Cushing’s Disease - Testing

What is Cushing's disease?

Cushing's disease is caused by the adrenal glands' increased production of cortisol (stress hormone). Increased cortisol production may be related to:

• Excessive stimulation of the adrenal glands by the pituitary gland, usually because of a pituitary tumor (pituitary origin).
• The unregulated production of cortisol by the adrenal glands (adrenal origin).
• Occasionally, administration of steroid-containing medications may result in the development of Cushing's disease (iatrogenic or caused by medication).

The most common type of Cushing's disease is of pituitary origin. The clinical signs of Cushing's disease are similar, regardless of the underlying cause of the disease. However, it is essential to identify the type of Cushing's disease as the treatment and prognosis differ slightly depending upon the form of the disease.

What initial screening tests help diagnose Cushing's disease?

There are characteristic laboratory changes related to Cushing's disease, which may be noted in the evaluation of your pet's blood and urine samples. Not only will the documentation of such changes help diagnose Cushing's disease, but these laboratory tests may also uncover other conditions related or unrelated to excessive cortisol production. Common initial screening tests using blood and urine samples include:

Complete blood count (CBC) (see handout "Complete Blood Count" for more information). The red and white blood cell counts and platelet components in a blood sample are evaluated. Often, an increase in the number of white blood cells (neutrophils and monocytes) may be noted with Cushing's disease. This increase is usually related directly to the effects of excessive cortisol, but occasionally, an increase in these cells may indicate underlying inflammation or infection.

Serum biochemistry profile (see handout "Serum Biochemistry" for more information). Serum (the liquid portion of blood) contains enzymes, proteins, lipids (fats), glucose (sugar), and metabolites. These serum components are derived from different organs, such as the liver, kidneys, and pancreas, and can indicate how these organs are functioning. Specific changes in the serum biochemistry profile may support the diagnosis of Cushing's disease and give an indication of overall organ function. Typical findings with Cushing's disease may include increased blood glucose and liver-related enzymes. A specific liver enzyme, steroid alkaline phosphatase (S-ALP), increases directly in response to increased blood cortisol levels in dogs but not in cats. Increases in blood lipid and cholesterol are also often noted.

Urinalysis (see handout "Urinalysis" for more information). Because dogs with Cushing's disease typically drink large amounts of water, the urine is dilute. Cats with Cushing's disease may not have dilute urine. A special instrument called a refractometer assesses urine concentration. Infection of the bladder (cystitis) is a common finding in Cushing's disease. The presence of infection can be assessed by looking at the cells voided into the urine and by sending a urine sample for bacterial culture (checking for the growth of bacteria). Some dogs and many cats with Cushing's disease also have concurrent diabetes. The presence of diabetes may be detected by glucose in the urine and by an increased concentration of glucose in the serum biochemistry profile.

"Some dogs and many cats with Cushing's disease also have concurrent diabetes."

Urine cortisol/creatinine ratio. Cushing's disease increases the amount of cortisol being lost in the urine. This cortisol can be measured and compared to the amount of creatinine, a normally occurring urinary component produced at a constant rate. The urine cortisol/creatinine ratio is usually elevated with Cushing's disease. However, stress and other illnesses may also increase this ratio. While a high urine cortisol/creatinine ratio suggests Cushing's disease, it cannot confirm the diagnosis.

If these initial screening tests show a number of changes characteristic of Cushing's disease, more extensive diagnostic tests will be required to confirm the diagnosis and establish the type of Cushing's disease that your pet has. Knowing the type of disease is essential to formulate an effective treatment plan and to predict possible outcomes. These tests may include an adrenocorticotropic hormone (ACTH) stimulation test, low-dose dexamethasone suppression tests (LDDST), and/or endogenous ACTH concentrations.

How does the ACTH stimulation test work?

ACTH is a hormone naturally produced by the pituitary gland (a gland located in the brain). The purpose of ACTH is to tell the adrenal glands when and how much cortisol to produce. As the blood level of cortisol increases, it causes the pituitary gland to decrease its production of ACTH, which results in a lowering of the cortisol level. The ACTH stimulation test mimics this natural stimulatory pathway and demonstrates the capacity of the pet's adrenal glands to produce cortisol in response to an injection of synthetic ACTH. 

This test requires two blood samples in dogs and three in cats. In dogs, the first blood sample is taken, then an injection of synthetic ACTH is given, and the second blood sample is taken two hours following the injection. In cats, the timing of blood samples may vary depending on the type of synthetic ACTH used, but often taken pre-injection, then again at 30 minutes and one hour.  When the cortisol levels in the samples are compared, they may show a normal response, an exaggerated response, or very little response.  This test is less predictable in cats than dogs, so normal results do not rule out disease.

With pituitary origin Cushing's disease, we usually see a markedly increased cortisol level following the synthetic ACTH injection. This exaggerated response occurs because the adrenal glands have been over-stimulated with naturally occurring ACTH secreted by the diseased pituitary (pituitary origin Cushing's disease). These adrenal glands are, therefore, highly responsive to stimulation by the synthetic ACTH. With adrenal origin, Cushing's disease, an exaggerated cortisol level may also be seen after the synthetic ACTH injection.

Since the ACTH stimulation test may be positive in both types of Cushing's disease, the test confirms that your pet has Cushing's disease but does not confirm the type or cause. Some pets with Cushing's disease of either pituitary or adrenal origin do not respond to the ACTH stimulation test, and additional diagnostic testing is required to confirm a diagnosis.

Pets showing signs of Cushing's disease because of corticosteroid-containing medications (called iatrogenic Cushing's syndrome) will show either a very mild response or no response to synthetic ACTH injection. This type of muted response, in conjunction with a history of medication use, confirms iatrogenic Cushing's syndrome.

"As your pet responds to treatment, the increases in cortisol will become less exaggerated in response to the injection of synthetic ACTH."

Another use of the ACTH stimulation test is to monitor a pet's response to therapy for Cushing's disease. Periodic ACTH stimulation tests during the initiation and maintenance phases of treatment will be required. As your pet responds to treatment, the increases in cortisol will become less exaggerated in response to the injection of synthetic ACTH.

How does the low-dose dexamethasone suppression (LDDST) test work?

In a healthy pet, ACTH is secreted by the pituitary gland and stimulates the adrenal glands to produce or secrete cortisol. As the blood cortisol level increases, it causes the pituitary gland to lower its production of ACTH, which results in lowering the blood cortisol level; this is called a negative feedback loop. This feedback loop can cause rapid changes in blood cortisol levels in response to the body's needs. Mimicking what happens naturally, when healthy pets are given an injection of synthetic cortisol (dexamethasone), the production of ACTH and cortisol is suppressed.

The LDDST requires three blood samples. The first sample is taken before dexamethasone injection, and the second and third samples are taken four and eight hours after injection.

"Cushing's disease diminishes or loses this negative feedback loop because cortisol is always high."

Cushing's disease diminishes or loses this negative feedback loop because cortisol is always high. If the patient has pituitary origin Cushing's disease, the negative feedback mechanism is only diminished, so a slight decrease in the four or eight-hour cortisol levels from the pre-injection sample is expected. With adrenal origin Cushing's disease, the negative feedback loop is lost, so there is no reduction in blood cortisol concentrations at either the four or eight-hour cortisol levels.

In most instances, the low-dose dexamethasone suppression test will allow the diagnosis of Cushing's disease and indicate the type of Cushing's disease (pituitary versus adrenal origin) present.

How does the high-dose dexamethasone suppression test work?

This test works on the same principle as the low-dose test and is used when no reductions in cortisol levels are seen on the low-dose test. The purpose is to determine which type of Cushing's disease is present. In rare instances with pituitary origin Cushing's disease, the negative feedback loop is more resistant to the effects of dexamethasone. A higher dosage of this synthetic cortisol may be required to overcome this resistance and demonstrate the negative feedback mechanism. If there is a reduction in cortisol levels at four or eight hours after the higher dose of dexamethasone, the diagnosis is pituitary origin Cushing's disease. If the levels do not budge from the high values of the pre-injection sample, the diagnosis is adrenal origin Cushing's disease.

"The purpose is to determine which type of Cushing's disease is present."

Endogenous (originating from within the body) ACTH concentration can be measured in blood samples to distinguish pituitary from adrenal origin Cushing's disease. With pituitary origin Cushing's disease, an increased concentration of endogenous ACTH will be found in the blood. With adrenal origin Cushing's disease or iatrogenic Cushing's disease (caused by cortisol-containing medications), the amount of endogenous ACTH in the blood will decrease. Unfortunately, the concentrations of ACTH found in blood with these different types of Cushing's disease may overlap and not allow clear differentiation of the cause of the disease.

In most instances, a combination of these tests will allow for a diagnosis of Cushing's disease or will rule out the disease in your pet. Additional testing, such as abdominal ultrasound, may be required to determine whether an adrenal tumor is present.

In rare instances, referral to a veterinary facility with MRI (magnetic resonance imaging) or CT (computed tomography) imaging may be needed to rule out or demonstrate the presence of pituitary or adrenal tumors.

Your veterinarian will determine which testing methods are best for your pet.