At a Glance

Adrenal adenomas are benign tumors of the adrenal glands. Most adrenal adenomas are spotted by chance at the time of computed tomography (CT) or magnetic resonance imaging (MRI) verifying some other symptoms. They are classified as either functional (hormone secreting) or nonfunctional (incidentalomas) and as either benign or malignant.

The majority of adrenocortical adenomas are benign and nonfunctional. Others are benign hormone secreting (cortisol, aldosterone, androgens) independently from the adrenocorticotropic hormone (ACTH) or renin-angiotensin system, causing Cushing’s syndrome (10%), primary aldosteronism (rare), and, to a lesser extent, virilization (usually malignant).

Careful history and physical examination should be performed to exclude signs and symptoms of pheochromocytoma, hyperaldosteronism, and Cushing’s syndrome.

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Diagnosis of Cushing’s syndrome is based on patient symptoms and signs, such as unusual features for age (osteoporosis, hypertension [HTN]), multiple and progressive features like easy bruising, facial plethora, proximal muscle weakness, striae, and, in children, weight gain wtih decreasing growth velocity and the presence of excess cortisol in serum, saliva, or urine.

Cushing’s syndrome can be ACTH dependent or independent:

ACTH-dependent Cushing’s syndrome can be due to pituitary hypersecretion of ACTH (Cushing’s disease 65-70%), ectopic secretion of ACTH by nonpituitary tumors (10-15%), ectopic secretion of CRH by nonhypothalamic tumors (<1%), and factitious Cushing’s syndrome due to exogenous administration of ACTH (<1%).

ACTH-independent Cushing’s syndrome is mainly factitious (administration of ACTH drugs), adrenocortical adenomas and carcinomas (18-20%), bilateral adrenal micronodular hyperplasia (<1%), and bilateral ACTH-independent macronodular hyperplasia (<1%).

Primary aldosteronism (PA) is a generic term for a group of disorders in which there is an excess production of aldosterone independent of the regulation of renin-angiotensin system. PA consists of 5 clinical entities:

Solitary aldosterone-producing adenoma (APA)

Idiopathic hyperaldosteronism (IHA) (bilateral hyperplasia of zona glomerulosa)

Primary adrenal hyperplasia

Adrenal carcinoma

Glucocorticoid remediable aldosteronism

Common manifestations of PA are HTN, hypokalemia, metabolic alkalosis, and hypomagnesemia. Effects occur more in women than in men and occur most commonly in the third to fifth decade of life. Spontaneous hypokalemia (<3.5 mmol/L) occurs in 80% of cases, and most symptoms are manifested by weakness, muscle cramps, paresthesias, headaches, palpitations, polyuria, and polydipsia. Glucose intolerance due to insulinopenia occurs in 25% of patients.

PA is more prevalent in patients with:

Moderate to severe HTN

Resistent HTN (defined as SBP >140, DBP >90 despite treatment with 3 hypertensive medications)

Hypertensive patients with spontaneous and diuretic induced hypokalemia

HTN with adrenal incidentalomas

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

The following initial screening tests are useful in determining the secretory activity of the tumors:

24-hour Urinary Measurements of Free Cortisol (UFC)

Late-night Salivary Cortisol

1 mg-Overnight Dexamethasone Suppression Test (DST)

Longer Low Dose Dexamethasone Suppression Test (2 mg/day for 48 hours)

Random serum cortisol, plasma ACTH, urinary 17-ketosteroids, insulin intolerance, and loperamide tests are not recommended.

In PA suspected cases, meaurements of plasma aldosterone-renin ratio (ARR) is recommended. This is superior to the measurements of potassium, aldosterone, or renin in isolation.

Test Results indicative of the Disorder

Because the hypercortisolism of Cushing’s syndrome can be variable, it is recommended that at least 2 measurements of urine or salivary cortisol be obtained. This strategy increases confidence in the test results, if consistently normal or abnormal results are obtained.

24-hour UFC

Twenty-four-hour UFC is increased in 95% of cases of Cushing’s syndrome. Values less than 100 μg per 24 hours exclude the diagnosis, and values greater than 300 μg per 24 hours confirm the diagnosis. If values are intermediate, a DST is indicated.

Salivary Cortisol

Serum free cortisol freely diffuses into saliva. Therefore, measurements of salivary cortisol more accurately reflect the serum free cortisol. The salivary cortisol concentration is independent of salivary flow rate.

Levels vary diurnally, with concentrations about 5.6 ng/mL at 8:00-9:00 AM and about 1 ng/mL at 11:00 PM. A significant increase from the normal range of Cushing’s syndrome is likely. This can be used in assessing cortisol secretion serially in ambulatory patients and cyclical Cushing’s syndrome.


Dexamethasone (DXM) is a potent synthetic glucocorticoid not detected by serum, urine, and salivary cortisol assays. DXM should not fully suppress ACTH and, therefore, should not decrease adrenal secretion of cortisol.

Low-dose DST

This test is a good standard screening test to differentiate patients with Cushing’s syndrome of any cause from patients who do not have Cushing’s syndrome. If the hypothalamic-pituitary axis is normal, any supraphysiologic dose of dexamethasone is sufficient to suppress pituitary ACTH secretion. This should lead to reductions in cortisol secretion and its concentration in serum and saliva, as well as in the 24-hour urine excretion.

Overnight 1 mg screening test and standard 2-day, 2 mg test

High-dose suppression tests are based on the fact that ACTH secretion in Cushing’s disease is only relatively resistant to glucocorticoid negative feedback inhibition and does not suppress normally with either the overnight 1 mg or the 2-day low-dose test. By increasing the dose of dexamethasone 4- to 8-fold, ACTH secretion can be suppressed in most patients with Cushing’s disease. Therefore, this test is used to distinguish patients with Cushings disease (Cushing’s syndrome caused by pituitary hypersecretion of ACTH) from most patients with ectopic ACTH syndrome (Cushing’s syndrome caused by nonpituitary ACTH-secreting tumors).

  • Overnight screening test is a quick screening test for nonsuppressible cortisol production and subclinical or clinical Cushing’s syndrome and should not be used as the sole criterion for excluding the diagnosis of Cushing’s syndrome. Dexamethasone (1 mg) is taken orally between 11 PM and 12 AM, and a single blood sample is drawn at 8 AM for assay of serum cortisol. The 2008 Endocrine Society Guidelines suggest a diagnostic serum cortisol criterion of 1.8 mcg/dL. The salivary cortisol concentration at 8 AM after 1 mg DXM given at 12 AM was 0.8±0.4 ng/mL (range, 0.6-1.1 ng/mL) in 101 normal subjects, which gave a sensitivity and specificity of 100%.

  • Standard 2-day, 2 mg test is used to assess suppressibility in patients with an equivocal overnight test or in patients who have not had an overnight test. Dexamethasone, 0.5 mg, is taken orally every 6 hours, usually at 8 AM, 2 PM, 8 PM, and 2 AM, for a total of 8 doses. Blood is drawn 2 or 6 hours after the last dose for measurement of cortisol. The normal response to the 2-day test consists of the following: urinary cortisol excretion should fall to less than 10 mcg per 24 hours on the second day of DXM administration; serum cortisol concentration is less than 5 mcg/dL; a plasma ACTH concentration is less than 5 pg/mL; and a serum DXM concentration is between 2.0 and 6.5 ng/mL. In a recent meta-analysis, the 1 mg test and the 2-day, 2 mg test were both accurate, but the 2-day, 2 mg test had slightly less diagnostic accuracy.


Aldosterone is primarily used in the diagnosis of PA. Normal ranges include:

8-10 AM: Supine: 2-19 ng/dL

8-10 AM: Upright Sitting: 3-34 ng/dL

4-6 PM: Upright Sitting: 2-23 ng/dL

Plasma Renin Activity (PRA)

Renin activity is measured indirectly by the ability of the patient’s plasma to generate angiotensin. Normal values depend on the laboratory and the patient’s prevailing Na and K, status of hydration, and posture. Only stimulated values are of practical value in evaluating hypertensive patients.

The reference ranges for PRA for adults on a normal sodium diet are:

Supine: 0.2-1.6 ng Angiotensin I/mL/hr3

Standing: 0.7-3.3 ng Angiotensin I/mL/hr3

Plasma Aldosterone Renin Ratio (ARR)

Currently, plasma ARR is the most reliable and available means of screening for PA. This test is most sensitive when used in patients with whom samples are collected in the morning, after patients have been out of bed for at least 2 hours and usually after patients have been seated for 5-15 minutes. Patients should have unrestricted dietary salt intake prior to testing. An ARR greater than 25 is suggestive of hyperaldosteronism.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

Several collection and assay methods are available for the measurement of cortisol, and results for a single sample measured in various assays could be quite different. Assays differ widely in their accuracy; results near the cutoff value on a single measurement can often be explained by assay variability. Normal ranges vary substantially, depending on the method used, so it is essential to interpret test results in the context of the appropriate normal range. Antibody-based immunoassays, such as unextracted radioimmunoassay (RIA) and enzyme immunoassay (EIA), can be affected by cross-reactivity with cortisol metabolites and synthetic glucocorticoids. In contrast, structurally based assays, such as high pressure liquid chromatography (HPLC) and tandem mass spectrometry, do not pose this problem and are used with increasing frequency.

Drugs, such as carbamazepine and fenofibrate, may interfere with some of these chromatographic methods, thereby causing falsely elevated values. Upper limits of normal are much lower with HPLC or liquid chromatography-tandem mass spectrometry (LC-MS/MS) than in antibody-based assays. Whenever possible, estrogen-containing drugs should be withdrawn for 6 weeks before testing or retesting. Conversely, decreases in cortisol binding globulin (CBG) or albumin, which occur in the critically ill or nephrotic patients, are associated with decreased serum cortisol values.

24-hour UFC

Urinary cortisol may be detected by antibody-based (immunoassays) or structurally based (HPLC-MS) tests. Immunoassays may be less specific, because antibodies may crossreact with similar steroids. Increased values may occur in depression, chronic alcoholism, eating disorders, and polycystic ovary syndrome but do not exceed 300 μg per 24 hours. Various drugs (e.g., carbamazepine, phenytoin, phenobarbital, and primidone) will falsely elevate free cortisol levels. Acute and chronic illnesses can increase free cortisol levels. Renal disease due to decreased excretion may falsely lower the levels of free cortisol.

Low-dose DST

This test has a significant false-positive rate when sensitivity is maximized. Using a serum cortisol criterion of less than 3.6 mcg/dL, the test has a 12-15% false-positive rate. If, however, the criterion for suppression of serum cortisol is increased to less than 7.2 mcg/dL, the false-positive rate falls to 7%. This suggests that the use of multiple criteria may be useful in interpreting the test.

False-positive results may occur in acute and chronic illness, alcoholism, and depression, as well as because of certain drugs (e.g., phenytoin, phenobarbital, primidone, carbamazepine, rifampicin, and spironolactone); estrogens may cause a false-positive overnight DST. Atypical or false-positive responses may also occur because of alcohol, estrogens, birth-control pills, pregnancy, obesity, acute illness, stress, and severe depression. This test is not a good choice for patients in whom CBG levels may be abnormal. Some patients (e.g., patients with psychiatric illness) with large ACTH-producing pituitary adenomas have marked resistance to high-dose DXM suppression. In long-standing cases, nodular hyperplasia of adrenal may develop, causing autonomous cortisol production and resistance to DST. Urine and plasma cortisol are not decreased after high or low doses of DXM in adrenal adenoma or carcinoma or ectopic ACTH syndrome.


There are many physiological factors that affect plasma aldosterone. Posture, salt intake, use of antihypertensive drugs, steroids, and oral contraceptives, age, menstrual cycle, and pregnancy can all have a strong influence on aldosterone results.


PRA levels cannot be interpreted if the patient is being treated with spironolactone (Aldactone). Spironolactone (Aldactone) should be discontinued for 4-6 weeks before testing. Angiotensin-converting enzyme (ACE) inhibitors have the potential to “falsely elevate” PRA. Therefore, in a patient treated with an ACE inhibitor, the findings of a detectable PRA level or a low SA/PRA ratio do not exclude the diagnosis of primary aldosteronism. In addition, a strong predictor for primary aldosteronism is a PRA level undetectably low in a patient taking an ACE inhibitor. This test is not useful for determination of plasma renin concentration. This test should not be requested in patients who have recently received radioisotopes, therapeutically or diagnostically, because of potential assay interference. A recommended time period before collection cannot be made, because such a time period depends on the isotope administered, the dose given, and the clearance rate in the individual patient.


Many medications falsely increase ARR, including beta adrenergic blockers, central alpha-2 drugs, and nonsteroidal anti-inflammatory drugs (NSAIDs). False negative ARRs occur in potassium wasting and sparing drugs, ACE-inhibitors, calcium blockers, and renin inhibitors.

What Lab Results Are Absolutely Confirmatory?

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

For adrenal incidentalomas, UFC is less sensitive than 1 mg DST or late-night salivary cortisol for the identification of Cushing’s syndrome. No consensus exists on on best algorithm or best diagnosis criteria for 1 mg DST. Suppressed ACTH or DHEA sulfate supports the diagnosis of Cushing’s syndrome in patients with adrenal masses (Table 1).

Table 1.
Test Procedure Interpretation
Oral Sodium Glucose Test 24-hour urinary aldosterone is measured on days 3-4, following increased sodium intake (~6 g/day) for 3 days. Urinary aldosterone levels of <10 mcg/24 hr in the absence of renal disease, PA may coexist.
Saline Infusion Test Blood sample for renin, aldosterone, cortisol, and potassium are measured at 0 and 4 hours during the infusion of 2 liters of IV 0.9% saline for 4 hours. Postinfusion aldosterone of >10 ng/dL, PA is likely.
<5 ng/dL, PA is unlikely.
Levels between 5 and 10 ng/dL are inconclusive.
Fludrocortisone Suppresssion Test Plasma aldosterone, PRA, and cortisol are measured on day 4, following 0.1 mg of oral fludrocortisone every 6 hours for 4 days. Plasma aldosterone levels >6 ng/dL on day 4 confirms PA.
Captopril Challenge Test PRA, aldosterone, and cortisol are measured at 0, 1, and 2 hours after 25-50 mg of captopril orally. Plama aldosterone is normally suppressed by 30% by captopril. If it remains elevated and PRA remains suppressed, PA is likely.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

The ARR test for the diagnosis of PA is most sensitive in the blood samples collected the morning after patients have been out of bed for at least 2 hours, usually after patients have been seated for 5–15 minutes. Patients should have unrestricted dietary salt intake prior to testing. Spironolactone, eplerenone, amiloride, and triamterene, potassium-wasting diuretics, products derived from licorice root (e.g., confectionary licorice), and chewing tobacco will affect ARR testing.