsys ® Cortisol II 2.5 th 313 nmol/L 406 nmol/L 88 (87-90) nmol/L <0.001 5 th 324 nmol/L 414 nmol/L 76 (74-78) nmol/L <0.001 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 01 Dec, 2025 Read the published version in Pituitary → Version 1 posted Editorial decision: Revision requested 28 Sep, 2025 Reviews received at journal 27 Sep, 2025 Reviews received at journal 23 Sep, 2025 Reviewers agreed at journal 22 Sep, 2025 Reviewers agreed at journal 20 Sep, 2025 Reviewers invited by journal 20 Sep, 2025 Editor assigned by journal 19 Sep, 2025 Submission checks completed at journal 19 Sep, 2025 First submitted to journal 18 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7651143","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":521704528,"identity":"ba428426-fccf-4641-ae25-94d6db36cd9b","order_by":0,"name":"Sema Okutan","email":"","orcid":"","institution":"Copenhagen University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sema","middleName":"","lastName":"Okutan","suffix":""},{"id":521704529,"identity":"bc03a3c4-fc8a-4a1f-8e9c-f520e038c3dc","order_by":1,"name":"Stina Willemoes Borresen","email":"","orcid":"","institution":"Copenhagen University 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Klose","email":"data:image/png;base64,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","orcid":"","institution":"Copenhagen University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Marianne","middleName":"","lastName":"Klose","suffix":""}],"badges":[],"createdAt":"2025-09-18 15:23:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7651143/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7651143/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11102-025-01608-y","type":"published","date":"2025-12-01T15:57:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":92584856,"identity":"59c4a426-8269-445a-8629-b4574a2aa179","added_by":"auto","created_at":"2025-10-01 10:17:46","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":401636,"visible":true,"origin":"","legend":"","description":"","filename":"SemaITTarticleFinalversion.docx","url":"https://assets-eu.researchsquare.com/files/rs-7651143/v1/341461a37437a5b4fbae7174.docx"},{"id":92584220,"identity":"142eae5e-9027-4e42-a7e3-b917d0b4596e","added_by":"auto","created_at":"2025-10-01 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10:09:46","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90437,"visible":true,"origin":"","legend":"","description":"","filename":"b57aede3be5949828c7a5924822ccb9d1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7651143/v1/b7b5119f0ce1ef4c7c603166.xml"},{"id":92584234,"identity":"22b2dabe-79b2-495a-aa2c-5748161af0c6","added_by":"auto","created_at":"2025-10-01 10:09:46","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":99181,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7651143/v1/6d05f1bdb17bd5e202a31a13.html"},{"id":92584853,"identity":"fd824181-aee7-4b34-9fe4-813387f0370d","added_by":"auto","created_at":"2025-10-01 10:17:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":115252,"visible":true,"origin":"","legend":"\u003cp\u003eThe histogram illustrates the distribution of the peak cortisol concentrations from the 87 healthy participants measured during the insulin tolerance test using both Roche Elecsys\u003csup\u003e®\u003c/sup\u003eCortisol I and II assays.\u0026nbsp;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7651143/v1/735c80f8edb05de911196e39.png"},{"id":92585476,"identity":"21bb3bce-76b1-42cb-a99c-2a3d10d2c643","added_by":"auto","created_at":"2025-10-01 10:25:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":104079,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e The Passing-Bablok regression plot shows the relationship between Elecsys\u003csup\u003e®\u003c/sup\u003eCortisol I and II for all serum cortisol measurements obtained during the insulin tolerance test from the 87 participants.\u003cstrong\u003e B) \u003c/strong\u003eThe Bland-Altman plot shows the difference between peak cortisol concentrations measured during the insulin tolerance test with Elecsys\u003csup\u003e®\u003c/sup\u003eCortisol I and II. The black stippled line represents the bias line (mean difference between the two methods), and the dotted lines indicate the upper and lower limits of agreement (LOA, mean difference ± 1.96 × standard deviation). Men; triangles. Women; dots.\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7651143/v1/912b8ccc9a1548e3be41a6c6.png"},{"id":92584222,"identity":"f74a7e5e-7721-42f3-a6d3-8b68ae950f3a","added_by":"auto","created_at":"2025-10-01 10:09:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32867,"visible":true,"origin":"","legend":"\u003cp\u003eThe percentage of patients who failed the insulin tolerance test with the currently used cortisol cut-off limits derived from the ACTH-test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7651143/v1/a9f3871cdaa69eda8092a6de.png"},{"id":97723823,"identity":"37c916ce-ea91-4c2b-8b9b-7068fd82648a","added_by":"auto","created_at":"2025-12-08 16:08:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":908862,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7651143/v1/9a6d599f-aa49-4a40-a3ca-5bd0613fb3a6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Defining normative cortisol responses to the insulin tolerance test: A comparison of Elecsys®Cort I and II assays","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe insulin tolerance test (ITT) is considered the gold standard for assessing the function of the hypothalamic-pituitary-adrenal axis (HPA-axis), with assessment of cortisol response to hypoglycemia as a driver of central stimulation of the HPA-axis. It is therefore superior in detecting central adrenal insufficiency and in cases of hypothalamic-pituitary dysfunction or impairment, for example, following radiotherapy and shortly after pituitary damage [1]. After the introduction of second-generation cortisol immunoassays, cortisol cut-off limits for adrenal insufficiency were determined for the corticotropin (ACTH) stimulation test, whereas similar assessments have not been performed for the ITT yet. Previous research indicates that the diagnostic cortisol cut-off limit for adrenal insufficiency depends on the type of stimulation test [2, 3], and the immunoassay used for the analysis of serum cortisol [4, 5]. Studies have evaluated different contemporary second-generation cortisol immunoassays, which use more specific antibodies than previous assays. They have suggested cortisol cut-off values for the ACTH test in the range of 350-460 nmol/L [6–12], which is approximately 20-30% lower than the previously recommended cut-off values of 500-550 nmol/L [11–13]. The switch to new and more specific assays, such as Elecsys\u003csup\u003e®\u003c/sup\u003eCortisol II, thereby has significant clinical implications, as it entails the need for an adjustment of the cut-off limits defining adrenal insufficiency, which guide the clinicians’ decision whether to treat or not.\u0026nbsp;A similar change in the cortisol cut-off remains\u0026nbsp;to be defined\u0026nbsp;for the ITT.\u003c/p\u003e\n\u003cp\u003eThis study aimed to define the normative cortisol response to the ITT measured with thefirst- and second-generation Roche Elecsys\u003csup\u003e®\u003c/sup\u003eCortisol immunoassays (Elecsys\u003csup\u003e®\u003c/sup\u003eCort I and II), respectively.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eParticipants\u003c/em\u003e\u003c/strong\u003e:\u0026nbsp;The participants were recruited by newspaper advertisements or web-based recruiting http://www.forsogsperson.dk [14]. All the participants gave their written informed consent before enrollment. A total of 90 participants were included in the study (Table 1). The inclusion criterion was age \u0026gt;18 years. The exclusion criteria were malignant disease, pituitary or adrenal disease, epilepsy, ischaemic heart disease, or cerebrovascular disease, ongoing treatment with glucocorticoids, spironolactone, or opioids, steroid injection within 3 months before the test, pregnancy, breastfeeding, and alcohol or drug abuse. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSample collection and handling\u003c/em\u003e\u003c/strong\u003e:All 90 participants underwent an ITT starting between 8:00 and 10:00 AM after an overnight fast. An intravenous (i.v.) line was established, followed by i.v. administration of 0.05– 0.15 U/kg insulin (Actrapid\u003csup\u003e®\u003c/sup\u003e; Novo Nordisk). Blood was drawn from the i.v. line 15 minutes before insulin administration, immediately before administration (t = 0 min) and 15, 30, 45, 60, 75, and 90 minutes after administration of insulin. During the test, 87 out of 90 participants achieved adequate hypoglycaemia, with a nadir blood glucose level below 2.2 mmol/L, accompanied by clinical symptoms, confirming appropriate testing. Blood samples were centrifuged, and serum was stored at -80°C until analysis of cortisol. All samples from each participant were measured in the same assay series.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLaboratory analysis\u003c/em\u003e\u003c/strong\u003e:Cortisol was quantified by the immunoassay Elecsys\u003csup\u003e®\u003c/sup\u003eCort I and II on Cobas 8000 e-modules (Roche GmbH, Germany), which uses electro-chemiluminescent detection and has interassay coefficients of variation (CVs) of 2.1%, 1.6%, and 1.8% at concentrations of 161, 532, and 837 nmol/L, respectively[15].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistics\u003c/em\u003e\u003c/strong\u003e:\u0026nbsp;Statistical analyses were performed using R Version 4.3.1 (R Foundation for Statistical Computing, Austria) and MicrosoftExcel Version 16.75.2 (Microsoft Corporation, USA). The agreement between the two immunoassays for measuring cortisol was assessed using Passing-Bablok regression and Bland-Altman plot with limits of agreement (LOA) defined as mean difference (bias) ± 2 SD (standard deviation). The cut-off limit for a normal adrenal function was calculated both as the 2.5\u003csup\u003eth\u003c/sup\u003e percentile of the peak s-cortisol concentration and as the 5\u003csup\u003eth\u003c/sup\u003e percentiles for the peak s-cortisol concentration. Differences in peak s-cortisol concentrations measured by the two assays were assessed using a paired t-test, while differences between sexes for each assay were evaluated using unpaired t-tests. The associations of age, sex, and body composition with cortisol concentrations during the ITT were examined using univariate and multivariate regression analyses. A difference was deemed statistically significant when \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt;0.05. Moreover, the percentage of false-positive cases relative to the recommended cortisol cut-off based on the ACTH test values was calculated.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunoassay comparison\u003c/em\u003e\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe mean peak cortisol was 608 ± 102 nmol/L measured by Elecsys\u003csup\u003e®\u003c/sup\u003eCort I and 501 ± 89 nmol/L by Elecsys\u003csup\u003e®\u003c/sup\u003eCort II, respectively (Table 1). Histograms of the distribution of the peak cortisol concentrations of the participants analysed by both immunoassays illustrated that Elecsys\u003csup\u003e®\u003c/sup\u003eCort\u0026nbsp;II systematically measured lower cortisol concentrations compared to Elecsys\u003csup\u003e®\u003c/sup\u003eCort\u0026nbsp;I (Fig. 1). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePassing-Bablok regression confirmed that Elecsys\u003csup\u003e®\u003c/sup\u003eCort II measured cortisol concentrations 11% (95% CI: 4-19%) lower compared with Elecsys\u003csup\u003e®\u003c/sup\u003eCort\u0026nbsp;I (Fig. 2A). The regression equation indicated both a fixed difference (intercept: −37 nmol/L) and a proportional difference (slope: 0.89).\u003c/p\u003e\n\u003cp\u003eComparison of the variability between the two immunoassays using Bland-Altman plots demonstrated a mean cortisol difference (bias) of 107 nmol/L (95% CI: 35-178 nmol/L), with higher values measured by Elecsys\u003csup\u003e®\u003c/sup\u003eCort I (Fig. 2B). The variability between the two immunoassays was 143 nmol/L.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCortisol cut-off limits\u003c/em\u003e\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe 2.5\u003csup\u003eth\u003c/sup\u003e percentile of peak cortisol\u0026nbsp;was 404 nmol/L with\u0026nbsp;Elecsys\u003csup\u003e®\u003c/sup\u003eCort\u0026nbsp;I and 320 nmol/L with\u0026nbsp;Elecsys\u003csup\u003e®\u003c/sup\u003eCort\u0026nbsp;II\u0026nbsp;(Table 2). The 5\u003csup\u003eth\u003c/sup\u003e percentiles of peak cortisol with Elecsys\u003csup\u003e®\u003c/sup\u003eCort I was 451 nmol/L, while it was 348 nmol/L with Elecsys\u003csup\u003e®\u003c/sup\u003eCort\u0026nbsp;II. Both the 2.5\u003csup\u003eth\u003c/sup\u003e and 5\u003csup\u003eth\u003c/sup\u003e percentiles were significantly lower when measured by Elecsys\u003csup\u003e®\u003c/sup\u003eCort II\u0026nbsp;(P \u0026lt; 0.001) (Table 2). The gender specific 2.5\u003csup\u003eth\u003c/sup\u003e percentile cortisol for men (Elecsys\u003csup\u003e®\u003c/sup\u003eCort\u0026nbsp;I 390 nmol/L; Elecsys\u003csup\u003e®\u003c/sup\u003eCort\u0026nbsp;II 313 nmol/L) and women (Elecsys I 474 nmol/L; Elecsys II 406 nmol/L) were also significantly different, with mean differences of 76 nmol/L (95% CI: 74–78) for Elecsys\u0026nbsp;Cortisol I and 88 nmol/L (95% CI: 87–90) for Elecsys Cortisol II, respectively (Table 2).\u003c/p\u003e\n\u003cp\u003eThe percentage of patients who failed the ITT based on assay-dependent cortisol cut-off limits derived from the ACTH-test was 15% with a peak cortisol below 500 nmol/L with Elecsys\u003csup\u003e®\u003c/sup\u003eCort I (Fig. 3), while 18% had a peak cortisol below 420 nmol/L with Elecsys\u003csup\u003e®\u003c/sup\u003eCort II, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFactors influencing the insulin tolerance test results:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeither sex, age nor body composition measurements (BMI, weight, height, waist, and hip) were significantly associated with peak s-cortisol in univariate and multivariate regression analyses (data not shown).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study established test- and assay-specific 2.5\u003csup\u003eth\u003c/sup\u003e and 5\u003csup\u003eth\u003c/sup\u003e percentiles for peak cortisol measured during the ITT with thefirst- and second-generation Roche Elecsys\u003csup\u003e®\u003c/sup\u003eCortisol immunoassays (Elecsys\u003csup\u003e®\u003c/sup\u003eCortisol I and II) to allow comparison with findings from previous research. The 2.5\u003csup\u003eth\u003c/sup\u003e and 5\u003csup\u003eth\u003c/sup\u003e percentiles of peak cortisol were 320 nmol/L and 348 nmol/L, respectively with Elecsys\u003csup\u003e®\u003c/sup\u003eCort II, which\u0026nbsp;systematically reported 11% (95% CI: 4-19%) lower cortisol concentrations compared with Elecsys\u003csup\u003e®\u003c/sup\u003eCort I. Applying the cortisol cut-off limits recommended for diagnosing adrenal insufficiency determined with the ACTH test to the ITT led to misdiagnosis of nearly 20% of the healthy participants.\u0026nbsp;These findings address a critical gap in the current knowledge regarding the diagnosis of adrenal insufficiency.\u003c/p\u003e\n\u003cp\u003eThe explanation for lower cortisol concentration measured by Elecsys\u003csup\u003e®\u003c/sup\u003eCort II is the change from using polyclonal antibodies in Elecsys\u003csup\u003e®\u003c/sup\u003eCort I to more specific monoclonal antibodies in Elecsys\u003csup\u003e®\u003c/sup\u003eCort II, which has reduced the variability and enhanced the specificity of the immunoassay as cortisol now binds to a single site on the target antigen reducing the possibility of cross-reactivity with other substances. [16]. Accordingly, studies have shown that cortisol concentrations and cut-off limits are lower when measured with monoclonal antibody-based immunoassays compared with polyclonal immunoassays and radioimmunoassays [2, 5, 6, 9]. The increased specificity has made the second-generation\u0026nbsp;immunoassays move closer to the gold standard method for steroid measurements, liquid chromatography–tandem mass spectrometry (LC-MS/MS)\u0026nbsp;[5, 9, 12]. The finding of lower cortisol concentrations and cut-off limits with the\u0026nbsp;second-generation\u0026nbsp;assay compared to the first-generation assay was consistent with earlier research in this field [5, 6, 10–12]. Previous studies, however, have assessed assay performances for the ACTH stimulation test, not the ITT.\u003c/p\u003e\n\u003cp\u003eThe present data suggest a cut-off limit for the ITT from healthy controls that is significantly lower than the one suggested for the ACTH stimulation tests. Previous studies of cortisol cut-off limits defining adrenal insufficiency for the ITT were performed two decades ago by less specific methods and reported cut-off limits ranging from 500-600 nmol/L, which was very similar to those determined for the ACTH test [17–21]. The way of defining cut-off points has varied between studies. Some defined the cortisol cut-off limit as the 5\u003csup\u003eth\u003c/sup\u003e percentile from healthy controls [2, 20, 22]. Cho et al. argued for using the 5\u003csup\u003eth\u003c/sup\u003e percentile due to the significant variation in peak cortisol levels in response to the ITT [2]. They determined a cortisol cut-off limit for the ITT of 15 μg/dL (414 nmol/L) which was\u0026nbsp;lower compared with cut-off limits determined for the low-dose and high-dose ACTH tests [2]. Similarly, Simsek et al. defined a cortisol cut-off for the ITT of \u0026lt;300 nmol/L based on concordant results from low-dose ACTH and glucagon stimulation tests [3]. They also found that the cut-off limit in response to ITT was significantly lower than those obtained for both low-dose ACTH and glucagon stimulation tests, in accordance with the present findings. Such data have resulted in the suggestion of implementation of stimulation test-specific cortisol cut-off limits [2, 23] to improve the diagnostic accuracy. The lower cortisol cut-off limits observed with the ITT compared to the ACTH test might be explained by the ITT’s mechanism of action. The ITT generates a natural stress response in the body due to hypoglycaemia-induced endogenous cortisol release, while the ACTH stimulation test generates a supra-physiological response to the injection of a high dose of synthetic ACTH, acting directly on the adrenal glands and exceeding the body’s natural production.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMean peak cortisol was not significantly different between sexes, which is in concordance with previous findings [3, 24]. Our data indicated a lower cut-off point in men as compared to women, although gender differences are not usually considered [5, 12]. The reason for the difference remains unknown, and validation in a larger study is required.\u003c/p\u003e\n\u003cp\u003eFailure to pass a cut-off limit for diagnosing adrenal insufficiency based on healthy controls should not stand alone in the diagnosis of adrenal insufficiency. While such cut-offs provide useful reference data, their clinical utility depends on validation in patient populations with varying pre-test probabilities, allowing the determination of threshold with acceptable predictive values. A recent study comparing the overnight metyrapone test with the ACTH stimulation test demonstrated that in patients with low pre-test probability, a normal response to ACTH test may suffice. However, in patients with higher likelihood of central adrenal insufficiency, confirmation by a reference standard test such as the overnight metyrapone test or ITT remains necessary [25]. Thus, in accordance with clinical experience, patient outcomes play a crucial role for optimizing the diagnosis [24]. Further research is needed to establish evidence-based cut-offs for the ITT, ideally by correlating test results with clinical outcomes in terms of hydrocortisone need, Addison crises etc.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe main limitation of this study is the relatively small number of healthy controls, which reflects the labour-intensive and time-consuming nature of the ITT. Additionally, data from patients with a high pre-test probability of HPA axis deficiency are needed to validate an appropriate cut-off that balances sensitivity and specificity.\u003c/p\u003e\n\u003cp\u003eNevertheless, our findings provide substantial evidence to support the reevaluation of the existing cortisol cut-off limit when changing assays due to new technology and the evolution and differences in clinical practice. These findings support the transition towards test- and assay-specific guidelines in endocrine testing protocols, particularly in settings where accurate diagnosis of adrenal insufficiency carries critical clinical implications.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eUsing different assays and different stimulation tests requires different cortisol cut-off limits to define adrenal insufficiency. Based on healthy controls, we defined 2.5\u003csup\u003eth\u003c/sup\u003e and 5\u003csup\u003eth\u003c/sup\u003e percentile normative cortisol response measured with first- and second-generation Roche Elecsys\u003csup\u003e®\u003c/sup\u003eCortisol immunoassays\u0026nbsp;for the ITT, which were considerably lower than commonly applied cut-off limits determined for the ACTH test measured with the same assays to confirm or reject adrenal insufficiency. Our data emphasise the importance of the clinician's understanding of cut-offs depending on the technique used in the laboratory, as well as the type of stimulation test. This is particularly essential in patients with low \u003cem\u003ea priory\u003c/em\u003e likelihood of adrenal insufficiency, because the medical decision-making is more challenging due to the risk of error, when the biochemical outcome lies in the proximity of a chosen cut-off limit. \u0026nbsp;The risk is a false positive outcome according to Bayes' Theorem [26], thereby compromising and concealing an accurate, clinically meaningful diagnosis.\u0026nbsp;\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003eThe study was approved by the local ethical committee (J.nr. H-B-2008–122) and the Danish Data Protection Agency (J.nr. 30–0330). The authors declare no conflict of interest. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAcknowledgments:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank laboratory technician Casper Kok for his valuable assistance. The study was funded by unrestricted grants from: The A.P. Moller Foundation for the Advancement of Medical Science, Arvid Nilsson’s Foundation, Christenson-Ceson’s Foundation, Axel-Muusfeldt’s Foundation and Else and Mogens Wedell-Wedellsborg’s Foundation. MK’s research salary was sponsored by Skibsreder Per Henriksen, R. \u0026amp; Hustrus Fund. UFR’s research salary was sponsored by a grant from Kirsten and Freddy Johansen’s Fund. SWB’s research salary was sponsored by\u0026nbsp;the Novo Nordisk Foundation: NNF20OC0063280.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmmari, F., Issa, B.G., Millward, E., Scanlon, M.F.: A comparison between short ACTH and insulin stress tests for assessing hypothalamo-pituitary-adrenal function. Clin Endocrinol (Oxf). 44, 473\u0026ndash;476 (1996)\u003c/li\u003e\n\u003cli\u003eCho, H.Y., Kim, J.H., Kim, S.W., Shin, C.S., Park, K.S., Kim, S.W., Jang, H.C., Kim, S.Y.: Different cut-off values of the insulin tolerance test, the high-dose short Synacthen test (250 \u0026mu;g) and the low-dose short Synacthen test (1 \u0026mu;g) in assessing central adrenal insufficiency. Clin Endocrinol (Oxf). 81, 77\u0026ndash;84 (2014). https://doi.org/10.1111/cen.12397\u003c/li\u003e\n\u003cli\u003eSimsek, Y., Karaca, Z., Tanriverdi, F., Unluhizarci, K., Selcuklu, A., Kelestimur, F.: A comparison of low-dose ACTH, glucagon stimulation and insulin tolerance test in patients with pituitary disorders. Clin Endocrinol (Oxf). 82, 45\u0026ndash;52 (2015). https://doi.org/10.1111/cen.12528\u003c/li\u003e\n\u003cli\u003eEl-Farhan, N., Pickett, A., Ducroq, D., Bailey, C., Mitchem, K., Morgan, N., Armston, A., Jones, L., Evans, C., Rees, D.A.: Method-specific serum cortisol responses to the adrenocorticotrophin test: Comparison of gas chromatography-mass spectrometry and five automated immunoassays. Clin Endocrinol (Oxf). 78, 673\u0026ndash;680 (2013). https://doi.org/10.1111/cen.12039\u003c/li\u003e\n\u003cli\u003eJavorsky, B.R., Raff, H., Carroll, T.B., Algeciras-Schimnich, A., Singh, R.J., Col\u0026oacute;n-Franco, J.M., Findling, J.W.: New Cutoffs for the Biochemical Diagnosis of Adrenal Insufficiency after ACTH Stimulation using Specific Cortisol Assays. J Endocr Soc. 5, (2021). https://doi.org/10.1210/jendso/bvab022\u003c/li\u003e\n\u003cli\u003eHusni, H., Abusamaan, M.S., Dinparastisaleh, R., Sokoll, L., Salvatori, R., Hamrahian, A.H.: Cortisol values during the standard-dose cosyntropin stimulation test: Personal experience with Elecsys cortisol II assay. Front Endocrinol (Lausanne). 13, (2022). https://doi.org/10.3389/fendo.2022.978238\u003c/li\u003e\n\u003cli\u003eZha, L., Li, J., Krishnan, S.M., Brennan, M.R., Zhang, Y.V., Povse, P., Kerlin, R., Shively, K., Oleksik, F., Williams, J.A., Sykes, E., Sun, Q.: New Diagnostic Cutoffs for Adrenal Insufficiency After Cosyntropin Stimulation Using Abbott Architect Cortisol Immunoassay. Endocrine Practice. 28, 684\u0026ndash;689 (2022). https://doi.org/10.1016/J.EPRAC.2022.04.003\u003c/li\u003e\n\u003cli\u003eUeland, G.\u0026Aring;., Methlie, P., \u0026Oslash;ksnes, M., Thordarson, H.B., Sagen, J., Kellmann, R., Mellgren, G., Raeder, M., Dahlqvist, P., Dahl, S.R., Thorsby, P.M., L\u0026oslash;v\u0026aring;s, K., L\u0026oslash;v\u0026aring;s, L., Husebye, E.S.: The Short Cosyntropin Test Revisited: New Normal Reference Range Using LC-MS/MS. J Clin Endocrinol Metab. 103, 1696\u0026ndash;1703 (2018). https://doi.org/10.1210/jc.2017-02602\u003c/li\u003e\n\u003cli\u003eGrassi, G., Morelli, V., Ceriotti, F., Polledri, E., Fustinoni, \u0026amp; S., D\u0026rsquo;agostino, S., Mantovani, G., Chiodini, I., Arosio, \u0026amp; M.: Minding the gap between cortisol levels measured with second-generation assays and current diagnostic thresholds for the diagnosis of adrenal insufficiency: a single-center experience. Hormones. 19, 425\u0026ndash;431 (2020). https://doi.org/10.1007/s42000-020-00185-y/Published\u003c/li\u003e\n\u003cli\u003eKline, G.A., Buse, J., Krause, R.D.: Clinical implications for biochemical diagnostic thresholds of adrenal sufficiency using a highly specific cortisol immunoassay. Clin Biochem. 50, 475\u0026ndash;480 (2017). https://doi.org/10.1016/J.CLINBIOCHEM.2017.02.008\u003c/li\u003e\n\u003cli\u003eRaverot, V., Richet, C., Morel, Y., Raverot, G., Borson-Chazot, F.: Establishment of revised diagnostic cut-offs for adrenal laboratory investigation using the new Roche Diagnostics Elecsys\u0026reg; Cortisol II assay. Ann Endocrinol (Paris). 77, 620\u0026ndash;622 (2016). https://doi.org/10.1016/j.ando.2016.05.002\u003c/li\u003e\n\u003cli\u003eOkutan, S., J\u0026oslash;rgensen, N.T., Pedersen, L.E., Borresen, S.W., Hilsted, L., Hansen, L.F., Feldt-Rasmussen, U., Klose, M.: Determination of cortisol cut-off limits and steroid dynamics in the ACTH stimulation test: a comparative analysis using Roche Elecsys Cortisol II immunoassay and LC-MS/MS. Endocrine. 85, 321\u0026ndash;330 (2024). https://doi.org/10.1007/s12020-024-03752-0\u003c/li\u003e\n\u003cli\u003eBeuschlein, F., Else, T., Bancos, I., Hahner, S., Hamidi, O., Van Hulsteijn, L., Husebye, E.S., Karavitaki, N., Prete, A., Vaidya, A., Yedinak, C., Dekkers, O.M.: European Society of Endocrinology and Endocrine Society Joint Clinical Guideline: Diagnosis and Therapy of Glucocorticoid-induced Adrenal Insufficiency. J Clin Endocrinol Metab. 109, 1657 (2024). https://doi.org/10.1210/CLINEM/DGAE250\u003c/li\u003e\n\u003cli\u003eKlose, M., Stochholm, K., Janukonyt\u0026eacute;, J., Christensen, L.L., Frystyk, J., Andersen, M., Laurberg, P., Christiansen, J.S., Feldt-Rasmussen, U.: Prevalence of posttraumatic growth hormone deficiency is highly dependent on the diagnostic set-up: Results from the danish national study on posttraumatic hypopituitarism. Journal of Clinical Endocrinology and Metabolism. 99, 101\u0026ndash;110 (2014). https://doi.org/10.1210/jc.2013-2397\u003c/li\u003e\n\u003cli\u003eKlose, M.C., Lange, M., Rasmussen, A.K., Skakkeb\u0026aelig;k, N.E., Hilsted, L., Haug, E., Andersen, M., Feldt-Rasmussen, U.: Factors influencing the adrenocorticotropin test: Role of contemporary cortisol assays, body composition, and oral contraceptive agents. Journal of Clinical Endocrinology and Metabolism. 92, 1326\u0026ndash;1333 (2007). https://doi.org/10.1210/jc.2006-1791\u003c/li\u003e\n\u003cli\u003eChoi, M.H.: Clinical and Technical Aspects in Free Cortisol Measurement, (2022)\u003c/li\u003e\n\u003cli\u003eMayenknecht, J., Diederich, S., B\u0026auml;hr, V., Pl\u0026ouml;ckinger, U., Oelkers, W.: Comparison of Low and High Dose Corticotropin Stimulation Tests in Patients with Pituitary Disease. Journal of Clinical Endocrinology and Metabolism. 83, 1558\u0026ndash;1562 (1998)\u003c/li\u003e\n\u003cli\u003eHurel, S.J., Thompson, C.J., Watson, M.J., Harris, M.M., Baylis, P.H., Kendall-Taylor, P.: The short Synacthen and insulin stress tests in the assessment of the hypothalamic-pituitary-adrenal axis. Clin Endocrinol (Oxf). 44, 141\u0026ndash;146 (1996)\u003c/li\u003e\n\u003cli\u003eAbdu, T.A.M., Elhadd, T.A., Neary, R., Clayton, R.N.: Comparison of the Low Dose Short Synacthen Test (1 g), the Conventional Dose Short Synacthen Test (250 g), and the Insulin Tolerance Test for Assessment of the Hypothalamo-Pituitary-Adrenal Axis in Patients with Pituitary Disease. Journal of Clinical Endocrinology and Metabolism. 84, 838\u0026ndash;843 (1999)\u003c/li\u003e\n\u003cli\u003eClark, P.M., Neylon, I., Raggatt, P.R., Sheppard, M.C., Stewart, P.M.: Defining the normal cortisol response to the short Synacthen test: Implications for the investigation of hypothalamic-pituitary disorders. Clin Endocrinol (Oxf). 49, 287\u0026ndash;292 (1998). https://doi.org/10.1046/j.1365-2265.1998.00555.x\u003c/li\u003e\n\u003cli\u003eBangar, V., Clayton, R.N.: How reliable is the short synacthen test for the investigation of the hypothalamic-pituitary-adrenal axis? Eur J Endocrinol. 139, 580\u0026ndash;583 (1998)\u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lbez, Villabona, Ram\u0026oacute;n, Navarro, Gim\u0026eacute;nez, Ricart, Soler: Establishment of reference values for standard dose short synacthen test (250 \u0026mu;g), low dose short synacthen test (1 \u0026mu;g) and insulin tolerance test for assessment of the hypothalamo-pituitary-adrenal axis in normal subjects. Clin Endocrinol (Oxf). 53, 199\u0026ndash;204 (2000). https://doi.org/10.1046/j.1365-2265.2000.01028.x\u003c/li\u003e\n\u003cli\u003eSuliman, A.M., Smith, T.P., Labib, M., Fiad, T.M., McKenna, T.J.: The low-dose ACTH test does not provide a useful assessment of the hypothalamic-pituitary-adrenal axis in secondary adrenal insufficiency. Clin Endocrinol (Oxf). 56, 533\u0026ndash;539 (2002). https://doi.org/10.1046/j.1365-2265.2002.01509.x\u003c/li\u003e\n\u003cli\u003eFinucane, F.M., Liew, A., Thornton, E., Rogers, B., Tormey, W., Agha, A.: Clinical insights into the safety and utility of the insulin tolerance test (ITT) in the assessment of the hypothalamo-pituitary-adrenal axis. Clin Endocrinol (Oxf). 69, 603\u0026ndash;607 (2008). https://doi.org/10.1111/J.1365-2265.2008.03240.X;JOURNAL:JOURNAL:13652265;PAGEGROUP:STRING:PUBLICATION\u003c/li\u003e\n\u003cli\u003eHaberbosch, L., Meyer, N.M.T., Lechner, L., Jensen, M., Soll, D., Kozarzewski, L., Hesse, L., Blankenstein, O., Hubertus, V., Vajkoczy, P., Strasburger, C.J., Spranger, J., Maurer, L., Mai, K.: Optimizing diagnostic strategies for central adrenal insufficiency in pituitary disease. Eur J Endocrinol. 192, 100\u0026ndash;109 (2025). https://doi.org/10.1093/ejendo/lvaf002\u003c/li\u003e\n\u003cli\u003eWebb, M.P.K., Sidebotham, D.: Bayes\u0026rsquo; formula: a powerful but counterintuitive tool for medical decision-making. BJA Educ. 20, 208\u0026ndash;213 (2020). https://doi.org/10.1016/j.bjae.2020.03.002\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eBaseline characteristics of the participants.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"642\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAll participants\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMen\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWomen\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP-\u003c/strong\u003e\u003cstrong\u003evalue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGender\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003en = 87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e57 (66%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e30 (34%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e35 (19\u0026minus;66)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e35 (19\u0026minus;65)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e34 (20\u0026minus;66)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeight (kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e78 (43\u0026minus;137)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e81 (62\u0026minus;119)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e64 (43\u0026minus;137)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHeight (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e176 \u0026plusmn; 10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e181 \u0026plusmn; 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e167 \u0026plusmn; 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e24 (17\u0026minus;47)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e24 (19\u0026minus;35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e23 (17\u0026minus;47)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWaist (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e87 \u0026plusmn; 13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e89 (74\u0026minus;118)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e78 (62\u0026minus;126)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHip (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e102.7 \u0026plusmn; 11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e101 \u0026plusmn; 8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e103 \u0026plusmn; 15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeak S-cortisol (nmol/L), Elecsys\u003c/strong\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003cstrong\u003eCortisol I\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e608 \u0026plusmn; 102\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e601 \u0026plusmn; 103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e622 \u0026plusmn; 97\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeak S-cortisol (nmol/L), Elecsys\u003c/strong\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003cstrong\u003eCortisol II\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e501 \u0026plusmn; 89\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e490 \u0026plusmn; 89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e523 \u0026plusmn; 85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlood glucose, nadir\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e1.6 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e1.7 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e1.5 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Data is presented as mean \u0026plusmn; SD or median (range). One outlier was removed before analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eAssay specific\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ecortisol cut-off limits based on normative cortisol responses to the insulin tolerance test.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"642\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eElecsys Cortisol I\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eElecsys Cortisol II\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean difference (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAll participants (N=87)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.5\u003csup\u003eth\u003c/sup\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e404 nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e320 nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e89 (86-91) nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003csup\u003eth\u003c/sup\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e451 nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e348 nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e95 (93-98) nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMen (N=57)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWomen (N=30)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean difference (95% CI)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eElecsys\u003c/strong\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003cstrong\u003eCortisol I\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.5\u003csup\u003eth\u003c/sup\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e390 nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e474 nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e76 (74-78) nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003csup\u003eth\u003c/sup\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e425 nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e488 nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e56 (54-59) nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eElecsys\u003c/strong\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003cstrong\u003eCortisol II\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.5\u003csup\u003eth\u003c/sup\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e313 nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e406 nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e88 (87-90) nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003csup\u003eth\u003c/sup\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e324 nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e414 nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e76 (74-78) nmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pituitary","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pitu","sideBox":"Learn more about [Pituitary]()","snPcode":"11102","submissionUrl":"https://submission.nature.com/new-submission/11102/3","title":"Pituitary","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cortisol, insulin tolerance test, cut-off, immunoassay, adrenal insufficiency, and HPA-axis","lastPublishedDoi":"10.21203/rs.3.rs-7651143/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7651143/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and aim:\u003c/strong\u003e Monoclonal immunoassays offer greater specificity than polyclonal assays for cortisol measurement, necessitating assay-specific cut-off limits, which have not yet been defined for the insulin tolerance test (ITT). The study aim was to define the normative cortisol response to the ITT with the\u003csup\u003e \u003c/sup\u003efirst- and second-generation Roche Elecsys\u003csup\u003e®\u003c/sup\u003eCortisol immunoassays (Elecsys\u003csup\u003e®\u003c/sup\u003eCort I and II).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Ninety participants underwent an ITT after an overnight fast. Blood samples were analysed by both Elecsys\u003csup\u003e®\u003c/sup\u003eCort I and II. The normative cortisol cut-off limit to the ITT was defined as the 2.5\u003csup\u003eth\u003c/sup\u003e percentile of the peak cortisol level for each assay. The percentage of healthy participants with peak cortisol below the cut-off limits defined for the ACTH test, 500 nmol/L for Elecsys\u003csup\u003e®\u003c/sup\u003eCort I and 420 nmol/L for Elecsys\u003csup\u003e®\u003c/sup\u003eCort II, respectively, was calculated as the false-positive rate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The mean peak cortisol was 608 ± 102 nmol/L with Elecsys\u003csup\u003e®\u003c/sup\u003eCort I and 501 ± 89 nmol/L with Elecsys\u003csup\u003e®\u003c/sup\u003eCort II. The 2.5\u003csup\u003eth\u003c/sup\u003e percentile was 404 nmol/L with Elecsys\u003csup\u003e®\u003c/sup\u003eCort I and 320 nmol/L with Elecsys\u003csup\u003e®\u003c/sup\u003eCort II; 5\u003csup\u003eth\u003c/sup\u003e percentiles were 451 nmol/L and 348 nmol/L, respectively. False-positive rates were 15% with Elecsys\u003csup\u003e®\u003c/sup\u003eCort I and 18% with Elecsys\u003csup\u003e®\u003c/sup\u003eCort II.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Elecsys\u003csup\u003e®\u003c/sup\u003eCort II demonstrated a lower normative cortisol response to the ITT compared with Elecsys\u003csup\u003e®\u003c/sup\u003eCort I, and the commonly used ACTH test thresholds. These data underline the need for assay- and test-specific cut-off limits to confirm or reject adrenal insufficiency.\u003c/p\u003e","manuscriptTitle":"Defining normative cortisol responses to the insulin tolerance test: A comparison of Elecsys®Cort I and II assays","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-01 10:09:41","doi":"10.21203/rs.3.rs-7651143/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-28T08:29:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-27T21:26:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-23T09:42:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26028709438952658907325409208358764208","date":"2025-09-22T15:30:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217424172951689780954220587319934121401","date":"2025-09-20T14:54:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-20T14:27:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-19T10:44:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-19T10:43:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pituitary","date":"2025-09-18T15:18:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pituitary","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pitu","sideBox":"Learn more about [Pituitary]()","snPcode":"11102","submissionUrl":"https://submission.nature.com/new-submission/11102/3","title":"Pituitary","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a74c1ecb-b4c7-4320-8a8d-1fe5ff82856c","owner":[],"postedDate":"October 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:00:47+00:00","versionOfRecord":{"articleIdentity":"rs-7651143","link":"https://doi.org/10.1007/s11102-025-01608-y","journal":{"identity":"pituitary","isVorOnly":false,"title":"Pituitary"},"publishedOn":"2025-12-01 15:57:31","publishedOnDateReadable":"December 1st, 2025"},"versionCreatedAt":"2025-10-01 10:09:41","video":"","vorDoi":"10.1007/s11102-025-01608-y","vorDoiUrl":"https://doi.org/10.1007/s11102-025-01608-y","workflowStages":[]},"version":"v1","identity":"rs-7651143","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7651143","identity":"rs-7651143","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}