Endocrine Abstracts

Introduction: Increased CBG (corticosteroid binding globulin) concentrations can mask low free cortisol levels by showing normal total cortisol values, potentially leading to missed diagnoses of adrenal insufficiency (AI). Free cortisol estimates in serum and saliva may provide a more accurate assessment of adrenal function in these situations. The aim of this study is to describe the dynamic responses of calculated and measured free cortisol, salivary cortisol, and salivary cortisone in healthy individuals during the insulin tolerance test (ITT) and 250μg short Synacthen test (SST), and to evaluate the degree of discordance. Due to lower total cortisol values measured by modern immunoassays, a new cut-off value of 13.5 μg/dl (instead of 18 μg/dl) for endocrine dynamic function tests has been suggested in the literature to prevent overdiagnosis of AI. We aimed to compare the peak total cortisol levels (measured by electrochemiluminescence immunoassay and liquid chromatography–mass spectrometry (LC-MS/MS)) with the new proposed cut-off value of 13.5 μg/dl.

Methods: 24 healthy individuals underwent an ITT with collection of plasma and salivary samples at baseline and 15, 30, 45 and 60 minutes after insulin injection. 23 healthy individuals underwent an SST with sample collection at baseline, 30 and 60 minutes after Synacthen injection. Free cortisol was measured using equilibrium dialysis followed by LC-MS/MS and calculated by using an in-house formula based on the law of mass-action, comparable to the Vermeulen formula (1) for free testosterone. During the ITT, total cortisol was measured using electrochemiluminescence immunoassay and LC-MS/MS for method comparison.

Results: Calculated free cortisol values were significantly lower than measured free cortisol values in both dynamic tests. Salivary cortisol peaked at significantly different time points during the ITT compared to other variables (with earlier peaks in a substantial proportion of participants). We observed a significant higher relative increase of salivary cortisol and free cortisol in serum compared to total cortisol. During the ITT, 8/24 individuals (LC-MS/MS) and 3/24 (immunoassay) did not reach the 18 μg/dl total cortisol cutoff, whereas only one fell below the 13.5 μg/dl threshold (using both immunoassay and LC-MS/MS). During the SST (using LC-MS/MS), 1/23 missed the 18 μg/dl cutoff, and 23/23 reached a peak value above 13.5 μg/dl.

Conclusion: Free cortisol estimates may be a potential alternative to total cortisol; however further research is required to assess their role in individuals with altered cortisol-binding states as well as a clinical suspicion of adrenal insufficiency. Larger studies are needed to establish universally accepted cutoff values that consider the specific analytical method used.

References: 1. Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of.2. free testosterone in serum. J Clin Endocrinol Metab 84, pages 3666–3672 (1999).

Keywords: Adrenal insufficiency, endocrine dynamic function tests, free cortisol