Clinical Utility of Salivary Cortisol for Diagnosis and Therapeutic Monitoring in Adrenal Insufficiency

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Methods: This prospective study included adult patients with clinical suspicion of primary or secondary AI who underwent ACTH stimulation testing (ACTH-t) and provided ambulatory CS samples at five time points (08:00, 13:00, 18:00, 24:00, and 08:00 the following day). CS was quantified via electrochemiluminescence immunoassay. Diagnostic performance, optimal thresholds, and correlations with morning serum cortisol (CB) and HC dosing were analyzed. Results: Out of 60 patients included, 11 (18.3%) were diagnosed with AI. CS at 08:00 (CS8) showed superior diagnostic performance over CB (AUC 0.804 vs. 0.739, P < 0.01). A CS8 threshold 0.708 µg/dL achieved 100% negative predictive value. Afternoon and evening CS levels were significantly higher in HC-treated patients, with elevated diurnal 08:00–24:00 cortisol exposure (AUC 7.82[2.57–11.14] vs. 2.73[2.19–3.79] ng·h/mL; p = 0.017) and a trend toward lower morning CS, suggesting overtreatment during the day and under-replacement at night 24:00–8:00 (AUC 2.06[0.67–6.13] vs. 1.6 [1.33–2.43] ng·h/mL; ns) . Conclusions: CS is a reliable, non-invasive biomarker for both screening and monitoring of AI. Its ability to reflect circadian cortisol dynamics offers clinical value in assessing replacement adequacy. Salivary profiling may guide individualized HC dose titration and help avoid overtreatment. Larger studies including salivary cortisone are warranted to refine monitoring strategies. Salivary cortisol adrenal insufficiency hydrocortisone replacement ACTH stimulation test glucocorticoid monitoring Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Adrenal insufficiency (AI) is a critical condition characterized by decreased endogenous cortisol production [ 1 ]. These patients require prompt and lifelong glucocorticoid replacement therapy[ 2 ]. However, due to the nonspecific nature of its clinical manifestations, including fatigue, anorexia, weight loss, arthralgia, myalgia, nausea, vomiting, and abdominal pain, diagnosis is often delayed or made during an adrenal crisis [ 3 ]. Conversely, a misdiagnosis of AI may result in unnecessary glucocorticoid therapy, leading to increased morbidity and mortality [ 2 ]. Traditionally, the most widely used tool in clinical practice for initial screening is the measurement of morning serum cortisol (CB), typically performed between 08:00 and 09:00 hours[ 2 ]. However, this method presents several limitations: its interpretation depends on circulating levels of corticosteroid-binding globulin (CBG), it does not accurately reflect the free cortisol fraction, and it may be affected by various systemic conditions or concomitant medications [ 4 , 5 ]. Additionally, it requires patient attendance at a specialized facility with trained personnel. Monitoring glucocorticoid replacement therapy in patients with AI also remains challenging, with a growing need for more individualized and less invasive approaches. Traditionally, monitoring has relied on clinical assessment of suggestive symptoms, measurement of basal cortisol levels, or 24-hour urinary free cortisol [ 2 , 6 , 7 ]. Nevertheless, all of these methods present significant limitations and fail to provide a reliable estimation of free cortisol levels or to assess proper replication of the physiological circadian rhythm [ 8 , 9 ]. In this context, salivary cortisol (CS) measurement may represent a valuable diagnostic tool. This method quantifies exclusively the free fraction of cortisol, is unaffected by circulating CBG concentrations, and can be obtained non-invasively under ambulatory conditions, thereby eliminating potential interference from acute stress related to venipuncture or the hospital environment. Nevertheless, the role of salivary cortisol in monitoring glucocorticoid replacement therapy remains controversial. While some studies have found no clinical utility due to poor correlation with serum cortisol levels [ 10 , 11 ] others have demonstrated good agreement and support its use for evaluating treatment efficacy [ 8 , 9 , 12 ]. Despite its advantages, the use of CS as a tool within the diagnostic and therapeutic algorithm for AI has not yet been standardized. Moreover, its utility as a screening test compared to morning serum cortisol requires broader clinical validation [ 2 ] The aim of the present study was to assess the diagnostic utility of CS as a screening tool for AI, in comparison with CB. Additionally, the study aimed to evaluate the functional integrity of the hypothalamic–pituitary–adrenal (HPA) axis by analyzing the circadian rhythm of CS, its correlation with plasma cortisol levels, and the influence of exogenous hydrocortisone (HC) administration in patients receiving replacement therapy. MATERIALS AND METHODS A prospective study was conducted including adult patients (≥ 18 years) with clinical suspicion of primary or secondary AI who were requested to undergo ACTH stimulation test (ACTH-t), and were evaluated at the Endocrinology Department of a tertiary care hospital between January 2016 and December 2022. The indication for performing the ACTH-t was determined by the attending endocrinologist based on clinical and biochemical criteria, in accordance with current international guidelines [ 2 ]. In parallel with the standard diagnostic ACTH-t protocol, patients were also asked to undergo ambulatory CS sampling to assess its circadian rhythm. Each participant was instructed on how to collect four salivary samples at home during a typical day using Salivette® Cortisol devices (Sarstedt, Germany). Samples were collected at the following times: 08:00 (CS8), 13:00 (CS13), 18:00 (CS18), 24:00 (CS24), and again at 08:00 the following day (CS8bis), following a written instruction sheet provided by the study team in accordance with the manufacturer’s guidelines. To avoid interference with cortisol measurement, participants were advised to refrain from eating, drinking, engaging in intense physical activity, or brushing their teeth for at least 60 minutes prior to each collection. Additionally, patients receiving HC replacement therapy were instructed to take their HC dose only after saliva sampling had been completed. Participants were also advised to avoid alcohol, chewing gum, and caffeinated beverages starting the day before sample collection. The Salivette swab was placed in the mouth for 1–2 minutes without chewing, then returned to the tube, which was sealed tightly. Samples were stored in a household refrigerator (2–8°C) and delivered to the hospital within 48 hours. Upon arrival at the laboratory, the samples were centrifuged at 3000 rpm for 10 minutes and stored frozen at − 20°C until analysis. Salivary cortisol quantification was performed using an electrochemiluminescence immunoassay (ECLIA) on the Elecsys Cortisol II® system (Roche Diagnostics, Mannheim, Germany), previously validated for salivary matrices. The analytical range of the method was 0.054 to 63.4 µg/dL, defined by the detection limit and the upper range established by the assay’s calibration curve. Additionally, each patient underwent baseline venous blood sampling between 08:00 and 09:00 hours, in a fasting state and at rest, for the determination of CB. Subsequently, 250 µg of intravenous tetracosactide was administered to perform the ACTH-t, with blood samples collected at 30 and 60 minutes. A post-stimulation plasma cortisol concentration ≥ 18 µg/dL was considered a normal response to the test. Clinical, anthropometric, and biochemical variables were collected, as well as laboratory parameters such as blood glucose, HbA1c, electrolytes, and renal and liver function. Hormonal measurements were also included, such as TSH, free T4, LH, FSH, testosterone, estrogens, DHEA, IGF-1, and prolactin, among others. In all cases, the presence of relevant metabolic comorbidities such as hypertension, diabetes mellitus, or dyslipidemia were documented. Use of medications potentially interfering with the hypothalamic–pituitary–adrenal axis was also recorded, including exogenous glucocorticoids (with details on hydrocortisone and fludrocortisone regimen and dosage), estrogens, and opioids. Patients with recent exposure to these treatments or with conditions that could impair the interpretation of adrenal function were excluded from the analysis. Statistical Analysis The sample size was calculated for a comparison of two independent means to detect a difference of 0.27 µg/dL in CS levels between two patient groups (AI vs. non-AI). A statistical power of 90% (β = 0.10) and a significance level of 0.05 (α = 0.05) were used. Based on these parameters and the expected unequal distribution of participants, the required sample sizes for the AI and non-AI groups were determined to be 9 and 45 participants, respectively. Quantitative variables were expressed as mean ± standard deviation (SD). Normality was assessed using the Kolmogorov–Smirnov test. Group comparisons were performed using Student’s t -test or the Wilcoxon test, as appropriate. Qualitative variables were expressed as percentages and analyzed using the Chi-square test or Fisher’s exact test, as required. The diagnostic performance of CS at each time point was evaluated against the ACTH-t result using receiver operating characteristic (ROC) curve analysis and area under the curve (AUC) calculation, identifying optimal cutoff points to achieve maximum sensitivity and specificity, respectively. Correlations between CS values (at different times) and baseline cortisol CB were assessed using Pearson’s correlation coefficient. Additionally, CS levels were compared between patients with and without HC replacement therapy at each time point to assess the potential use of CS as a tool for outpatient therapy monitoring. To evaluate cortisol exposure over the course of the day, three AUCs were calculated based on CS concentrations at 08:00, 13:00, 18:00, 24:00, and 08:00 the following day. AUCs were estimated using the trapezoidal rule and represented total 24-hour exposure (AUC 08:00–08:00), daytime exposure (AUC 08:00–24:00), and nighttime exposure (AUC 24:00–08:00). A p -value < 0.05 was considered statistically significant. Analyses were performed using SPSS® version 23.0 (IBM Corp., Armonk, NY, USA) and RStudio® version 2022 (RStudio, PBC, Boston, MA, USA). Ethical Considerations The study was approved by the Clinical Research Ethics Committee (PI 23-3203), and written informed consent was obtained from all participants. The study protocol was developed in accordance with the ethical principles of the Declaration of Helsinki and applicable data protection regulations. RESULTS A total of 60 patients were included (60% female; mean age 50.2 ± 15.7 years). Four patients (6.6%) were ultimately excluded from the analysis due to improper CS sample collection, evidenced by abnormally elevated values (> 11.0 µg/dL) suggestive of contamination. The most frequent indication for performing the ACTH-t was a history of pituitary surgery (42.9%; n = 24), in the context of HPA axis evaluation following neurosurgical intervention. This was followed by the presence of symptoms consistent with AI associated with low baseline cortisol levels without a clearly established etiology (37.5%; n = 21). Less common indications included active pituitary disease (10.6%; n = 6) and unilateral adrenalectomy (8.9%; n = 5) (Table 1 ). Table 1 Baseline characteristics of the study population values are presented as mean ± standard deviation, frequency (%), or median [interquartile range] Variable No adrenal insufficiency (n = 45) Adrenal insufficiency (n = 11) p-value Women 53.3% 90.9% 0.037 Age (years) 48.7 ± 15.8 56.4 ± 11.3 0.112 Diabetes 20.5% (n = 9) 9,1% (n = 1) 0.434 Hypertension 29.5% (n = 13) 18,2% (n = 2) 0.491 Dyslipidemia 25.0%(n = 11) 18,2%(n = 2) 0.734 Sodium (mEq/L) 141.1 ± 2.1 140.9 ± 2.9 0.812 Potassium (mEq/L) 4.45 ± 0.45 4.30 ± 0.49 0.419 Glucose (mg/dL) 98.9 ± 21.8 95.0 ± 26.4 0.684 Mean hydrocortisone dose (mg/day) — 22.2 ± 9.4 — Basal ACTH (pg/mL) 11.7 [7.2–22.0] 6.50 [2.85–37.62] 0.169 Basal Cortisol (µg/dL) 12.5 ± 5.2 6.39 ± 4.84 < 0.001 Cortisol 30 min post-ACTH (µg/dL) 21.5 ± 5.5 11.12 ± 5.59 < 0.001 Cortisol 60 min post-ACTH (µg/dL) 23.9 ± 6.3 12.66 ± 5.88 < 0.001 Cortisol saliva 8h (µg/dL) 0.33 [0.23–0.48] 0.26 [0.08–0.36] 0.058 Cortisol saliva 13h (µg/dL) 0.17 [0.13–0.23] 0.51 [0.24–1.36] < 0.01 Cortisol saliva 18h (µg/dL) 0.11 [0.05–0.15] 0.18 [0.11–0.78] < 0.01 Cortisol saliva 24h (µg/dL) 0.07 [0.05–0.11] 0.10 [0.07–0.52] < 0.01 Cortisol saliva 8h bis (µg/dL) 0.32 [0.22–0.46] 0.23 [0.08–0.34] 0.061 DHEA (µg/dL) 69.0 [9.1–222.0] 7.50 [5.50–118.23] 0.086 TSH (µUI/mL) 2.37 ± 1.63 1.74 ± 1.19 0.278 T4L (ng/dL) 1.17 ± 0.25 1.24 ± 0.33 0.537 LH (mUI/mL) 6.800 [3.250–24.400] 15.0 [3.7–50.1] 0.092 FSH (mUI/mL) 5.270 [2.530–22.700] 17.3 [5.3–74.8] 0.017 Testosterone (ng/dL) 260.0 [21.8–604.0] 81.5 [14.9–351.3] 0.045 Estrogens (pg/mL) 11.5 ± 8.6 15.4 ± 11.2 0.248 IGF-1 (ng/mL) 144.2 ± 61.8 149.2 ± 69.2 0.813 Prolactin (ng/mL) 15.0 ± 8.7 16.4 ± 13.5 0.688 Post-pituitary surgery 42.2% (n = 19) 45.5% (n = 5) Suspected primary AI 42.2% (n = 19) 18.2% (n = 2) Active pituitary disease 4.4% (n = 2) 36.4% (n = 4) Unilateral adrenalectomy 11.1% (n = 5) 0% (n = 0) A total of 11 patients (18.3%) were diagnosed with AI based on the ACTH-t response, and were receiving a mean oral HC dose of 22.2 ± 9.4 mg/day at their last follow-up visit. The mean follow-up duration in the Endocrinology outpatient clinic was 6.1 ± 4.9 years. In the total sample (N = 56), mean baseline CB and ACTH levels were 11.3 ± 5.2 µg/dL and 30.5 ± 25.8 pg/mL, respectively. Following ACTH-t, mean cortisol concentrations at 30 and 60 minutes were 20.7 ± 5.8 µg/dL and 23.0 ± 6.6 µg/dL, respectively. The diurnal CS profile showed a decreasing pattern throughout the day, with mean values of 0.38 ± 0.18 µg/dL for CS8, 0.31 ± 0.22 µg/dL for CS13, 0.19 ± 0.13 µg/dL for CS18, 0.13 ± 0.09 µg/dL for CS24, and 0.35 ± 0.16 µg/dL for CS8bis. A comparison of these parameters between patients with and without AI, along with other baseline clinical and biochemical characteristics, is presented in Table 1 . When analyzing the correlation between CS and CB levels, a moderate and statistically significant positive correlation was observed between CS8 and CB (r = 0.446; p = 0.002). However, no significant correlations were found for the other CS time points analyzed. The diagnostic performance of CS8 versus CB for the identification of AI was evaluated using ROC curve analysis, with ACTH-t results considered the gold standard (Fig. 1 ). CS8 showed an area under the curve (AUC) of 0.804 (95% CI: 0.578–1.000; p < 0.01), demonstrating good diagnostic performance. In contrast, CB showed a lower discriminative capacity, with an AUC of 0.739 (95% CI: 0.463–1.000; p < 0.05). However, CS levels at 13:00, 18:00, and 24:00 were not useful for diagnosing adrenal insufficiency, with significantly lower AUC values, all below 0.5: 0.202 (95% CI: 0.000–0.451; p < 0.05), 0.280 (95% CI: 0.000–0.590; p = 0.062), and 0.283 (95% CI: 0.041–0.524; p = 0.065), respectively (Fig. 2 ). The use of CS8 as a diagnostic tool enabled the identification of two clinically relevant cutoff points for the diagnosis of AI. A lower threshold of < 0.0975 µg/dL correctly identified all patients with AI, with a sensitivity of 100%, specificity of 57.1%, and a positive predictive value (PPV) of 75.0%. Conversely, a threshold of 0.708 µg/dL showed a negative predictive value (NPV) of 100%, reliably ruling out the disease. Applying these thresholds in our cohort would have avoided the need for 21.4% of ACTH stimulation tests. CS levels were also compared between patients receiving HC replacement therapy and those not on treatment. Significant differences were observed throughout the day, particularly in the afternoon and evening samples. Patients on HC therapy had significantly higher CS concentrations at CS13 (0.51 [0.24–1.36] vs. 0.17 [0.13–0.23] µg/dL; p < 0.01), CS18 (0.18 [0.11–0.78] vs. 0.11 [0.05–0.15] µg/dL; p < 0.01), and CS24 (0.10 [0.07–0.52] vs. 0.07 [0.05–0.11] µg/dL; p < 0.01), compared to untreated patients. No statistically significant differences were observed at CS8 (0.26 [0.08–0.36] vs. 0.33 [0.23–0.48] µg/dL; p = 0.058), although a trend toward higher concentrations in untreated patients (Table 1 ). Finally, as shown in Table 2 , salivary cortisol exposure, calculated using the trapezoidal rule, was significantly greater in patients receiving HC compared to those not on treatment, for both total 24-hour AUC (8.49 [3.74–23.71] vs. 4.49 [3.72–5.94] ng·h/mL; p = 0.039) and daytime AUC (7.82 [2.57–11.14] vs. 2.73 [2.19–3.79] ng·h/mL; p = 0.017). No significant differences were observed in nighttime exposure (2.06 [0.67–6.13] vs. 1.67 [1.33–2.43] ng·h/mL; p = 0.564). Daytime AUC values by treatment group are illustrated in Fig. 3 . Table 2 Salivary cortisol exposure (AUC) in patients with and without hydrocortisone treatment Period HC-treated Median [IQR] Non-treated Median [IQR] p-value Total 24h AUC (ng·h/mL) 8,49 [3,74–23,71] 4,49 [3,72–5,94] .039 Daytime AUC (ng·h/mL) 7,82 [2,57–11,14] 2.73 [2.19–3.79] .017 Nighttime AUC (ng·h/mL) 2,06 [0,67–6,13] 1.67 [1.33–2.43] .564 DISCUSSION This study demonstrates the diagnostic utility of CS as a screening tool for AI, emphasizing its superior diagnostic performance compared to CB and its potential value in assessing circadian rhythm and the adequacy of HC dosing in patients receiving replacement therapy. Furthermore, it evaluates the performance of CS in comparison to the ACTH-t and its applicability in outpatient monitoring. The diagnostic performance of CS8 for detecting AI showed superior accuracy compared to CB, yielding an AUC of 0.804 versus 0.739, respectively. These findings are consistent with previously published data showing improved diagnostic accuracy of CS over CB in evaluating AI [ 12 – 14 ]. Our study supports CS as a non-invasive, patient-friendly method that can be easily implemented in ambulatory settings, offering clear logistical advantages over CB. Moreover, CS is unaffected by venipuncture-induced stress and remains independent of variations in cortisol-binding proteins such as CBG and albumin [ 12 , 15 , 16 ]. Notably, sample collection failure occurred in only 6.6% of cases in a real-world clinical practice cohort. However, the AUC values below 0.5 observed for afternoon and nighttime CS measurements may reflect an inverse biomarker–disease relationship, potentially related to overtreatment. All patients with confirmed AI in our cohort were receiving HC replacement therapy, which resulted in supraphysiological CS levels during these time points, thereby affecting diagnostic accuracy and further supporting the presence of overtreatment. We identified a CS8 threshold of < 0.0975 µg/dL, which achieved 100% sensitivity, 57.1% specificity, and a positive predictive value (PPV) of 75% for AI, accurately identifying all true AI cases without false negatives—even in a cohort with relatively low disease prevalence. These results are consistent with previously reported thresholds ranging between < 0.032 and 0.708 µg/dL yielded an NPV of 100%, comparable to previously reported cutoffs around 0.61 µg/dL that achieved 100% specificity [ 17 ]. Other studies have proposed substantially lower upper thresholds [ 12 ]. These discrepancies may be explained, at least in part, by differences in assay methodology. While previous studies employed LC-MS/MS, our study used immunoassays, which are more susceptible to cross-reactivity and reduced precision at low concentration [ 18 ]. Although salivary cortisone measured by LC-MS/MS is considered the gold standard analytical approach for assessing adrenal function, our study supports the clinical validity of immunoassay-based CS measurements—the most widely used laboratory technique in routine clinical settings—when interpreted cautiously and under appropriate conditions[ 19 ]. Beyond its diagnostic performance, the relationship between CS and CB levels provides further insights into its utility The use of CS as a screening tool to evaluate AI relies in part on the correlation between CS and CB levels. Although under physiological conditions CS and CB exhibit a strong positive correlation [ 20 , 21 ], this relationship may weaken in clinical contexts where cortisol levels are low, such as in AI [ 10 , 11 ]. In our cohort, a moderate but statistically significant correlation was observed between morning CS8 and CB (r = 0.446; p = 0.002), supporting this association. These findings are consistent with previous reports showing moderate correlations between CS and CB[ 13 , 22 ], although lower than those reported in studies demonstrating stronger associations [ 9 ]. These differences in correlation, which may impact the diagnostic utility of CS as a more practical and non-invasive alternative to CB for diagnosing AI, are likely attributable to both methodological and population-related factors. Some studies have suggested a non-linear relationship between CB and CS [ 9 , 23 ], particularly across different concentration ranges. Notably, the strongest correlations have been observed at lower CS values, which are the most relevant for diagnosing AI. Our cohort reflects real-world clinical practice and mirrors the typical context in which the ACTH stimulation test (ACTH-t) is primarily used to rule out AI in patients with a relatively low pre-test probability, often yielding normal results and few pathological findings. However, at higher cortisol concentrations (above 18 µg/dL), CBG saturation may further weaken the correlation between serum and salivary cortisol [ 9 , 20 , 23 ]. The consistent observation of correlations in studies with low AI prevalence and linear analytical methods reinforces the clinical utility of CS in routine diagnostic settings. Patients receiving HC therapy showed significantly higher CS concentrations in the afternoon and evening samples, with median values of 0.51, 0.18, and 0.10 µg/dL at 13:00, 18:00, and 24:00 h, respectively, compared to 0.17, 0.11, and 0.07 µg/dL in untreated patients ( p < 0.01 for all comparisons). These findings are consistent with a previous study [ 8 ], which conducted detailed evaluations of salivary steroid profiles and demonstrated that no current glucocorticoid replacement regimen (including conventional HC, cortisone acetate, or dual-release formulations) successfully replicates either the physiological circadian rhythm or overall cortisol bioavailability. A similar pattern of cortisol overexposure during the daytime period (08:00–24:00 h) was observed in both the previous study and the present cohort, as evidenced by a significantly higher diurnal AUC in HC-treated patients, with no significant differences in nocturnal AUC (24:00–08:00 h). Interestingly, morning cortisol levels (CS8 and CS8bis) tended to be lower in treated patients, although not statistically significant, suggesting daytime overtreatment combined with relative nocturnal under-replacement. This pattern reinforces the role of CS as a dynamic biomarker of glucocorticoid exposure [ 8 ], with serial salivary measurements enabling detection of overdosing and other non-physiological replacement patterns, particularly in the context of conventional HC regimens Based on these findings, individualized adjustment of HC dosing appears warranted. In this study, patients receiving an average dose of 22.2 ± 9.4 mg/day showed clear biochemical signs of overexposure, particularly during the morning and afternoon, as reflected in elevated CS concentrations and increased daytime AUC. These results suggest that lower daily doses, potentially below 15 mg/day, may be sufficient for achieving physiological cortisol replacement in many patients. Current clinical guidelines and recent reviews [ 1 , 2 ] recommend a daily HC dose of 15 to 25 mg in divided doses for otherwise healthy adults with adrenal insufficiency. However, the evidence of overtreatment at a mean dose of 22 mg/day in this cohort supports consideration of dose reduction. Previous studies have suggested that patients with secondary AI may require lower doses[ 24 , 25 ]. In all cases, titration should be tailored to the individual, integrating both clinical assessment and objective parameters such as CS profiles to optimize efficacy and safety. Among the main strengths of our study is the use of serial CS sampling, which enabled us to characterize circadian cortisol exposure in real-life, outpatient conditions using a simple, non-invasive method. This approach provides a practical and physiological view of HC replacement therapy, reflecting not only morning values but also late-day cortisol levels, which are often overlooked in clinical practice. The inclusion of a control group not receiving treatment further strengthens the comparison of circadian profiles. However, several limitations should be acknowledged. First, the sample size was relatively small despite being adequate according to the sample size calculation. Second, the cross-sectional design and short duration of follow-up did not allow us to assess the long-term clinical consequences of cortisol overexposure, including its impact on metabolic or cardiovascular outcomes. Third, salivary cortisone was not measured, which may be a more reliable biomarker in certain situations. Finally, while saliva sampling was standardized, home collection introduces variability that cannot be entirely eliminated. In conclusion, this study supports the clinical utility of CS as a dynamic, non-invasive biomarker for both the screening and monitoring of adrenal insufficiency. Beyond its diagnostic value, salivary profiling provides essential insights into glucocorticoid exposure patterns during replacement therapy, allowing detection of overtreatment that may not be evident through conventional methods. Given the observed biochemical overexposure in patients receiving standard HC doses, individualized dose titration—guided by both clinical assessment and objective salivary markers—appears necessary to optimize physiological replacement and avoid long-term adverse effects. Future prospective studies including larger, well-characterized cohorts and salivary cortisone measurement will be essential to refine monitoring strategies and improve patient outcomes. Declarations Author Contribution P.F.V. was involved in investigation, statistical analysis, writing—original draft, writing—review and editing; I.A.M., S.R.L., B.T.T., A.O.B., E.G.H., W.T.F., D.C.N., D.d.L.R were involved in investigation; G.D.S. was involved in investigation, supervision, statistical analysis, writing—review and editing. References Husebye ES, Pearce SH, Krone NP, Kämpe O (2021) Adrenal insufficiency. 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European Journal of Endocrinology 169:31–36 El-Farhan N, Tennant S, Rees SE, Evans C, Rees DA (2024) Salivary Cortisol Response to ACTH Stimulation Is a Reliable Alternative to Serum Cortisol in Evaluating Hypoadrenalism. The Journal of Clinical Endocrinology & Metabolism 109:e579–e588 Perogamvros I, Owen LJ, Newell-Price J, Ray DW, Trainer PJ, Keevil BG (2009) Simultaneous measurement of cortisol and cortisone in human saliva using liquid chromatography–tandem mass spectrometry: Application in basal and stimulated conditions. Journal of Chromatography B 877:3771–3775 Hammarstrand C, Ragnarsson O, Hallén T, Andersson E, Skoglund T, Nilsson AG, Johannsson G, Olsson DS (2017) Higher glucocorticoid replacement doses are associated with increased mortality in patients with pituitary adenoma. European Journal of Endocrinology 177:251–256 Graziadio C, Hasenmajer V, Venneri MA, Gianfrilli D, Isidori AM, Sbardella E (2018) Glycometabolic Alterations in Secondary Adrenal Insufficiency: Does Replacement Therapy Play a Role? Front Endocrinol 9:434 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 15 Oct, 2025 Read the published version in Endocrine → Version 1 posted Editorial decision: Revision requested 22 Sep, 2025 Reviews received at journal 19 Sep, 2025 Reviews received at journal 06 Sep, 2025 Reviewers agreed at journal 01 Sep, 2025 Reviewers agreed at journal 01 Sep, 2025 Reviewers agreed at journal 25 Aug, 2025 Reviewers invited by journal 25 Aug, 2025 Editor assigned by journal 13 Aug, 2025 Submission checks completed at journal 13 Aug, 2025 First submitted to journal 12 Aug, 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. 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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-7354873","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":508280375,"identity":"94d35a43-4ddd-4096-b768-5cf020a43891","order_by":0,"name":"Pablo Fernández Velasco","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYFAC5gYQyc9wgPkAkJaQIUILI1iLZMMBtgSQFh5StPAYgBiEtRgcb2z8+KPGRoLv/JnPr27UWPAwsLc/wK/lzMFmaZ5jaRKSB85us845BnQYzxkDvFokZyQ2SDM2HK4zONi7zTiHDahFIge/wyTnP2z++bPhsITBYZ5nxjn/gFrkn+N3GL8EY5sEL0jLMR7mx7ltIFsY8DuMnyexzRrslzNsZsy5fRI8bDw5+LWwsR8+fBMSYocff875VifHz34cv8NQtEuASaLVAwHzB1JUj4JRMApGwcgBAC7aRAKIbPf+AAAAAElFTkSuQmCC","orcid":"","institution":"University of Valladolid","correspondingAuthor":true,"prefix":"","firstName":"Pablo","middleName":"Fernández","lastName":"Velasco","suffix":""},{"id":508280376,"identity":"1a78d15d-9a74-4922-a336-ef0279463853","order_by":1,"name":"Inés Aníbarro Miralles","email":"","orcid":"","institution":"Hospital Clínico Universitario de Valladolid","correspondingAuthor":false,"prefix":"","firstName":"Inés","middleName":"Aníbarro","lastName":"Miralles","suffix":""},{"id":508280377,"identity":"36cf4794-c4e8-45bf-bcd3-febf2376f3e5","order_by":2,"name":"Sara Rubio Lanchas","email":"","orcid":"","institution":"Hospital Clínico Universitario de Valladolid","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"Rubio","lastName":"Lanchas","suffix":""},{"id":508280378,"identity":"a8fd9d20-31e9-4b14-acbe-04ef0f35f0ae","order_by":3,"name":"Beatriz Torres Torres","email":"","orcid":"","institution":"University of Valladolid","correspondingAuthor":false,"prefix":"","firstName":"Beatriz","middleName":"Torres","lastName":"Torres","suffix":""},{"id":508280379,"identity":"3e16e690-12d8-47ca-b690-17afc5c42fd8","order_by":4,"name":"Ana Ortolá Buigues","email":"","orcid":"","institution":"University of Valladolid","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Ortolá","lastName":"Buigues","suffix":""},{"id":508280380,"identity":"72fccc9c-e584-45a1-81c0-5ca533175228","order_by":5,"name":"Emilia Gómez Hoyos","email":"","orcid":"","institution":"University of Valladolid","correspondingAuthor":false,"prefix":"","firstName":"Emilia","middleName":"Gómez","lastName":"Hoyos","suffix":""},{"id":508280381,"identity":"21c88646-34fa-45e1-a414-01349df85786","order_by":6,"name":"Wysali Trapiello Fernández","email":"","orcid":"","institution":"Hospital Clínico Universitario de Valladolid","correspondingAuthor":false,"prefix":"","firstName":"Wysali","middleName":"Trapiello","lastName":"Fernández","suffix":""},{"id":508280382,"identity":"06a89e44-0f5b-4481-b1a5-84cd64723332","order_by":7,"name":"Dolores Calvo Nieves","email":"","orcid":"","institution":"Hospital Clínico Universitario de Valladolid","correspondingAuthor":false,"prefix":"","firstName":"Dolores","middleName":"Calvo","lastName":"Nieves","suffix":""},{"id":508280383,"identity":"d34bf266-1ee8-44d7-94bf-fafc04d2e3b8","order_by":8,"name":"Daniel De Luís Román","email":"","orcid":"","institution":"University of Valladolid","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"De Luís","lastName":"Román","suffix":""},{"id":508280384,"identity":"17b61211-4b0d-4bfa-aaf5-a896b516684d","order_by":9,"name":"Gonzalo Díaz Soto","email":"","orcid":"","institution":"Hospital Clínico Universitario de Valladolid","correspondingAuthor":false,"prefix":"","firstName":"Gonzalo","middleName":"Díaz","lastName":"Soto","suffix":""}],"badges":[],"createdAt":"2025-08-12 10:53:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7354873/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7354873/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12020-025-04455-w","type":"published","date":"2025-10-15T15:57:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90526819,"identity":"142f4f93-ada9-4442-823e-432c72dae312","added_by":"auto","created_at":"2025-09-03 17:12:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":35780,"visible":true,"origin":"","legend":"\u003cp\u003eDiagnostic performance of salivary cortisol at 08:00 (CS8) and morning serum cortisol (CB) for the identification of adrenal insufficiency\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7354873/v1/80d36659835dd1d866d8eae7.png"},{"id":90526820,"identity":"1ac008bd-62de-431d-9619-62fa3ee7fe3b","added_by":"auto","created_at":"2025-09-03 17:12:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45763,"visible":true,"origin":"","legend":"\u003cp\u003eDiagnostic performance of salivary cortisol at later time points (CS13, CS18, and CS24) for the diagnosis of adrenal insufficiency.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7354873/v1/dc26a5db0846aeebd0811849.png"},{"id":90526821,"identity":"cebb5e58-046f-4a62-b642-ad7f8f90b240","added_by":"auto","created_at":"2025-09-03 17:12:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66708,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of diurnal salivary cortisol exposure (AUC 08:00–24:00) in patients with and without hydrocortisone (HC) replacement therapy.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7354873/v1/d70296c26259f464b0d3f0a4.png"},{"id":93956003,"identity":"19057a74-9668-4af8-91d1-784384fb48ba","added_by":"auto","created_at":"2025-10-20 16:09:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":983128,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7354873/v1/915fd4de-4fd9-4b55-bbe1-750a2b6ad195.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eClinical Utility of Salivary Cortisol for Diagnosis and Therapeutic Monitoring in Adrenal Insufficiency\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAdrenal insufficiency (AI) is a critical condition characterized by decreased endogenous cortisol production [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These patients require prompt and lifelong glucocorticoid replacement therapy[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, due to the nonspecific nature of its clinical manifestations, including fatigue, anorexia, weight loss, arthralgia, myalgia, nausea, vomiting, and abdominal pain, diagnosis is often delayed or made during an adrenal crisis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Conversely, a misdiagnosis of AI may result in unnecessary glucocorticoid therapy, leading to increased morbidity and mortality [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTraditionally, the most widely used tool in clinical practice for initial screening is the measurement of morning serum cortisol (CB), typically performed between 08:00 and 09:00 hours[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, this method presents several limitations: its interpretation depends on circulating levels of corticosteroid-binding globulin (CBG), it does not accurately reflect the free cortisol fraction, and it may be affected by various systemic conditions or concomitant medications [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Additionally, it requires patient attendance at a specialized facility with trained personnel.\u003c/p\u003e\u003cp\u003eMonitoring glucocorticoid replacement therapy in patients with AI also remains challenging, with a growing need for more individualized and less invasive approaches. Traditionally, monitoring has relied on clinical assessment of suggestive symptoms, measurement of basal cortisol levels, or 24-hour urinary free cortisol [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Nevertheless, all of these methods present significant limitations and fail to provide a reliable estimation of free cortisol levels or to assess proper replication of the physiological circadian rhythm [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this context, salivary cortisol (CS) measurement may represent a valuable diagnostic tool. This method quantifies exclusively the free fraction of cortisol, is unaffected by circulating CBG concentrations, and can be obtained non-invasively under ambulatory conditions, thereby eliminating potential interference from acute stress related to venipuncture or the hospital environment. Nevertheless, the role of salivary cortisol in monitoring glucocorticoid replacement therapy remains controversial. While some studies have found no clinical utility due to poor correlation with serum cortisol levels [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] others have demonstrated good agreement and support its use for evaluating treatment efficacy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite its advantages, the use of CS as a tool within the diagnostic and therapeutic algorithm for AI has not yet been standardized. Moreover, its utility as a screening test compared to morning serum cortisol requires broader clinical validation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eThe aim of the present study was to assess the diagnostic utility of CS as a screening tool for AI, in comparison with CB. Additionally, the study aimed to evaluate the functional integrity of the hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal (HPA) axis by analyzing the circadian rhythm of CS, its correlation with plasma cortisol levels, and the influence of exogenous hydrocortisone (HC) administration in patients receiving replacement therapy.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eA prospective study was conducted including adult patients (\u0026ge;\u0026thinsp;18 years) with clinical suspicion of primary or secondary AI who were requested to undergo ACTH stimulation test (ACTH-t), and were evaluated at the Endocrinology Department of a tertiary care hospital between January 2016 and December 2022. The indication for performing the ACTH-t was determined by the attending endocrinologist based on clinical and biochemical criteria, in accordance with current international guidelines [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In parallel with the standard diagnostic ACTH-t protocol, patients were also asked to undergo ambulatory CS sampling to assess its circadian rhythm.\u003c/p\u003e\u003cp\u003e Each participant was instructed on how to collect four salivary samples at home during a typical day using Salivette\u0026reg; Cortisol devices (Sarstedt, Germany). Samples were collected at the following times: 08:00 (CS8), 13:00 (CS13), 18:00 (CS18), 24:00 (CS24), and again at 08:00 the following day (CS8bis), following a written instruction sheet provided by the study team in accordance with the manufacturer\u0026rsquo;s guidelines. To avoid interference with cortisol measurement, participants were advised to refrain from eating, drinking, engaging in intense physical activity, or brushing their teeth for at least 60 minutes prior to each collection. Additionally, patients receiving HC replacement therapy were instructed to take their HC dose only after saliva sampling had been completed. Participants were also advised to avoid alcohol, chewing gum, and caffeinated beverages starting the day before sample collection. The Salivette swab was placed in the mouth for 1\u0026ndash;2 minutes without chewing, then returned to the tube, which was sealed tightly. Samples were stored in a household refrigerator (2\u0026ndash;8\u0026deg;C) and delivered to the hospital within 48 hours.\u003c/p\u003e\u003cp\u003eUpon arrival at the laboratory, the samples were centrifuged at 3000 rpm for 10 minutes and stored frozen at \u0026minus;\u0026thinsp;20\u0026deg;C until analysis. Salivary cortisol quantification was performed using an electrochemiluminescence immunoassay (ECLIA) on the Elecsys Cortisol II\u0026reg; system (Roche Diagnostics, Mannheim, Germany), previously validated for salivary matrices. The analytical range of the method was 0.054 to 63.4 \u0026micro;g/dL, defined by the detection limit and the upper range established by the assay\u0026rsquo;s calibration curve.\u003c/p\u003e\u003cp\u003eAdditionally, each patient underwent baseline venous blood sampling between 08:00 and 09:00 hours, in a fasting state and at rest, for the determination of CB. Subsequently, 250 \u0026micro;g of intravenous tetracosactide was administered to perform the ACTH-t, with blood samples collected at 30 and 60 minutes. A post-stimulation plasma cortisol concentration\u0026thinsp;\u0026ge;\u0026thinsp;18 \u0026micro;g/dL was considered a normal response to the test.\u003c/p\u003e\u003cp\u003eClinical, anthropometric, and biochemical variables were collected, as well as laboratory parameters such as blood glucose, HbA1c, electrolytes, and renal and liver function. Hormonal measurements were also included, such as TSH, free T4, LH, FSH, testosterone, estrogens, DHEA, IGF-1, and prolactin, among others. In all cases, the presence of relevant metabolic comorbidities such as hypertension, diabetes mellitus, or dyslipidemia were documented.\u003c/p\u003e\u003cp\u003eUse of medications potentially interfering with the hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal axis was also recorded, including exogenous glucocorticoids (with details on hydrocortisone and fludrocortisone regimen and dosage), estrogens, and opioids. Patients with recent exposure to these treatments or with conditions that could impair the interpretation of adrenal function were excluded from the analysis.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eThe sample size was calculated for a comparison of two independent means to detect a difference of 0.27 \u0026micro;g/dL in CS levels between two patient groups (AI vs. non-AI). A statistical power of 90% (β\u0026thinsp;=\u0026thinsp;0.10) and a significance level of 0.05 (α\u0026thinsp;=\u0026thinsp;0.05) were used. Based on these parameters and the expected unequal distribution of participants, the required sample sizes for the AI and non-AI groups were determined to be 9 and 45 participants, respectively.\u003c/p\u003e\u003cp\u003eQuantitative variables were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Normality was assessed using the Kolmogorov\u0026ndash;Smirnov test. Group comparisons were performed using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test or the Wilcoxon test, as appropriate. Qualitative variables were expressed as percentages and analyzed using the Chi-square test or Fisher\u0026rsquo;s exact test, as required.\u003c/p\u003e\u003cp\u003eThe diagnostic performance of CS at each time point was evaluated against the ACTH-t result using receiver operating characteristic (ROC) curve analysis and area under the curve (AUC) calculation, identifying optimal cutoff points to achieve maximum sensitivity and specificity, respectively. Correlations between CS values (at different times) and baseline cortisol CB were assessed using Pearson\u0026rsquo;s correlation coefficient.\u003c/p\u003e\u003cp\u003eAdditionally, CS levels were compared between patients with and without HC replacement therapy at each time point to assess the potential use of CS as a tool for outpatient therapy monitoring. To evaluate cortisol exposure over the course of the day, three AUCs were calculated based on CS concentrations at 08:00, 13:00, 18:00, 24:00, and 08:00 the following day. AUCs were estimated using the trapezoidal rule and represented total 24-hour exposure (AUC 08:00\u0026ndash;08:00), daytime exposure (AUC 08:00\u0026ndash;24:00), and nighttime exposure (AUC 24:00\u0026ndash;08:00).\u003c/p\u003e\u003cp\u003eA \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Analyses were performed using SPSS\u0026reg; version 23.0 (IBM Corp., Armonk, NY, USA) and RStudio\u0026reg; version 2022 (RStudio, PBC, Boston, MA, USA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEthical Considerations\u003c/h3\u003e\n\u003cp\u003eThe study was approved by the Clinical Research Ethics Committee (PI 23-3203), and written informed consent was obtained from all participants. The study protocol was developed in accordance with the ethical principles of the Declaration of Helsinki and applicable data protection regulations.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eA total of 60 patients were included (60% female; mean age 50.2\u0026thinsp;\u0026plusmn;\u0026thinsp;15.7 years). Four patients (6.6%) were ultimately excluded from the analysis due to improper CS sample collection, evidenced by abnormally elevated values (\u0026gt;\u0026thinsp;11.0 \u0026micro;g/dL) suggestive of contamination.\u003c/p\u003e\u003cp\u003eThe most frequent indication for performing the ACTH-t was a history of pituitary surgery (42.9%; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24), in the context of HPA axis evaluation following neurosurgical intervention. This was followed by the presence of symptoms consistent with AI associated with low baseline cortisol levels without a clearly established etiology (37.5%; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;21). Less common indications included active pituitary disease (10.6%; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6) and unilateral adrenalectomy (8.9%; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBaseline characteristics of the study population values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, frequency (%), or median [interquartile range]\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNo adrenal insufficiency\u0026nbsp; (n\u0026thinsp;=\u0026thinsp;45)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAdrenal insufficiency\u0026nbsp; (n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eWomen\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e53.3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e90.9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.037\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAge (years)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e48.7\u0026thinsp;\u0026plusmn;\u0026thinsp;15.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56.4\u0026thinsp;\u0026plusmn;\u0026thinsp;11.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.112\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDiabetes\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20.5% (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9,1% (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.434\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHypertension\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e29.5% (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18,2% (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.491\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDyslipidemia\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25.0%(n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18,2%(n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.734\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSodium (mEq/L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e141.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e140.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.812\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePotassium (mEq/L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.419\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGlucose (mg/dL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e98.9\u0026thinsp;\u0026plusmn;\u0026thinsp;21.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e95.0\u0026thinsp;\u0026plusmn;\u0026thinsp;26.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.684\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMean hydrocortisone dose (mg/day)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22.2\u0026thinsp;\u0026plusmn;\u0026thinsp;9.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBasal ACTH (pg/mL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.7 [7.2\u0026ndash;22.0]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.50 [2.85\u0026ndash;37.62]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.169\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBasal Cortisol (\u0026micro;g/dL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.39\u0026thinsp;\u0026plusmn;\u0026thinsp;4.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCortisol 30 min post-ACTH (\u0026micro;g/dL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.12\u0026thinsp;\u0026plusmn;\u0026thinsp;5.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCortisol 60 min post-ACTH (\u0026micro;g/dL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.66\u0026thinsp;\u0026plusmn;\u0026thinsp;5.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCortisol saliva 8h (\u0026micro;g/dL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.33\u0026nbsp;[0.23\u0026ndash;0.48]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.26\u0026nbsp;[0.08\u0026ndash;0.36]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.058\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCortisol saliva 13h (\u0026micro;g/dL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.17\u0026nbsp;[0.13\u0026ndash;0.23]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.51\u0026nbsp;[0.24\u0026ndash;1.36]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCortisol saliva 18h (\u0026micro;g/dL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.11\u0026nbsp;[0.05\u0026ndash;0.15]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.18\u0026nbsp;[0.11\u0026ndash;0.78]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCortisol saliva 24h (\u0026micro;g/dL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.07\u0026nbsp;[0.05\u0026ndash;0.11]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.10\u0026nbsp;[0.07\u0026ndash;0.52]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCortisol saliva 8h bis (\u0026micro;g/dL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.32 [0.22\u0026ndash;0.46]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.23 [0.08\u0026ndash;0.34]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.061\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDHEA (\u0026micro;g/dL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e69.0 [9.1\u0026ndash;222.0]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.50 [5.50\u0026ndash;118.23]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.086\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTSH (\u0026micro;UI/mL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.278\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eT4L (ng/dL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.537\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLH (mUI/mL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.800 [3.250\u0026ndash;24.400]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.0 [3.7\u0026ndash;50.1]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.092\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFSH (mUI/mL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.270 [2.530\u0026ndash;22.700]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.3 [5.3\u0026ndash;74.8]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.017\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTestosterone (ng/dL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e260.0 [21.8\u0026ndash;604.0]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e81.5 [14.9\u0026ndash;351.3]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.045\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eEstrogens (pg/mL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;11.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.248\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIGF-1 (ng/mL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e144.2\u0026thinsp;\u0026plusmn;\u0026thinsp;61.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e149.2\u0026thinsp;\u0026plusmn;\u0026thinsp;69.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.813\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eProlactin (ng/mL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;8.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.4\u0026thinsp;\u0026plusmn;\u0026thinsp;13.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.688\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePost-pituitary surgery\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e42.2% (n\u0026thinsp;=\u0026thinsp;19)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45.5% (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSuspected primary AI\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e42.2% (n\u0026thinsp;=\u0026thinsp;19)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.2% (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eActive pituitary disease\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.4% (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e36.4% (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eUnilateral adrenalectomy\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.1% (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0% (n\u0026thinsp;=\u0026thinsp;0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eA total of 11 patients (18.3%) were diagnosed with AI based on the ACTH-t response, and were receiving a mean oral HC dose of 22.2\u0026thinsp;\u0026plusmn;\u0026thinsp;9.4 mg/day at their last follow-up visit. The mean follow-up duration in the Endocrinology outpatient clinic was 6.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9 years.\u003c/p\u003e\u003cp\u003eIn the total sample (N\u0026thinsp;=\u0026thinsp;56), mean baseline CB and ACTH levels were 11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2 \u0026micro;g/dL and 30.5\u0026thinsp;\u0026plusmn;\u0026thinsp;25.8 pg/mL, respectively. Following ACTH-t, mean cortisol concentrations at 30 and 60 minutes were 20.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8 \u0026micro;g/dL and 23.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.6 \u0026micro;g/dL, respectively. The diurnal CS profile showed a decreasing pattern throughout the day, with mean values of 0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 \u0026micro;g/dL for CS8, 0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 \u0026micro;g/dL for CS13, 0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u0026micro;g/dL for CS18, 0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 \u0026micro;g/dL for CS24, and 0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 \u0026micro;g/dL for CS8bis. A comparison of these parameters between patients with and without AI, along with other baseline clinical and biochemical characteristics, is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eWhen analyzing the correlation between CS and CB levels, a moderate and statistically significant positive correlation was observed between CS8 and CB (r\u0026thinsp;=\u0026thinsp;0.446; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). However, no significant correlations were found for the other CS time points analyzed.\u003c/p\u003e\u003cp\u003eThe diagnostic performance of CS8 versus CB for the identification of AI was evaluated using ROC curve analysis, with ACTH-t results considered the gold standard (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). CS8 showed an area under the curve (AUC) of 0.804 (95% CI: 0.578\u0026ndash;1.000; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), demonstrating good diagnostic performance. In contrast, CB showed a lower discriminative capacity, with an AUC of 0.739 (95% CI: 0.463\u0026ndash;1.000; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, CS levels at 13:00, 18:00, and 24:00 were not useful for diagnosing adrenal insufficiency, with significantly lower AUC values, all below 0.5: 0.202 (95% CI: 0.000\u0026ndash;0.451; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), 0.280 (95% CI: 0.000\u0026ndash;0.590; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.062), and 0.283 (95% CI: 0.041\u0026ndash;0.524; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.065), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe use of CS8 as a diagnostic tool enabled the identification of two clinically relevant cutoff points for the diagnosis of AI. A lower threshold of \u0026lt;\u0026thinsp;0.0975 \u0026micro;g/dL correctly identified all patients with AI, with a sensitivity of 100%, specificity of 57.1%, and a positive predictive value (PPV) of 75.0%. Conversely, a threshold of 0.708 \u0026micro;g/dL showed a negative predictive value (NPV) of 100%, reliably ruling out the disease. Applying these thresholds in our cohort would have avoided the need for 21.4% of ACTH stimulation tests.\u003c/p\u003e\u003cp\u003eCS levels were also compared between patients receiving HC replacement therapy and those not on treatment. Significant differences were observed throughout the day, particularly in the afternoon and evening samples. Patients on HC therapy had significantly higher CS concentrations at CS13 (0.51 [0.24\u0026ndash;1.36] vs. 0.17 [0.13\u0026ndash;0.23] \u0026micro;g/dL; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), CS18 (0.18 [0.11\u0026ndash;0.78] vs. 0.11 [0.05\u0026ndash;0.15] \u0026micro;g/dL; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and CS24 (0.10 [0.07\u0026ndash;0.52] vs. 0.07 [0.05\u0026ndash;0.11] \u0026micro;g/dL; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), compared to untreated patients.\u003c/p\u003e\u003cp\u003eNo statistically significant differences were observed at CS8 (0.26 [0.08\u0026ndash;0.36] vs. 0.33 [0.23\u0026ndash;0.48] \u0026micro;g/dL; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.058), although a trend toward higher concentrations in untreated patients (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFinally, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, salivary cortisol exposure, calculated using the trapezoidal rule, was significantly greater in patients receiving HC compared to those not on treatment, for both total 24-hour AUC (8.49 [3.74\u0026ndash;23.71] vs. 4.49 [3.72\u0026ndash;5.94] ng\u0026middot;h/mL; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.039) and daytime AUC (7.82 [2.57\u0026ndash;11.14] vs. 2.73 [2.19\u0026ndash;3.79] ng\u0026middot;h/mL; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017). No significant differences were observed in nighttime exposure (2.06 [0.67\u0026ndash;6.13] vs. 1.67 [1.33\u0026ndash;2.43] ng\u0026middot;h/mL; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.564). Daytime AUC values by treatment group are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSalivary cortisol exposure (AUC) in patients with and without hydrocortisone treatment\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePeriod\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHC-treated\u003c/p\u003e\u003cp\u003eMedian [IQR]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNon-treated\u003c/p\u003e\u003cp\u003eMedian [IQR]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal 24h AUC (ng\u0026middot;h/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8,49 [3,74\u0026ndash;23,71]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4,49 [3,72\u0026ndash;5,94]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e.039\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDaytime AUC (ng\u0026middot;h/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7,82 [2,57\u0026ndash;11,14]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.73 [2.19\u0026ndash;3.79]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e.017\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNighttime AUC (ng\u0026middot;h/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2,06 [0,67\u0026ndash;6,13]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.67 [1.33\u0026ndash;2.43]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e.564\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study demonstrates the diagnostic utility of CS as a screening tool for AI, emphasizing its superior diagnostic performance compared to CB and its potential value in assessing circadian rhythm and the adequacy of HC dosing in patients receiving replacement therapy. Furthermore, it evaluates the performance of CS in comparison to the ACTH-t and its applicability in outpatient monitoring.\u003c/p\u003e\u003cp\u003eThe diagnostic performance of CS8 for detecting AI showed superior accuracy compared to CB, yielding an AUC of 0.804 versus 0.739, respectively. These findings are consistent with previously published data showing improved diagnostic accuracy of CS over CB in evaluating AI [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our study supports CS as a non-invasive, patient-friendly method that can be easily implemented in ambulatory settings, offering clear logistical advantages over CB. Moreover, CS is unaffected by venipuncture-induced stress and remains independent of variations in cortisol-binding proteins such as CBG and albumin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Notably, sample collection failure occurred in only 6.6% of cases in a real-world clinical practice cohort. However, the AUC values below 0.5 observed for afternoon and nighttime CS measurements may reflect an inverse biomarker\u0026ndash;disease relationship, potentially related to overtreatment. All patients with confirmed AI in our cohort were receiving HC replacement therapy, which resulted in supraphysiological CS levels during these time points, thereby affecting diagnostic accuracy and further supporting the presence of overtreatment.\u003c/p\u003e\u003cp\u003eWe identified a CS8 threshold of \u0026lt;\u0026thinsp;0.0975 \u0026micro;g/dL, which achieved 100% sensitivity, 57.1% specificity, and a positive predictive value (PPV) of 75% for AI, accurately identifying all true AI cases without false negatives\u0026mdash;even in a cohort with relatively low disease prevalence. These results are consistent with previously reported thresholds ranging between \u0026lt;\u0026thinsp;0.032 and \u0026lt;\u0026thinsp;0.035 \u0026micro;g/dL, which demonstrated similar sensitivity and PPV values in other clinical cohorts [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConversely, our upper threshold of \u0026gt;\u0026thinsp;0.708 \u0026micro;g/dL yielded an NPV of 100%, comparable to previously reported cutoffs around 0.61 \u0026micro;g/dL that achieved 100% specificity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Other studies have proposed substantially lower upper thresholds [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These discrepancies may be explained, at least in part, by differences in assay methodology. While previous studies employed LC-MS/MS, our study used immunoassays, which are more susceptible to cross-reactivity and reduced precision at low concentration [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Although salivary cortisone measured by LC-MS/MS is considered the gold standard analytical approach for assessing adrenal function, our study supports the clinical validity of immunoassay-based CS measurements\u0026mdash;the most widely used laboratory technique in routine clinical settings\u0026mdash;when interpreted cautiously and under appropriate conditions[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Beyond its diagnostic performance, the relationship between CS and CB levels provides further insights into its utility\u003c/p\u003e\u003cp\u003eThe use of CS as a screening tool to evaluate AI relies in part on the correlation between CS and CB levels. Although under physiological conditions CS and CB exhibit a strong positive correlation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], this relationship may weaken in clinical contexts where cortisol levels are low, such as in AI [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In our cohort, a moderate but statistically significant correlation was observed between morning CS8 and CB (r\u0026thinsp;=\u0026thinsp;0.446; p\u0026thinsp;=\u0026thinsp;0.002), supporting this association. These findings are consistent with previous reports showing moderate correlations between CS and CB[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], although lower than those reported in studies demonstrating stronger associations [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese differences in correlation, which may impact the diagnostic utility of CS as a more practical and non-invasive alternative to CB for diagnosing AI, are likely attributable to both methodological and population-related factors. Some studies have suggested a non-linear relationship between CB and CS [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], particularly across different concentration ranges. Notably, the strongest correlations have been observed at lower CS values, which are the most relevant for diagnosing AI. Our cohort reflects real-world clinical practice and mirrors the typical context in which the ACTH stimulation test (ACTH-t) is primarily used to rule out AI in patients with a relatively low pre-test probability, often yielding normal results and few pathological findings. However, at higher cortisol concentrations (above 18 \u0026micro;g/dL), CBG saturation may further weaken the correlation between serum and salivary cortisol [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The consistent observation of correlations in studies with low AI prevalence and linear analytical methods reinforces the clinical utility of CS in routine diagnostic settings.\u003c/p\u003e\u003cp\u003ePatients receiving HC therapy showed significantly higher CS concentrations in the afternoon and evening samples, with median values of 0.51, 0.18, and 0.10 \u0026micro;g/dL at 13:00, 18:00, and 24:00 h, respectively, compared to 0.17, 0.11, and 0.07 \u0026micro;g/dL in untreated patients (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for all comparisons). These findings are consistent with a previous study [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], which conducted detailed evaluations of salivary steroid profiles and demonstrated that no current glucocorticoid replacement regimen (including conventional HC, cortisone acetate, or dual-release formulations) successfully replicates either the physiological circadian rhythm or overall cortisol bioavailability. A similar pattern of cortisol overexposure during the daytime period (08:00\u0026ndash;24:00 h) was observed in both the previous study and the present cohort, as evidenced by a significantly higher diurnal AUC in HC-treated patients, with no significant differences in nocturnal AUC (24:00\u0026ndash;08:00 h). Interestingly, morning cortisol levels (CS8 and CS8bis) tended to be lower in treated patients, although not statistically significant, suggesting daytime overtreatment combined with relative nocturnal under-replacement. This pattern reinforces the role of CS as a dynamic biomarker of glucocorticoid exposure [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], with serial salivary measurements enabling detection of overdosing and other non-physiological replacement patterns, particularly in the context of conventional HC regimens\u003c/p\u003e\u003cp\u003eBased on these findings, individualized adjustment of HC dosing appears warranted. In this study, patients receiving an average dose of 22.2\u0026thinsp;\u0026plusmn;\u0026thinsp;9.4 mg/day showed clear biochemical signs of overexposure, particularly during the morning and afternoon, as reflected in elevated CS concentrations and increased daytime AUC. These results suggest that lower daily doses, potentially below 15 mg/day, may be sufficient for achieving physiological cortisol replacement in many patients. Current clinical guidelines and recent reviews [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] recommend a daily HC dose of 15 to 25 mg in divided doses for otherwise healthy adults with adrenal insufficiency. However, the evidence of overtreatment at a mean dose of 22 mg/day in this cohort supports consideration of dose reduction. Previous studies have suggested that patients with secondary AI may require lower doses[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In all cases, titration should be tailored to the individual, integrating both clinical assessment and objective parameters such as CS profiles to optimize efficacy and safety.\u003c/p\u003e\u003cp\u003eAmong the main strengths of our study is the use of serial CS sampling, which enabled us to characterize circadian cortisol exposure in real-life, outpatient conditions using a simple, non-invasive method. This approach provides a practical and physiological view of HC replacement therapy, reflecting not only morning values but also late-day cortisol levels, which are often overlooked in clinical practice. The inclusion of a control group not receiving treatment further strengthens the comparison of circadian profiles.\u003c/p\u003e\u003cp\u003eHowever, several limitations should be acknowledged. First, the sample size was relatively small despite being adequate according to the sample size calculation. Second, the cross-sectional design and short duration of follow-up did not allow us to assess the long-term clinical consequences of cortisol overexposure, including its impact on metabolic or cardiovascular outcomes. Third, salivary cortisone was not measured, which may be a more reliable biomarker in certain situations. Finally, while saliva sampling was standardized, home collection introduces variability that cannot be entirely eliminated.\u003c/p\u003e\u003cp\u003eIn conclusion, this study supports the clinical utility of CS as a dynamic, non-invasive biomarker for both the screening and monitoring of adrenal insufficiency. Beyond its diagnostic value, salivary profiling provides essential insights into glucocorticoid exposure patterns during replacement therapy, allowing detection of overtreatment that may not be evident through conventional methods. Given the observed biochemical overexposure in patients receiving standard HC doses, individualized dose titration\u0026mdash;guided by both clinical assessment and objective salivary markers\u0026mdash;appears necessary to optimize physiological replacement and avoid long-term adverse effects. Future prospective studies including larger, well-characterized cohorts and salivary cortisone measurement will be essential to refine monitoring strategies and improve patient outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.F.V. was involved in investigation, statistical analysis, writing\u0026mdash;original draft, writing\u0026mdash;review and editing; I.A.M., S.R.L., B.T.T., A.O.B., E.G.H., W.T.F., D.C.N., D.d.L.R were involved in investigation; G.D.S. was involved in investigation, supervision, statistical analysis, writing\u0026mdash;review and editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHusebye ES, Pearce SH, Krone NP, K\u0026auml;mpe O (2021) Adrenal insufficiency. The Lancet 397:613\u0026ndash;629\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBornstein SR, Allolio B, Arlt W, et al (2016) Diagnosis and Treatment of Primary Adrenal Insufficiency: An Endocrine Society Clinical Practice Guideline. The Journal of Clinical Endocrinology \u0026amp; Metabolism 101:364\u0026ndash;389\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBleicken B, Ventz M, Quinkler M, Hahner S (2010) Delayed Diagnosis of Adrenal Insufficiency Is Common: A Cross-Sectional Study in 216 Patients. The American Journal of the Medical Sciences 339:525\u0026ndash;531\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVerbeeten KC, Ahmet AH (2018) The role of corticosteroid-binding globulin in the evaluation of adrenal insufficiency. Journal of Pediatric Endocrinology and Metabolism 31:107\u0026ndash;115\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDichtel LE, Schorr M, Loures De Assis C, Rao EM, Sims JK, Corey KE, Kohli P, Sluss PM, McPhaul MJ, Miller KK (2019) Plasma Free Cortisol in States of Normal and Altered Binding Globulins: Implications for Adrenal Insufficiency Diagnosis. The Journal of Clinical Endocrinology \u0026amp; Metabolism 104:4827\u0026ndash;4836\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHowlett TA (1997) An Assessment of Optimal Hydrocortisone Replacement Therapy. Clinical Endocrinology 46:263\u0026ndash;268\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMah PM, Jenkins RC, Rostami-Hodjegan A, Newell‐Price J, Doane A, Ibbotson V, Tucker GT, Ross RJ (2004) Weight‐related dosing, timing and monitoring hydrocortisone replacement therapy in patients with adrenal insufficiency. Clinical Endocrinology 61:367\u0026ndash;375\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTucci L, Fanelli F, Improta I, et al (2024) Monitoring adrenal insufficiency through salivary steroids: a pilot study. European Journal of Endocrinology 190:327\u0026ndash;337\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKvam Hellan K, Lyngstad M, Methlie P, L\u0026oslash;v\u0026aring;s K, Husebye ES, Ueland G\u0026Aring; (2025) Utility of Salivary Cortisol and Cortisone in the Diagnostics of Adrenal Insufficiency. The Journal of Clinical Endocrinology \u0026amp; Metabolism 110:1218\u0026ndash;1223\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaguire AM, Ambler GR, Moore B, Waite K, McLean M, Cowell CT (2007) The clinical utility of alternative, less invasive sampling techniques in the assessment of oral hydrocortisone therapy in children and adolescents with hypopituitarism. eur j endocrinol 156:471\u0026ndash;476\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThomson AH, Devers MC, Wallace AM, Grant D, Campbell K, Freel M, Connell JMC (2007) Variability in hydrocortisone plasma and saliva pharmacokinetics following intravenous and oral administration to Patients with adrenal insufficiency. Clinical Endocrinology 66:789\u0026ndash;796\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDebono M, Elder CJ, Lewis J, et al (2023) Home Waking Salivary Cortisone to Screen for Adrenal Insufficiency. NEJM Evidence. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1056/EVIDoa2200182\u003c/span\u003e\u003cspan address=\"10.1056/EVIDoa2200182\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim YJ, Kim JH, Hong AR, Park KS, Kim SW, Shin CS, Kim SY (2020) Stimulated Salivary Cortisol as a Noninvasive Diagnostic Tool for Adrenal Insufficiency. Endocrinol Metab 35:628\u0026ndash;635\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLangelaan MLP, Kisters JMH, Oosterwerff MM, Boer A-K (2018) Salivary cortisol in the diagnosis of adrenal insufficiency: cost efficient and patient friendly. Endocrine Connections 7:560\u0026ndash;566\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRaff H (2009) Utility of Salivary Cortisol Measurements in Cushing\u0026rsquo;s Syndrome and Adrenal Insufficiency. The Journal of Clinical Endocrinology \u0026amp; Metabolism 94:3647\u0026ndash;3655\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLaudat MH, Cerdas S, Fournier C, Guiban D, Guilhaume B, Luton JP (1988) Salivary Cortisol Measurement: A Practical Approach to Assess Pituitary-Adrenal Function. The Journal of Clinical Endocrinology \u0026amp; Metabolism 66:343\u0026ndash;348\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKalaria T, Buch H, Agarwal M, et al (2022) Morning serum cortisol is superior to salivary cortisone and cortisol in predicting normal adrenal function in suspected adrenal insufficiency. Clinical Endocrinology 96:916\u0026ndash;918\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJavorsky BR, Raff H, Carroll TB, Algeciras-Schimnich A, Singh RJ, Col\u0026oacute;n-Franco JM, Findling JW (2021) New Cutoffs for the Biochemical Diagnosis of Adrenal Insufficiency after ACTH Stimulation using Specific Cortisol Assays. Journal of the Endocrine Society 5:bvab022\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDebono M, Harrison RF, Whitaker MJ, Eckland D, Arlt W, Keevil BG, Ross RJ (2016) Salivary Cortisone Reflects Cortisol Exposure Under Physiological Conditions and After Hydrocortisone. The Journal of Clinical Endocrinology \u0026amp; Metabolism 101:1469\u0026ndash;1477\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWood P (2009) Salivary steroid assays \u0026ndash; research or routine? Ann Clin Biochem 46:183\u0026ndash;196\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCeccato F, Barbot M, Zilio M, et al (2013) Performance of salivary cortisol in the diagnosis of Cushing\u0026rsquo;s syndrome, adrenal incidentaloma, and adrenal insufficiency. European Journal of Endocrinology 169:31\u0026ndash;36\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl-Farhan N, Tennant S, Rees SE, Evans C, Rees DA (2024) Salivary Cortisol Response to ACTH Stimulation Is a Reliable Alternative to Serum Cortisol in Evaluating Hypoadrenalism. The Journal of Clinical Endocrinology \u0026amp; Metabolism 109:e579\u0026ndash;e588\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePerogamvros I, Owen LJ, Newell-Price J, Ray DW, Trainer PJ, Keevil BG (2009) Simultaneous measurement of cortisol and cortisone in human saliva using liquid chromatography\u0026ndash;tandem mass spectrometry: Application in basal and stimulated conditions. Journal of Chromatography B 877:3771\u0026ndash;3775\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHammarstrand C, Ragnarsson O, Hall\u0026eacute;n T, Andersson E, Skoglund T, Nilsson AG, Johannsson G, Olsson DS (2017) Higher glucocorticoid replacement doses are associated with increased mortality in patients with pituitary adenoma. European Journal of Endocrinology 177:251\u0026ndash;256\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGraziadio C, Hasenmajer V, Venneri MA, Gianfrilli D, Isidori AM, Sbardella E (2018) Glycometabolic Alterations in Secondary Adrenal Insufficiency: Does Replacement Therapy Play a Role? Front Endocrinol 9:434\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"endocrine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"endo","sideBox":"Learn more about [Endocrine](https://www.springer.com/journal/12020)","snPcode":"12020","submissionUrl":"https://submission.nature.com/new-submission/12020/3","title":"Endocrine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Salivary cortisol, adrenal insufficiency, hydrocortisone replacement, ACTH stimulation test, glucocorticoid monitoring","lastPublishedDoi":"10.21203/rs.3.rs-7354873/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7354873/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose:\u003c/h2\u003e\u003cp\u003eTo evaluate the diagnostic utility of salivary cortisol (CS) as a non-invasive screening and monitoring tool for adrenal insufficiency (AI), and to assess its capacity to reflect cortisol exposure in patients receiving hydrocortisone (HC) replacement therapy.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e\u003cp\u003eThis prospective study included adult patients with clinical suspicion of primary or secondary AI who underwent ACTH stimulation testing (ACTH-t) and provided ambulatory CS samples at five time points (08:00, 13:00, 18:00, 24:00, and 08:00 the following day). CS was quantified via electrochemiluminescence immunoassay. Diagnostic performance, optimal thresholds, and correlations with morning serum cortisol (CB) and HC dosing were analyzed.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e\u003cp\u003eOut of 60 patients included, 11 (18.3%) were diagnosed with AI. CS at 08:00 (CS8) showed superior diagnostic performance over CB (AUC 0.804 vs. 0.739, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). A CS8 threshold\u0026thinsp;\u0026lt;\u0026thinsp;0.0975 \u0026micro;g/dL yielded 100% sensitivity and 75% positive predictive value, while\u0026thinsp;\u0026gt;\u0026thinsp;0.708 \u0026micro;g/dL achieved 100% negative predictive value. Afternoon and evening CS levels were significantly higher in HC-treated patients, with elevated diurnal 08:00\u0026ndash;24:00 cortisol exposure (AUC 7.82[2.57\u0026ndash;11.14] vs. 2.73[2.19\u0026ndash;3.79] ng\u0026middot;h/mL;\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017) and a trend toward lower morning CS, suggesting overtreatment during the day and under-replacement at night 24:00\u0026ndash;8:00 (AUC 2.06[0.67\u0026ndash;6.13] vs. 1.6 [1.33\u0026ndash;2.43] ng\u0026middot;h/mL;\u003cem\u003ens)\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e\u003cp\u003eCS is a reliable, non-invasive biomarker for both screening and monitoring of AI. Its ability to reflect circadian cortisol dynamics offers clinical value in assessing replacement adequacy. Salivary profiling may guide individualized HC dose titration and help avoid overtreatment. Larger studies including salivary cortisone are warranted to refine monitoring strategies.\u003c/p\u003e","manuscriptTitle":"Clinical Utility of Salivary Cortisol for Diagnosis and Therapeutic Monitoring in Adrenal Insufficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 17:12:15","doi":"10.21203/rs.3.rs-7354873/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-22T08:15:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-19T15:05:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-06T13:57:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"127424812138636634417530885424706215973","date":"2025-09-01T06:06:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187770620598224450431181364450460371044","date":"2025-09-01T04:52:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26028709438952658907325409208358764208","date":"2025-08-25T11:11:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-25T07:45:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-13T06:50:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-13T06:49:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Endocrine","date":"2025-08-12T10:51:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"endocrine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"endo","sideBox":"Learn more about [Endocrine](https://www.springer.com/journal/12020)","snPcode":"12020","submissionUrl":"https://submission.nature.com/new-submission/12020/3","title":"Endocrine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ed9df909-a7a3-49cb-8699-e5176da86fbd","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T16:02:10+00:00","versionOfRecord":{"articleIdentity":"rs-7354873","link":"https://doi.org/10.1007/s12020-025-04455-w","journal":{"identity":"endocrine","isVorOnly":false,"title":"Endocrine"},"publishedOn":"2025-10-15 15:57:08","publishedOnDateReadable":"October 15th, 2025"},"versionCreatedAt":"2025-09-03 17:12:15","video":"","vorDoi":"10.1007/s12020-025-04455-w","vorDoiUrl":"https://doi.org/10.1007/s12020-025-04455-w","workflowStages":[]},"version":"v1","identity":"rs-7354873","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7354873","identity":"rs-7354873","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}