Bedtime Salivary Cortisol as a Screening Test for Cushing Syndrome in Children

Grethe Å. Ueland; Ralf Kellmann; Melissa Jørstad Davidsen; Kristin Viste; Eystein S. Husebye; Bjørg Almås; Helen L. Storr; Jørn V. Sagen; Gunnar Mellgren; Petur B. Júlíusson; Paal Methlie


J Endo Soc. 2021;5(5) 

In This Article

Abstract and Introduction


Background: Diagnosing Cushing syndrome (CS) can be challenging. The 24-hour urine free cortisol (UFC) measurement is considered gold standard. This is a laborious test, dependent on correct urine collection. Late-night salivary cortisol is easier and is used as a screening test for CS in adults, but has not been validated for use in children.

Objective: To define liquid chromatography tandem mass spectrometry (LC-MS/MS)-based cutoff values for bedtime and morning salivary cortisol and cortisone in children, and validate the results in children with and without CS.

Methods: Bedtime and morning salivary samples were collected from 320 healthy children aged 4 to 16 years. Fifty-four patients from the children's outpatient obesity clinic and 3 children with pituitary CS were used for validation. Steroid hormones were assayed by LC-MS/MS. Cutoff levels for bedtime salivary cortisol and cortisone were defined by the 97.5% percentile in healthy subjects.

Results: Bedtime cutoff levels for cortisol and cortisone were 2.4 and 12.0 nmol/L, respectively. Applying these cutoff levels on the verification cohort, 1 child from the obesity clinic had bedtime salivary cortisol exceeding the defined cutoff level, but normal salivary cortisone. All 3 children with pituitary CS had salivary cortisol and cortisone far above the defined bedtime cutoff levels. Healthy subjects showed a significant decrease in salivary cortisol from early morning to bedtime.

Conclusions: We propose that bedtime salivary cortisol measured by LC-MS/MS with a diagnostic threshold above 2.4 nmol/L can be applied as a screening test for CS in children. Age- and gender-specific cutoff levels are not needed.


The overall incidence of Cushing syndrome (CS) is approximately 2 to 5 new cases per million people per year, with only 5% to 10% of all cases occurring in children,[1] an incidence hampered by uncertainty as etiological classifications differ. In children, CS is most often diagnosed during infancy and in children above the age of 8 years. Delayed diagnosis may occur, with the condition first being diagnosed in adulthood.[2]

In healthy individuals, cortisol is subject to overall regulation of the pituitary/hypothalamus via ACTH. Cortisol has a diurnal profile, with highest levels early in the morning and low levels in the evening.[3] In CS, patients are exposed to higher circulating levels of cortisol, and the diurnal profile is often disrupted or absent.[4]

The most prominent symptom in children is growth stagnation and weight gain, unlike what is seen with alimentary obesity, in which there is increase in both height and body weight.[5,6] The symptoms of hypercortisolism are influenced by cortisol levels, duration of disease, age, and gender.[7–10]

The diagnosis of CS is largely based on laboratory testing and can be challenging to perform and interpret. Measurements of s-cortisol and p-ACTH in the morning and evening for evaluation of diurnal variation, the 1-mg dexamethasone suppression test, and 24-hour urine free cortisol (UFC) are the 3 most commonly used diagnostic tests in children.[11] The first 2 tests are not validated in children because age- and gender-specific reference limits or diagnostic cutoffs are lacking. For UFC, normal limits for children exist, and this test is considered the gold standard for the diagnosis of CS both in children and adults.[11,12]

Measurement of UFC is laborious. The caretaker must be trained in proper sampling or the child hospitalized for correct sampling. Even with appropriate preparation, the risk of sampling errors is substantial. Furthermore, fluid intake and renal function may affect cortisol excretion in the urine.[11] Thus, there is a need for a simple highly sensitive and specific screening test for CS in children.

In adults, late-night salivary cortisol is commonly used as a screening test for cortisol overproduction, and UFC obtained to confirm the diagnosis.[11] Data for diagnostic sensitivity and specificity in adults indicate that late-night salivary cortisol and UFC are equivalent.[13] Based on high activity of the enzyme 11β-HSD type II in salivary glands, which convert cortisol to cortisone, 1 study has suggested that salivary cortisone could be an even better biomarker for hypercortisolism.[14] There are a few studies that report on expected normal levels of salivary cortisol in children,[15–17] but none of these use highly specific analytical methods such as liquid chromatography tandem mass spectometry (LC-MS/MS).

In the present study, we have established LC-MS/MS based age- and gender-specific reference levels for bedtime salivary cortisol and cortisone to potentially pave the way for salivary cortisol and cortisone as a screening test for CS in children.