Introduction

The dexamethasone suppression test (DST) is used in the evaluation of endogenous Cushing syndrome (CS), by assessing for the lack of suppression of the hypothalamic-pituitary-adrenal (HPA) axis by exogenous corticosteroids. Dexamethasone is a potent synthetic corticosteroid (dexamethasone 0.75 mg = prednisone 5 mg = methylprednisolone 4 mg = hydrocortisone 20 mg) with high affinity for the glucocorticoid receptors and long duration of action (biological half-life 36 to 54 hours; plasma half-life 4 to 5 hours). It possesses minimal mineralocorticoid activity and unlike other glucocorticoids, it does not interfere with cortisol measurement in the plasma, urine or saliva. It is these characteristics which make dexamethasone, the steroid of choice for the evaluation of the HPA axis.

Pathophysiology

The HPA axis, a primary neuroendocrine system, helps maintain the body’s homeostatic function and stress response.[1] The neurons in the paraventricular nucleus of hypothalamus synthesize corticotropin-releasing hormone (CRH) which via the hypophysial portal blood is transported to the anterior pituitary, wherein it stimulates the production of adrenocorticotrophic hormone (ACTH). ACTH is then transported by the bloodstream to the adrenal glands and stimulates the synthesis and secretion of cortisol by zona fasciculata of the adrenal cortex. Serum cortisol, also called the stress hormone, in turn, exerts negative feedback on both the hypothalamus and the anterior pituitary; thus, inhibiting the secretion of CRH and ACTH, respectively. This positive/negative feedback mechanism helps regulate the body’s serum cortisol levels and stress response.When HPA axis is intact, exogenously administered corticosteroids exert feedback inhibition on the production of serum CRH and ACTH by binding to the hypothalamic and pituitary glucocorticoid receptors, respectively, that subsequently causes suppression of the synthesis and secretion of serum cortisol. However, in pathological hypercortisolism, the HPA axis becomes partially or entirely resistant to feedback inhibition by exogenous steroids.

Diagnostic Tests

Types of dexamethasone suppression test (DST):

1. Low-dose DST

• Overnight, 1 mg test
• Two-day, 2 mg test

2. High-dose DST

• Overnight, 8 mg test
• Two-day, 8 mg test

3. Intravenous DST

4. Dexamethasone-CRH test

Testing Procedures

1. Low-dose DST (LDDST)

LDDST helps in the initial diagnosis of Cushing syndrome, as a screening or a confirmatory test.[2] It can be performed either as an overnight or a two-day test.

• Overnight, 1 mg test: Due to ease and convenience, the overnight test is the most commonly used screening test. Dexamethasone 1 mg is administered orally between 11 PM and midnight. Serum cortisol levels are drawn the next morning between 8 and 9 AM.[3][4]

• Two-day, 2 mg test: Dexamethasone 0.5 mg is administered orally every 6 hours (9 AM, 3 PM, 9 PM, 3 AM) for two days (total dose 4 mg). Serum cortisol level is drawn 6 hours (9 AM) after the last administered dose.[5]

2. High-dose DST (HDDST)

Once the diagnosis of Cushing syndrome is confirmed, the next step is to categorize ACTH-independent vs. ACTH-dependent Cushing syndrome, by checking the plasma ACTH levels.  In ACTH-independent Cushing syndrome, the plasma ACTH is low or undetectable, indicating an adrenal etiology (causing pituitary suppression of ACTH). While, in ACTH-dependent Cushing syndrome, the plasma ACTH is inappropriately normal or high, suggesting either pituitary or an ectopic source.

In ACTH-dependent Cushing syndrome, HDDST can help distinguish pituitary (i.e., Cushing disease) from an ectopic source of ACTH overproduction. The principle behind the high-dose test is that overproduction of ACTH in Cushing disease (but not ectopic tumors) can undergo partial or full suppression by high doses of dexamethasone (approximately 8 mg). Like LDDST, it utilizes oral dexamethasone, either as an overnight or two-day test.

• Overnight, 8 mg test: Baseline morning serum cortisol is measured, and oral dexamethasone 8 mg is administered between 11 PM and midnight. Repeat serum cortisol is drawn the next morning (between 8 and 9 AM).[6]

• Two-day, 8 mg test: On day 1, a baseline morning serum cortisol or 24-hour urinary free cortisol (UFC) from the day before is obtained. Then oral dexamethasone 2 mg every 6 hours ((9 AM, 3 PM, 9 PM, 3 AM) is given for 2 days (total dose 16 mg between Day 1 and Day2) with simultaneous collection of a urine sample for UFC. Serum cortisol levels are checked 6 hours after the last dose (9 AM).[5]

3. Intravenous DST

This test helps in the initial diagnosis of Cushing syndrome while overcoming concerns of drug compliance and malabsorption. Additionally, it is also useful to differentiate Cushing disease (CD) from ACTH-dependent ectopic tumor and ACTH-independent adrenal etiology. After a baseline morning serum cortisol (8 to 9 AM) is obtained, an infusion of intravenous dexamethasone at 1 mg/hour for 4 to 7 hours is administered. Repeat serum cortisol levels are measured at the end of the infusion (Day-1) and 23 to 24 hours later (Day-2).[7][8][9][10]

4. Dexamethasone – CRH Test

Based on the rationale that glucocorticoid suppression of the HPA axis can be overcome by CRH stimulation in Cushing disease and not in pseudo-Cushing syndrome (physiologic hypercortisolism), this test helps distinguish the two entities. Dexamethasone 0.5 mg every 6 hours (12 PM, 6 PM, 12 AM, 6 AM) is given orally for 48 hours. Two hours after the last dose of dexamethasone, an intravenous CRH dose of 1 mcg/kg is administered (8 AM) and serum cortisol is drawn 15 minutes later [11].

Interfering Factors

1. Iatrogenic hypercortisolism can cause exogenous Cushing syndrome. Biochemical testing shows elevated serum cortisol levels (due to cross-reactivity of most exogenous steroids with cortisol immunoassays) and depressed ACTH level as it is usually seen in ACTH-independent Cushing syndrome. It is crucial that the individuals receiving exogenous corticosteroids (inhaled/topical/parenteral/intraarticular) are identified, before pursuing a work-up for pathological hypercortisolemia.

2. Pseudo-Cushing syndrome, also called physiological or non-neoplastic hypercortisolism is seen in conditions like alcoholism, obesity, insulin resistance and neuropsychiatric disorders due to HPA axis stimulation.[12] A thorough history and physical examination can play a pivotal role in recognizing many subjects with pseudo-Cushing syndrome. In equivocal cases, midnight serum cortisol, late-night salivary cortisol, dexamethasone-CRH, or desmopressin test can assist in the distinction.[13]

3. In any acute illness (emotional or physical) a stress response can be presented via the HPA axis, resulting in elevated ACTH and cortisol levels, that's why the evaluation for Cushing syndrome should take place after the resolution of acute stress.

4. Corticosteroid-binding globulin (CBG): About 90% of the circulating cortisol is protein-bound and currently available assays measure the total cortisol (free and protein-bound); therefore, conditions resulting in elevated levels of CBG (pregnancy, estrogen-pill, etc.), or reduced levels of CBG (nephrotic syndrome, malnutrition, etc.) may lead to spurious results on DSTs.[14] In these scenarios, late-night salivary cortisol, or UFC are the preferred tests. Patients receiving estrogen therapy should stop treatment at least for six-weeks before DST.

5. Dexamethasone bioavailability: Malabsorption, altered metabolism, or non-compliance with taking the medication can result in variable bioavailability of dexamethasone confounding the results.[15][16] Dexamethasone gets metabolized in the liver via CYP3A4. Thus, CYP3A4 inducers (phenytoin, carbamazepine, etc.), or CYP3A4 inhibitors (itraconazole, fluoxetine, ritonavir) may result in decreased or increased clearance of the dexamethasone, risking false positive or negative results, respectively. This error can be overcome by measuring serum dexamethasone levels at the same time as serum cortisol levels. Most laboratories that conduct this test, provide reference ranges based on dexamethasone dose and interval of blood drawn.

6. Improper urine collection: For UFC, an inadequately collected urine sample leads to diagnostic errors. For this reason, the urine sample should include testing for 24-hour urinary creatinine excretion, in addition to cortisol. In adults less than 50 years old, 24-hour urinary creatinine excretion is approximately 15-20 mg/kg/day in women and 20 to 25 mg/kg/day in men. In people older than 50 years of age, there is a progressive decline in muscle mass, hence, creatinine excretion can be as low as 10 mg/kg/day.

7. Others: Cyclic Cushing syndrome and glucocorticoid receptor polymorphisms are a few rare causes of false test results.[17][18]

Results, Reporting, Critical Findings

1. LDDST

In either of the LDDST (overnight, or two-day), serum cortisol level of 1.8 mcg/dl (50 nmol/L) is the recommended cut-off value that increases the diagnostic sensitivity of the test to approximately 95%.[19][20] However, at this cut-off value, the specificity of the two-day test is better compared to that of the overnight test (97 to 100% versus 86%). A recent meta-analysis showed that for the 1 mg overnight test, abnormal and normal results possessed a positive and negative likelihood ratio (LR) of 11.6 and 0.09, respectively. On the other hand, for the two-day 2 mg test, abnormal and normal results had a positive and negative LR of 7.3 and 0.8, respectively.[21] Serum cortisol level under 1.8 mcg/dl suggests adequate HPA axis suppression by dexamethasone and excludes CS. Levels over 1.8 mcg/dl should be verified with a second test (24-hour urinary free cortisol, or late-night salivary cortisol), before establishing a confirmed diagnosis of CS.[4]

2. HDDST

Reduction in UFC, or serum cortisol greater than 50% in the overnight, or two-day HDDST makes Cushing disease (CD) the likely source of ACTH-dependent Cushing syndrome. At a cut-off value of 50% suppression, HDDST provides a sensitivity and specificity of 60 to 100%. Increasing the cut-off to above 90% cortisol suppression increases the specificity of diagnosing Cushing disease to almost 100%, albeit at a much-reduced sensitivity.[22] Because of this limitation, HDDST is not the recommendation, unless, both the pituitary MRI and bilateral inferior petrosal venous sampling are negative or logistically challenging. Even in this scenario, HDDST is performed in conjunction with a CRH stimulation test, to enhance diagnostic accuracy.[23]

3. Intravenous DST

The diagnosis of Cushing syndrome is made if the day-2 serum cortisol level is above 20% of baseline [or greater than 4.7 mcg/dl (130 nmol/L)] with sensitivity 100% and specificity 96%.[7][8] Additionally, in Cushing disease as opposed to other etiologies of Cushing syndrome, the day-1 (i.e., the end of infusion) serum cortisol level shows greater than 70% suppression from the baseline, followed by rebound hypercortisolism in 24 hours.[8][9][10]

4. Dexamethasone - CRH

Serum cortisol levels over 1.4 mcg/dl (39 nmol/L) at 15 minutes suggest CD with a 90 to 100% sensitivity and 50 to 100% specificity.[11][24][25] Raising the cut-off value to over 3.8 mcg/dl (87 nmol/L) increases the specificity to 100%, at the cost of slightly reduced sensitivity (94%).[26] The cumbersomeness of this test limits its extensive application in the ambulatory setting.[27]

Clinical Significance

Iatrogenic hypercortisolism is the most common cause of Cushing syndrome; and it should be recognized before these individuals are subject to further diagnostic workup; instead, the focus should be on titrating down (or discontinuing, if feasible) the prescribed steroid dosages. It is crucial that the dexamethasone suppression test is performed and interpreted in the light of pretest probability that should be based on a thorough history and physical examination. Additionally, clinicians should be mindful of all the tests' diagnostic accuracy, limitations, and interfering factors. In subjects with high clinical suspicion of Cushing syndrome but equivocal or negative test results, repeat testing should take place in 3 to 6 months, as untreated hypercortisolemia has detrimental consequences.

Enhancing Healthcare Team Outcomes

Primary care providers initially evaluate the patients with suspected Cushing syndrome and they refer to an endocrinologist for further work-up. Clear communication and care coordination between physician, patient, and nurse are of paramount importance as the correct implementation of DST and sample collection can have a dramatic impact of these factors on subsequent results.