The current epidemic of obesity presents an increasing number of patients with clinical features of Cushing's syndrome with the majority not having Cushing's syndrome. A simple, efficient and highly specific screening test is necessary to avoid large numbers of false-positive results with associated cost and anxiety. Since mild hypercortisolism is also associated with increased morbidity, false-negative results should also be avoided.
Salivary cortisol has been identified as a less Invasive and convenient test that offers an equivalent performance to the current invasive and less convenient conventional tests. There is a considerable body of evidence to support the use of salivary cortisol as a routine screening diagnostic test for Cushing's syndrome.
Mechanism of cortisol entry into saliva
Saliva is a mixture of fluids secreted by the three major pairs of salivary glands, i.e. parotid, submaxillary and sublingual glands, with some contribution from 600 – 1000 minor salivary glands within the oral cavity. The parotid glands consist of serous acinar cells which produce a serous, watery secretion, while submaxillary glands consist of serous and mucous acinar cells and produce mixed serous and mucous saliva. Sublingual glands mainly produce mucous saliva.
Serum components that are soluble in the lipid-rich cell membrane of the salivary gland acinar cells can pass freely into the cell and diffuse through into saliva. This is called intracellular diffusion. It only applies to lipid-soluble, non-protein-bound unconjugated steroids.
Non-protein-bound, unconjugated serum components with a molecular weight less than 1900kDa, together with water, may pass between the acinar cells tight junctions. This process is called ultrafiltration.
The two mechanisms of serum components entry into saliva explain why the concentrations of salivary unconjugated steroids such as cortisol, oestradiol and testosterone are the same as their free concentrations in serum. A healthy adult salivary cortisol concentration is typically 6 – 30 nmol/L between 08h00 and 10h00 and < 5 nmol/L between 22h00 and midnight.
Diagnostic use of salivary cortisol
Salivary cortisol meets the following basic criteria required of a diagnostic salivary steroid test:
- Salivary cortisol has a constant and predictable correlation with total serum cortisol concentrations.
- The diagnostic accuracy of salivary cortisol is equal to that of serum cortisol.
- A single salivary cortisol measurement gives the same information as a single serum cortisol measurement.
Salivary cortisol diurnal rhythm parallels that of serum cortisol and late-night saliva cortisol measurements are Recommended for Cushing's syndrome screening. It is not recommended for adrenal insufficiency and glucocorticoid therapeutic monitoring.
Cushing's syndrome screening
Currently, the three main screening tests are:
- Assessment of diurnal rhythm in serum cortisol
- Morning serum cortisol following a low-dose overnight dexamethasone suppression test
- 24-hour urine cortisol measurement
Unfortunately, these tests have the following limitations:
- Late-night serum cortisol is an in-hospital procedure which has added admission costs and stress with subsequent activation of Hypothalamic-Pituitary-Adrenal axis for a few days after admission. The latter may affect the validity of the test.
- 1-mg overnight low-dose dexamethasone suppression test requires a cut-off limit of serum cortisol of 50 nmol/L to achieve a diagnostic sensitivity of more than 90%. At this low cut-off, specificity of this test is severely reduced which renders the test less useful for screening purposes. In addition medication and some common conditions such as obesity may alter dexamethasone metabolism which may result in erroneous results.
- 24-hour urine collection, even with the most compliant patients, can be very difficult. Once an accurate urine collection is achieved, the sensitivity of a single urinary free cortisol measurement is 45% – 71% at a set specificity of 100%.
Salivary cortisol specimens can be collected in a home environment, which is relatively stress-free, and sent to the laboratory for analysis. There are now several studies that confirm that salivary cortisol is a reliable test for Cushing's syndrome screening.
It is recommended that screening procedures for Cushing's syndrome should include collection of
08h00 to 10h00 and 22h00 to midnight saliva samples over 2- 3 days. Mild Cushing's syndrome may show a normal late-night cortisol if tested only once. Results greaterthan 5 nmol/L are generally considered abnormal.
Salivary cortisol measurement may be used in low- dose overnight dexamethasone suppression test.
Pitfalls of salivary cortisol measurements:
- For the evening cortisol, ensure that the patient has a normal sleep/wake cycle.
- If collected at home, ensure that this is done at the correct time, i.e. 22h00 to midnight.
- Patients with pseudo-Cushing's i.e. alcohol abuse, type2 diabetes and major depression may have positive results.
Cyclical Cushing's syndrome
Cyclical Cushing's syndrome may have periodicity of 12 hours to 85 days. This will be difficult to diagnose using the conventional 24-hour urine collection, especially for identifying the necessary three peaks and two troughs in cortisol secretion. Salivary testing is an ideal convenient test and can be undertaken several times.
NB: It is currently not recommended to use salivary cortisol for adrenal insufficiency. Further studies are necessary.
Precautions for saliva collection
Ensure that the mouth is rinsed with water before collection.
Salivary samples should not be collected within 30 minutes of brushing teeth, eating, drinking or chewing flavoured chewing gum.
No ingestion of any foods of animal origin within 3 hours prior collection.
Blood contamination must be avoided and any samples showing some red discolouration should be discarded.
It is recommended that all morning samples be collected within 2 hours of waking up. The circadian rhythm cortisol studies will require both early morning and late night samples.
- Findling JW et al. Cushing's syndrome: important issues in diagnosis and management. JCEM 2006, 91:3746-3753
- Nieman LK et al. The diagnosis of Cushing's syndrome: an Endocrine Society Clinical Practice Guideline. JCEM 2008, 93:1526-1540
- Yuko T et al. Evaluation of salivary cortisol measurements for the diagnosis of subclinical Cushing's syndrome. Endocr J 2012, 59; 4:283-289
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