Salivary Cortisol and Cortisone After Low-Dose Corticotropin Stimulation in the Diagnosis of Adrenal Insufficiency

Ingrid Yin Fung Mak; Benjamin Yick Toa Au Yeung; Ying Wai Ng; Cheung Hei Choi; Heidi Yan Ping Iu; Chi Chung Shek; Sau Cheung Tiu


J Endo Soc. 2017;1(2):96-108. 

In This Article

Subjects and Methods

A prospective study was carried out in a regional hospital in Hong Kong between January 2014 and September 2015. The study was approved by the Hospital's Research and Ethics Committee, and all participants provided written consent for the investigations.


Healthy subjects were recruited from hospital staff and their relatives. "Healthy" was defined as the absence of any symptoms and the absence of conditions or medications that were known to affect the hypothalamic-pituitary-adrenal axis, the CBG, or the albumin levels. Subjects suspected of having AI from the clinical context constituted the patient group. All subjects with acute hypocortisolism, hemodynamic instability at the time of testing, bleeding inside the oral cavity, pregnancy, or mental incapacity for informed consent were excluded.

Test Protocol

Patients receiving chronic hydrocortisone replacement had to stop the drug for at least 3 days before the test (Supplemental Data 1). A saliva sample for steroid measurement was collected by placing the cotton tubes (Salivette) in the mouth, chewing for 2 to 3 minutes, and then carefully putting the Salivette into a plastic container without touching it with hands. No drinking, eating, using mouthwash, or brushing teeth was allowed within 30 minutes before the collection of saliva samples, and subjects were advised to refrain from smoking or eating liquorice within 24 hours.

All subjects underwent the low-dose SST (LDSST) in the morning. Simultaneous saliva and serum samples were collected at baseline. A 1-μg bolus of Synacthen was then injected intravenously (Supplemental Data 1). At 30 and 60 minutes after the injection, 2 more pairs of saliva and serum samples were collected. The higher value of the tested parameters, at either 30 or 60 minutes, was regarded as the "peak" value.

Laboratory Methods

Serum total cortisol was assayed with the competitive chemiluminescent microparticle immunoassay using the Abbott Architect i2000SR system (Abbott Laboratories, Abbott Park, IL). The assay coefficient of variation (CV) was 4.0% to 6.2% at low levels and 3.3% to 4.3% at high levels. Salivary cortisol and cortisone were assayed with LC-MS/MS using the Waters Xevo TQ MS system (Waters, Milford, MA). The assay CV for salivary cortisol was ~5% to 7% across all ranges; that for salivary cortisone was ~7% to 10% across all ranges. The lower limit of detection was 0.5 nmol/L for both salivary cortisol and cortisone. Serum CBG was measured using a commercial human CBG enzyme-linked immunosorbent assay kit (BioVendor–Laboratorni Medicina, Brno, Czech Republic).

Statistical Analysis

The statistical analysis was performed using SPSS version 21.0. When the data were shown to be in a normal distribution by both the Kolmogorov–Smirnov and Shapiro–Wilk tests, the reference range was established by using 2 standard deviations (SDs) above or below the mean (mean ± 2 SDs); otherwise, the 95% reference level was used. Receiver operating characteristic (ROC) curves were drawn to compare the performance of serum cortisol, salivary cortisol, and salivary cortisone during LDSST in the patient group. Continuous data were expressed as mean ± SD and/or range. Correlations among continuous variables were assessed using Pearson correlation coefficients. A P value of <0.05 was considered as statistically significant.