In an adult being evaluated for possible hypercortisolism, how should I obtain and interpret a midnight cortisol level?

Midnight Cortisol in the Evaluation of Hypercortisolism

Obtain a sleeping midnight serum cortisol or late-night salivary cortisol (collected between 11 PM-midnight) as one of three first-line screening tests for Cushing's syndrome, with a sleeping midnight serum cortisol >50 nmol/L (>1.8 μg/dL) or late-night salivary cortisol >3.6 nmol/L indicating loss of normal circadian rhythm and supporting the diagnosis of hypercortisolism. 1, 2

Collection Methods and Timing

Sleeping Midnight Serum Cortisol

• The patient must be asleep when blood is drawn at midnight for serum cortisol measurement, as the test specifically evaluates loss of the normal cortisol nadir that occurs during sleep 1, 2
• A sleeping midnight serum cortisol ≥50 nmol/L (≥1.8 μg/dL) has 100% sensitivity and 60% specificity for Cushing's syndrome in pediatric populations, with similar performance in adults 1
• This test requires hospitalization or an indwelling catheter placed hours before collection to avoid stress-induced cortisol elevation 3

Late-Night Salivary Cortisol (Preferred Outpatient Method)

• Collect saliva between 2300-2400 hours (11 PM-midnight) using a commercial collection device while the patient is at home in their usual environment 2, 4
• Late-night salivary cortisol >3.6 nmol/L has 92-100% sensitivity and 93-100% specificity for Cushing's syndrome 2, 5
• Obtain 2-3 separate collections on different nights to account for test-to-test variability and detect cyclic Cushing's syndrome 2, 4

Diagnostic Algorithm

Step 1: Initial Screening (Use Multiple Tests)

• Order at least two of the following three screening tests to increase diagnostic accuracy 2, 5:
  • Late-night salivary cortisol (2-3 collections)
  • 24-hour urinary free cortisol (2-3 collections, with >193 nmol/24h or >70 μg/m² considered abnormal) 1, 2
  • Overnight 1-mg dexamethasone suppression test (cortisol ≥50 nmol/L or ≥1.8 μg/dL at 8 AM is abnormal) 2, 5

Step 2: Interpretation

• Two or more abnormal screening tests confirm hypercortisolism and warrant proceeding to determine the etiology 2, 5
• A single midnight serum cortisol >207 nmol/L (>7.5 μg/dL) correctly identifies Cushing's syndrome with 96% sensitivity at 100% specificity, superior to 24-hour urinary cortisol (45% sensitivity) 3
• In children and adolescents, sleeping midnight serum cortisol ≥50 nmol/L combined with elevated 24-hour urinary free cortisol has 89-100% sensitivity 1

Step 3: Determine Etiology After Confirming Hypercortisolism

• Measure 9 AM plasma ACTH: normal or elevated ACTH (>1.1 pmol/L or >5 ng/L) indicates ACTH-dependent Cushing's (pituitary or ectopic source) 1, 6
• Suppressed ACTH indicates adrenal source of hypercortisolism 6, 7

Critical Pitfalls to Avoid

Before Testing

• Eliminate all exogenous glucocorticoid sources (oral prednisone, dexamethasone, fluticasone inhalers, topical hydrocortisone, intra-articular/epidural injections) before biochemical testing, as these suppress endogenous cortisol and invalidate results 1, 2
• Verify the patient is not taking oral contraceptives or estrogen therapy, which increase cortisol-binding globulin and falsely elevate total cortisol without true hypercortisolism 2

Sample Collection Issues

• Do not test night-shift workers with late-night salivary cortisol due to disrupted circadian rhythm 2
• Avoid teeth brushing, dental work, or oral trauma within 1-2 hours of salivary collection to prevent blood contamination 2
• Ensure no topical hydrocortisone contamination of salivary samples 2

Medication Interactions

• CYP3A4 inducers (phenobarbital, carbamazepine, phenytoin) accelerate dexamethasone metabolism, causing false-positive dexamethasone suppression tests 2, 8
• Consider measuring dexamethasone levels concomitantly with cortisol during suppression testing to confirm adequate drug absorption 2

Pseudo-Cushing's States

• Severe obesity, depression, alcoholism, and polycystic ovary syndrome can cause mild hypercortisolism that mimics Cushing's syndrome 2, 5
• If screening tests are equivocal, consider Dex-CRH test or desmopressin test to distinguish true Cushing's from pseudo-Cushing's states 2

Special Considerations

Cyclic Cushing's Syndrome

• Patients may have weeks to months of normal cortisol secretion interspersed with hypercortisolism episodes 2
• Repeat testing during symptomatic periods if initial screening is normal but clinical suspicion remains high 2
• Extended monitoring over 3-6 months may be necessary to capture episodes of cortisol excess 2

Pediatric Populations

• In children with suspected Cushing's disease, late-night salivary cortisol has 95% sensitivity and 100% specificity 1
• Sleeping midnight serum cortisol ≥50 nmol/L combined with other screening tests provides optimal diagnostic accuracy in pediatric patients 1