Robert J. Kemppainen,     Auburn, Alabama

Ellen N. Behrend,     Auburn, Alabama

Adrenocorticotropic Hormone Stimulation Test

I am having difficulty locating a source for adrenocorticotropic hormone (ACTH) gel. What should I do?

We recommend the use of Cortrosyn (synthetic ACTH or cosyntropin). Cortrosyn is sold in packs of 10 or as a single vial of 0.25 mg (250 µg). The dose for dogs is 5 µg/kg IV (maximal dose, 250 µg; Kerl et al, 1999); for cats the dose is 125 µg IV. In dogs and cats a pre-ACTH sample is collected, ACTH is injected, and one post-ACTH sample is collected 1 hour later. Recent work indicates that the 5 µg/kg dose is equally effective in dogs when given IM, using the same sampling times (Behrend et al, 2006). Once a Cortrosyn vial is reconstituted, it can be stored in the refrigerator for up to 4 months and reused. We tested four ACTH formulations compounded by pharmacies and sold to veterinarians (Kemppainen, Behrend, and Busch, 2005). This study, performed using healthy laboratory dogs, showed that each of these products stimulated serum cortisol to an equivalent extent, as did Cortrosyn when samples were collected at 1 hour after ACTH injection; however, values at 2 hours after ACTH varied considerably. Therefore we recommend that users of compounded ACTH products collect two post-ACTH samples (at 1 and 2 hours after ACTH). To our knowledge no studies have yet assessed responses to various forms of compounded ACTH in dogs with adrenal disease nor have possible variations in responses related to lot-to-lot differences been conducted.

I performed an ACTH stimulation test to evaluate a dog for Addison’s disease, and the post-ACTH cortisol value was greater than normal. Could this dog actually have hyperadrenocorticism?

It is unlikely. It is not uncommon to find a mildly-to-moderately elevated post-ACTH cortisol concentration in dogs tested for Addison’s disease (in dogs not affected by the disease). Presumably a normal physiologic response to recent (or chronic) stress is the cause of the elevation; thus we consider this response to be indicative of a normal pituitary-adrenal axis. In true cases of Addison’s disease, cortisol concentrations both pre- and post-ACTH administration are usually very low (both less than 10 nmol/L).

Can recent steroid therapy affect the ACTH stimulation test?

Yes, glucocorticoids can affect the results in two ways. First, some glucocorticoids cross-react in the cortisol assay and can be detected as immunoreactive cortisol. In our assay, cross-reacting steroids include hydrocortisone, prednisone, and prednisolone. In general, we recommend waiting at least 12 hours after (oral) administration to allow these drugs to clear. Second, glucocorticoid therapy may suppress the pituitary-adrenal axis, causing reduced cortisol concentrations pre- and post-ACTH administration. The severity and duration of this suppression are related to dose, potency, frequency of use, and chemical form (e.g., repositol versus oral) of the glucocorticoid therapy.

I treated a dog suspected of having Addison’s disease with steroids yesterday, and now I want to perform an ACTH stimulation test. Will the results be meaningful?

Yes, they probably will be useful. A single injection (or infusion) of glucocorticoids may lower the cortisol values in the ACTH stimulation test. However, this type of short-term, nonrepositol treatment only moderately lowers the cortisol values (about 25%) relative to the normal range. Most dogs with Addison’s disease have very low plasma cortisol concentrations and fail to show any increase in cortisol in response to ACTH.

How do I monitor the efficacy of mitotane or trilostane therapy?

Periodic ACTH stimulation testing is recommended in all dogs with hyperadrenocorticism treated with mitotane or trilostane. “Ideal” cortisol concentrations in a dog receiving treatment are between 30 and 150 nmol/L in both pre- and post-ACTH samples. If values are below ideal for dogs receiving mitotane, the drug should be discontinued and the dog rechecked with ACTH stimulation periodically (i.e., about every 2 to 3 weeks at first). Once values rise into the ideal range, maintenance therapy can be instituted. If values are mildly-to-moderately greater than 150 nmol/L (e.g., in the 150- to 300-nmol/L range), one should consider increasing the dose (approximately 25%) if maintenance therapy is being given or continuing daily loading therapy for a few additional days. If values are higher, it is likely that 5 to 7 days of daily mitotane will be necessary to reduce the adrenal cortical mass to the target level. Clinical condition and recurrence of signs must be considered in the choice of therapy.

For patients receiving trilostane, ACTH stimulation testing is recommended 10 to 14 days after starting on the drug and 10 to 14 days after any dosage adjustment. The test should be performed 4 to 6 hours after a dose of the drug is given. Once a satisfactory dose has been established, ACTH stimulation testing should be performed 30 and 90 days later and then every 3 months. Dose adjustments should be based on clinical response and ACTH stimulation test results.

How long should I wait between performing a dexamethasone suppression test and an ACTH stimulation test?

We suggest waiting at least 48 hours after a low dose of dexamethasone (0.01 to 0.015 mg/kg) and 5 days after a high dose of dexamethasone (0.1 to 1.0 mg/kg). No delay is necessary when performing the combined dexamethasone suppression–ACTH stimulation test (Kemppainen and Zerbe, 1989). Dexamethasone suppression tests can be performed the day after ACTH stimulation testing.

Dexamethasone Suppression Testing

What form of dexamethasone should I use for a dexamethasone suppression test?

Dexamethasone in polyethylene glycol or dexamethasone sodium phosphate can be used, but the dose is based on the amount of active dexamethasone in solution. For example, a 4-mg/ml solution of dexamethasone sodium phosphate provides about 3 mg/ml of active dexamethasone. The dexamethasone can be given IV or IM, although we prefer the IV route.

I think I injected the dexamethasone outside the vein. What should I do?

It is best to wait 48 hours (low dose) or 72 hours (high dose) and repeat the test.

The calculated (low) dose of dexamethasone is only 0.03 ml for this poodle. How do I accurately administer such a small volume of dexamethasone?

It is important to administer the correct dose. Dilute the dexamethasone (e.g., make a 1 : 20 dilution by mixing 0.2 ml of dexamethasone with 3.8 ml of sterile saline or sterile water) so that a reasonable volume is given. (For Azium, a 1 : 20 dilution will make the solution 0.1 mg/ml.)

Can I use the results of the low-dose dexamethasone suppression test to both diagnose and differentiate hyperadrenocorticism in dogs?

Sometimes the results can strongly indicate a diagnosis of pituitary-dependent hyperadrenocorticism (but never adrenal-dependent hyperadrenocorticism). If the concentration in the 8-hour sample is greater than 30 nmol/L, the results are consistent with hyperadrenocorticism. If in addition the 4-hour postdexamethasone cortisol concentration is less than 30 nmol/L and/or the concentration in the 4- and/or 8-hour sample is less than 50% of the predexamethasone cortisol concentration, pituitary-dependent hyperadrenocorticism is likely.

General Questions Concerning the Diagnosis of Hyperadrenocorticism

Do normal results on an ACTH stimulation test or a low-dose dexamethasone suppression test rule out hyperadrenocorticism? Conversely, do abnormal results definitely diagnose the disease?

Results of the ACTH stimulation test are in the normal range in at least 20% to 30% of dogs with hyperadrenocorticism, whereas the false-negative rate with the low-dose dexamethasone test is approximately 5%. If results of one test are normal but clinical suspicion for the disease is high, one should consider performing the other screening test. It is important to recognize that each test also has false-positives, particularly when dogs with nonadrenal disease are tested. Interestingly, of the two screening procedures the low-dose dexamethasone suppression test seems more prone to this potential error. About 50% of dogs in one study with nonadrenal illness had abnormal results (inadequate suppression of plasma cortisol) when tested with a low dose of dexamethasone (Kaplan, Peterson, and Kemppainen, 1995). None of the dogs in that study had a clinical presentation or signs consistent with hyperadrenocorticism. Assessment of test results in dogs with nonadrenal illness in which a clinical suspicion of hyperadrenocorticism is also present is challenging. The predictive value for hyperadrenocorticism of a positive screening test result increases in direct proportion to the number and severity of clinical signs and biochemical changes typically occurring in the disease.

What is the best test for hyperadrenocorticism in cats?

We recommend the high-dose dexamethasone suppression test, administering the steroid at a dose of 0.1 mg/kg IV and collecting a sample before dexamethasone administration and two samples after dexamethasone administration (at 4 and 8 hours).

How and why should I try to differentiate pituitary- from adrenal-dependent hyperadrenocorticism in dogs?

Unless results of the low-dose dexamethasone suppression test support pituitary-dependent hyperadrenocorticism, a differentiating test is recommended. Results of ACTH stimulation testing cannot differentiate the types. The two endocrine tests for this purpose are the high-dose dexamethasone suppression test (0.1 or 1.0 mg/kg in dogs and 1 mg/kg in cats) and endogenous ACTH measurement. Timing of sample collection in the high-dose dexamethasone suppression test is the same as for the low-dose test. If cortisol concentrations at 4 and/or 8 hours post-dexamethasone suppression (>50% decline from baseline or decline to <30 nmol/L), a diagnosis of pituitary-dependent disease is made. However, in about 15% to 20% of dogs with pituitary-dependent hyperadrenocorticism, cortisol is not suppressed by dexamethasone. Considering that cortisol is not suppressed by dexamethasone in dogs with adrenal-dependent disease, and that pituitary-dependent disease is the most common form, a failure to suppress in response to dexamethasone means that the odds of having either form is about 50%. In other words, failure of cortisol concentrations to suppress in response to high-dose dexamethasone does not mean that a patient has an adrenal tumor. Endogenous ACTH measurement can positively differentiate the forms, since circulating ACTH concentrations are low to nondetectable in adrenal-dependent disease and normal to high in pituitary-dependent disease. Unfortunately, there is also a gray zone relative to the interpretation of endogenous ACTH measurements, and values that fall into this range are nondiagnostic. Accurate measurement of ACTH in plasma requires special sample handling (Kemppainen and Clark, 1995). Other means to differentiate hyperadrenocorticism include ultrasonography and other imaging modalities and surgical exploration. It is important to distinguish the forms because of differences in therapy and prognosis. Surgical removal of an adrenal tumor can be curative. If mitotane is used for medical management, the therapeutic approach is different, depending on the type of disease. In general, adrenal-dependent disease requires higher dosages and a longer induction phase (Kintzer and Peterson, 1989). Overall the prognosis varies between the two forms. Trilostane can also be used to treat functional adrenal tumors.

What is the value in measuring a urinary cortisol:creatinine ratio (UCCR)?

This test is best used to determine if a dog does not have hyperadrenocorticism. Nearly 100% of dogs with spontaneous hyperadrenocorticism have an elevated UCCR. However, many dogs with an elevated UCCR do not have hyperadrenocorticism. Consequently, a high ratio means that hyperadrenocorticism is possible, and a more definitive screening test (ACTH stimulation, low-dose dexamethasone suppression test) is indicated. Therefore a normal UCCR is valuable in ruling out hyperadrenocorticism.