Canine Hyperadrenocorticism (HAC – Cushing’s Disease)

Hyperadrenocorticism occurs in middle–aged and geriatric dogs. In around 80% of cases it is pituitary-dependent (PDH). In the remainder, the disease results from primary adrenal-dependent neoplasia or hyperplasia (ADH).

Individual tests

Cortisol – Basal cortisol levels are of no value in diagnosing HAC. There is significant overlap in cortisol concentration among healthy, stressed, sick and affected dogs.

Cortisol/Creatinine Ratio (CCR) – This is a very sensitive test to exclude HAC (very few false negatives) but must not be used to diagnose HAC as it is very poorly specific and non-adrenal illness commonly gives a positive result. A morning urine sample is collected in the animal’s home environment. This reflects the cortisol released over several hours. Normal dogs will have a CCR less than 30 x 10-6.

Dynamic test protocols

Which test should be used to diagnose HAC reliably in the dog?
Unfortunately, there is no perfect diagnostic test or test protocol for hyperadrenocorticism. The line between physiologic adrenal stress responses to non-adrenal illness and pathologic hyperfunction of the pituitary-adrenal axis can be fine indeed. As a consequence, false positive and false negative test results occur. The different available diagnostic protocols available have different properties, advantages and disadvantages but none is perfect. In establishing confidence that hyperadrenocorticism is confirmed or ruled out more than one diagnostic test protocol may be required.

ACTH stimulation is a good screening test in the first instance and is the test of choice for diagnosing iatrogenic Cushing’s and monitoring anti-adrenal therapy. It has a lower false positive rate than the low-dose dexamethasone suppression test but a significant false negative rate. It will reliably diagnose about 85% of PDH cases but only 50% of ADH cases. It is quick and simple to perform and is less affected by stress and non-adrenal illness. The initial values are useful as a reference to monitor effectiveness of treatment.

Low-dose Dexamethasone screening test is more sensitive than the ACTH stimulation test in confirming HAC. Diagnostic sensitivity is almost perfect (>95%) meaning that false negative results very seldom occur giving us tremendous confidence in a negative test result. Unfortunately, false positive results are common especially when there is concurrent non-adrenal illness or other sources of stress. Positive results of the low-dose dexamethasone test should be regarded with suspicion in dogs known to have significant non-adrenal illness. Ideally, the test should be postponed until any identified non-adrenal illnesses have been resolved or stabilised. See “Diagnosing Canine Hyperadrenocorticism” flowchart.

17-hydroxyprogesterone measurement in the diagnosis of adrenocortical disease
Certain functional adrenocortical tumours do not have cortisol as their principle secretory product but instead the tumour has developed in such a way that it is adrenal steroid precursors that are released into the circulation. Many dogs with this type of functional adrenal tumour will have clinical signs suggestive of hyperadrenocorticism. A common cortisol response to ACTH in these cases is a “flat-line, mid-range” pattern. Measuring 17OHP before and after ACTH stimulation (same sample as you would take for cortisol) can be very helpful in identifying these tumours.

Additionally, in cases suspected of hyperadrenocorticism which do demonstrate an exaggerated cortisol response to ACTH, the additional measurement of 17OHP can help improve the confidence in ruling hyperadrenocorticism in or out. Less than 10% of dogs with classic hyperadrenocorticism would be expected to have post-ACTH 17OHP <4.5 nmol/L (Chapman et al, 2003 Veterinary Record, 153(25):771-5) and fewer than 10% of dogs which do not have classic hyperadrenocorticism would be expected to have post-ACTH 17OHP >16.7 nmol/L.

Consider measuring 17OHP in addition to cortisol when:

  • Flat-line, mid-range cortisol response to ACTH
    • Investigate functional adrenocortical tumour
  • Mildly exaggerated cortisol response to ACTH and few appropriate clinical signs of HAC
    • Look for post-ACTH <4.5nmol/L to improve rule-out
  • Normal cortisol response to ACTH and clinical signs consistent with HAC
    • Investigate “atypical” hyperadrenocorticism (Ristic et al 2002 JVIM 16(4):433-9) and “Alopecia X” (see p32-33)

17OHP may also be useful in ruling out HAC where an exaggerated Cortisol response is seen which is mainly due to stress, often associated with other illness. The Cortisol levels may be borderline or high but the 17OHP response to ACTH is normal.

ACTH Stimulation Test

  1. Collect basal blood sample.
  2. Immediately inject 0.25 mg synthetic ACTH (Synacthen*) i/v or i/m
  3. Collect a second blood sample one hour later.
  4. Label samples clearly and request Cortisol analysis.

* If Synacthen is unavailable Dechra Veterinary Products Ltd have an alternative drug to Synacthen available on special prescription, contact for details.


Normal dogs will show an increase in cortisol levels of up to 450 nmol/L post stimulation.
An exaggerated response is expected in animals with PDH and cortisol concentrations rise above 600 nmol/L and often above 1000 nmol/L.
Dogs with an adrenal tumour often have basal cortisol above 250 nmol/L with little or no change after stimulation. However the ACTH stimulation test is not as sensitive at detecting adrenal tumours and negative results should be confirmed with a low dose dexamethasone test when strong clinical suspicion remains.

Low Dose Dexamethasone Screening Test

  1. Collect a basal blood sample
  2. Inject 0.01 mg/kg i/v of Dexamethasone.
  3. Collect two further blood samples 3 hours and 8 hours later
  4. Label sample times clearly on the tubes and request cortisol.


The low-dose dexamethasone screening test is interpreted in two stages. Firstly, the presence or absence of hyperadrenocorticism is established by examining the 8-hour result. A value above 40 nmol/L is positive. The next step applies in the positive cases and checks for evidence of suppression sufficient to identify the source of hyperadrenocorticism. Greater than 50% suppression from the baseline value at either 3 or 8 hours is consistent with pituitary dependent disease. When there is no, or minimal, evidence of suppression it is necessary to follow-up with a differentiation test to identify the source (high-dose dexamethasone suppression test, endogenous (plasma) ACTH, adrenal ultrasonography). Both dogs with an adrenal tumour and some dexamethasone “resistant” pituitary cases will not depress by 50%. In about 60% of hyperadrenocorticism cases, we will obtain differentiation from the low-dose test alone with no need to move on to the differentiation tests.

High Dose Dexamethasone Suppression Test

This test is used to help distinguish between PDH and an adrenal tumour. It must only be used after Cushing’s disease has been confirmed by other tests (low dose dex. or ACTH stim.)
The disadvantages to the HDDST are the 8 hour duration and that a small proportion of pituitary dependent cases will fail to suppress even at substantial doses of dexamethasone. An alternative differentiation test is the endogenous (plasma) ACTH which has the advantage of being a single point test.

  1. Collect a basal blood sample.
  2. Inject 0.1 – 1.0 mg/kg i/v of Dexamethasone.
  3. Collect two further blood samples 3 hours and 8 hours later.
  4. Label sample times clearly on the tubes and request cortisol.


Any suppression greater than 50% of the baseline concentration indicates a pituitary source. A failure to suppress by 50% is consistent with either an adrenal tumour or a “dexamethasone-resistant” pituitary lesion. When no suppression is observed, repeating the test with a higher dose of dexamethasone or diagnostic imaging of the adrenals and/or pituitary could be considered.

Endogenous (plasma) ACTH

Endogenous ACTH may be used as a reliable tool for differentiating PDH from ADH when hyperadrenocorticism as already been confirmed by ACTH stimulation test or low-dose dexamethasone screening test. However, it has no value as a diagnostic test for hyperadrenocorticism in dogs and cats. Special sample handling procedures apply.

ACTH Sample Preparation

Sample MUST be EDTA plasma or EDTA plasma with Aprotinin

  1. Request a transport pack and follow the instructions that come with the pack for sample preparation.
  2. Take the blood sample into the cooled Aprotinin EDTA tube (supplied in pack) which has been cooled in the fridge.
  3. Mix well but gently at least 10 times and centrifuge as quickly as possible.
  4. Transfer the plasma into a cooled plastic (not glass!) PLAIN tube kept in the fridge.
  5. Immediately freeze (<-10°C) the plasma sample and keep frozen until dispatch in the transport pack. Freeze the sample first, separately, before placing in the frozen pack for transport/li>
  6. Mark the tube with “EDTA plasma” and animal/owner name.

Cortisol/Creatinine Ratio (CCR) with Dexamethasone Suppression Test

When Cushing’s disease is strongly suspected, this test can be used when the dog is easily stressed or difficult to sample. The owner does all the sampling at home.

  1. Day 1, collect a first morning urine sample.
  2. Mix the urine and add some to Sample Tube 1, place in the fridge until dispatch.
  3. Day 2, collect a first morning urine sample.
  4. Mix the urine and add some to Sample Tube 2, place in the fridge until dispatch.
  5. Immediately after urine collection, note the time and give the dog the required number of Dexamethasone tablets (dose = 0.1mg/kg).
  6. 8 hours later give the dog a 2nd set of Dexamethasone tablets.
  7. 16 hours later give the dog a 3rd set of Dexamethasone tablets.
  8. Day 3, collect a first morning urine sample.
  9. Mix the urine and add some to Sample Tube 3, place in the fridge until dispatch.
  10. Send all 3 urine samples to the laboratory for CCR.


HAC is suspected if the CCR is greater than 30 x 10-6 in two consecutive morning urine samples.
If the CCR in the 3rd urine sample is depressed by 50% of the mean CCR of the first two samples, PDH is the likely diagnosis.
If the suppression is less than 50%, an endogenous ACTH is suggested to confirm an adrenal tumour
Normal dogs will have a CCR less than 30 x 10-6.
PDH or ADH dogs usually have a CCR greater than 50 x 10-6.

Monitoring Therapy

The standard ACTH stimulation test is the test of choice in monitoring anti-adrenal therapy (Lysodren, Vetoryl®). The aim of therapy is to achieve a modestly sub-normal ACTH response in order to abolish clinical signs.
Dogs receiving Vetoryl® must have the ACTH response test done 4 – 6 hours after the Vetoryl® tablet. The post ACTH cortisol level should be between 50 and 200 nmol/L to reflect good control. Experience in the field has shown that a post ACTH cortisol of up to 250 nmol/L may not require an increase in dosage for continued clinical effect. It is important that there is some stimulation from the pre to the post sample. Cortisol levels of <20 (pre) and <20 (post) are not acceptable. Clinical signs must be monitored closely. ACTH stimulation tests should be carried out at 10 days, 4 weeks, 12 weeks and then every 3 months post treatment with Vetoryl®. The UCCR can also be used to monitor therapy if necessary in dogs which are difficult to handle or which do not do well in the clinic setting. Whether the time of sampling is important depends on the therapeutic product being used.

Monitoring Therapy Using “Pre-Pill” Single Cortisol Samples

The standard ACTH stimulation test is the documented procedure in the data sheet to monitor Vetoryl treatment, however recent research carried out by Professor Ian Ramsey at Glasgow Vet School has shown that “pre-pill” single cortisol samples may be an effective alternative to ACTH stimulation tests when monitoring Vetoryl when used in conjunction with the animals condition (well or unwell) and any clinical signs of HAC. The following page gives some guidelines, based on the initial research, for the interpretation of basal cortisol measured before the Vetoryl capsule. Advice regarding pre-pill single cortisol testing may be obtained from