Diagnostic Evaluation of The Hypothalamic-Pituitary Adrenal Axis

• Canine Hyperadrenocorticism

• Feline Hyperadrenocorticism

The term ‘Cushing's syndrome’ relates to the constellation of clinical signs and clinicopathological changes resulting from chronic exposure to excessive glucocorticoids. Persistent hypercortisolaemia may be a result of excessive ACTH production from a micro- or macro-adenoma in the pituitary gland (Pituitary Dependant Hyperadrenocorticism (PDH)), a cortisol-secreting adrenal tumour (AT) or excessive administration of exogenous glucorticoids (iatrogenic hyperadrenocorticism).

While clinical signs typically occur together, some individuals exhibit one sign only (often polydipsia, alopecia or conformational changes) at presentation. As a consequence, the list of clinical indications in which testing of the pituitary-adrenal axis may be indicated is extensive (see Table 8). However, because of the potential for false positive and false negative results, which are inherent in all the currently available tests, it is important that testing be undertaken to support the clinical suspicion based on the history and physical findings taken overall. Similarly, while there are many clinicopathological changes associated with Cushing's (see Table 9), further endocrine testing is indicated only where the clinical picture is consistent.

Increased glucocorticoid release is an important part of the response to stress. Severe non-adrenal disease can be associated with chronic stress and abnormal responsiveness of the hypothalamic-pituitary-adrenal axis (HPA). An important part of the clinical evaluation of potential cases of Cushing's syndrome is the exclusion of other diseases before testing, which may otherwise lead to abnormal test results and erroneous diagnosis of Cushing's disease. It is also critical to review current and previous drug therapy for agents that may influence test results. Both topical and systemic glucocorticoids should be withdrawn for 14 days before diagnostic evaluation of the HPA. Testing should be delayed for 3 months following depot steroid or progestagen administration. In some cases, anticonvulsants can affect results of ACTH testing.

Table 8: Common findings in hyperadrenocorticism

Table 9: Clinicopathological changes in hyperadrenocorticism

Tests of the HPA in Hyperadrenocorticism

Basal Cortisol

Sample required: Separated serum

NB: Binding of cortisol to red blood cells will lead to artefactually low results where analysis is delayed.

Interpretation: There is a high degree of overlap between basal cortisol concentration in normal dogs and those with hyperadrenocorticism. Consequently, sensitivity of this test is low (50%). Stress and non-adrenal illness may result in increased basal cortisol values resulting in poor test specificity as well. Given these limitations, basal cortisol is considered of no diagnostic value in hyperadrenocorticism but can be useful in suspected hypoadrenocorticism.

Steroid-Induced Alkaline Phosphatase (SIALP)

Sample required: Separated serum. Haemolysis significantly inhibits all isoenzymes of Alkaline Phosphatase (ALP).

Interpretation: Although ALP is distributed widely (liver biliary endothelium, bone, intestinal mucosa, renal cortex, placenta), in the dog, there is a further isoenzyme whose synthesis is related to circulatory glucorticoid levels. Significant (>3 fold top normal) elevations of ALP usually reflect increased production of the liver specific or steroid induced ALP isoenzymes.

An increased percentage (>35%) of total ALP derived from SIALP is common in hyperadrenocorticism. However, an increased % SIALP also occurs with stress, primary liver disease and in other disease conditions.

Accuracy: SIALP can be sensitive (81-92%) but has poor specificity (55%). Therefore, a normal SIALP therefore makes hyperadrenocorticism unlikely. An increased SIALP is an indication for further diagnostic evaluation.

Urinary Cortisol:Creatinine Ratio

Sample required: Random plain voided urine sample (collected at home).

Interpretation: Hypercortisolaemia is associated with increased urine cortisol elimination when assessed in relation to urine creatinine as a marker of urine concentration. Increased ratios are very common in hyperdrenocorticism but are also seen with many non-adrenal diseases, including some associated with polydipsia.

Accuracy: One study showed high sensitivity (92%) and specificity (97%) amongst normal dogs but very poor specificity (21%) in dogs with non-adrenal illness. As a consequence, this test can be regarded as a low cost, easy screening test with a negative (normal) result making Cushing's unlikely. However, a positive result must be confirmed by ACTH or low-dose dexamethasone suppression test. This test is not suitable for monitoring dogs on therapy with lysodren.

ACTH Stimulation Test

Protocol:

• Obtain a basal serum sample
• Inject 250 µg Synacthen iv
• Collect a second serum sample 1-2 hours later

Indications: This test is commonly used as a screening test because of its speed and relatively low cost. It is the test of choice for iatrogenic hypercortisolism and for monitoring adrenal reserve in dogs on therapy. The short duration of the test also makes it suitable for use concurrently with insulin in diabetes mellitus.

Interpretation: See Table 10.

Accuracy: Sensitivity for PDH is good (83%) but poor in AT (60%). Further testing (usually low-dose dexamethasone suppression test) should therefore be undertaken in dogs with suggestive signs of hyperadrenocorticism but normal ACTH test results. Specificity is good in normal dogs but poor (63%) in dogs with severe non-adrenal disease.

Table 10: Interpretation of ACTH Stimulation Test

Low Dose Dexamethasone Suppression (LDDS)

Protocol:

• Obtain a baseline serum sample
• Inject 0.01 mg/kg of dexamethasone iv and collect further samples 3 and 8 hours post injection
• It is critical that the dog is otherwise left unstressed during the test and no other procedures are undertaken. Failure to do so may result in false positive results.

Interpretation: The eight-hour sample is assessed initially. Significant endogenous cortisol production at eight hours is diagnostic of hyperadrenocorticism. A reduction in cortisol to less than 50% of baseline at 3 hours or below a level of 40nmol/l is diagnostic for PDH and no further evaluation is required. However, AT and 50-60% of PDH cases do not suppress and require further evaluation (Table 11).

Accuracy: Reported sensitivities range from 90-95%, suggesting LDDS to be a more sensitive test than ACTH Stimulation Test. However, 5-10% of cases will still be negative when first evaluated and may require retesting after a few months. Specificity may be poor, however, (51%) in dogs with non-adrenal disease and particularly diabetes mellitus and renal failure. Positive test results must always be viewed in light of the history and clinical signs.

Table 11: Patterns of results on LDDS

High-Dose Dexamethasone Suppression Test

Protocol:

• Obtain baseline serum
• Inject 0.1 mg/kg dexamethasone iv
• Collect further samples at 3 and 8 hours post injection

Interpretation: Suppression to <50% of baseline cortisol or <40 nmol/l at 3 or 8 hours is diagnostic for PDH. Failure to suppress to this level may reflect AT and occurs in 15 – 50% of PDH cases. These individuals require further evaluation by abdominal radiography, ultrasound or CAT scanning, or endogenous ACTH determination (see below).

Endogenous ACTH

Protocol: Refer to submitting laboratory.

Interpretation: In dogs with previously confirmed hyperadrenocorticism, ACTH concentration below 20 pg/ml is consistent with AT, levels between 20-50 pg/ml are considered non-diagnostic and may be seen with AT or PDH. Concentrations in excess of 50 pg/ml are consistent with PDH.

Accuracy: ACTH is considered the most accurate test for differentiation of AT from PDH where other tests are non-discriminatory. However, until recently, the usefulness of the assay was severely limited by stringent sample requirements. A new approach to sample handling using the protein inhibitor approtinin has achieved stability for up to 4 days at 4°C, and this test is likely to be much more widely used in future.

Therapeutic monitoring of hyperadrenocorticism

Dogs on therapy with lysodren, ketoconazole or modrenal should have regular repeat ACTH stimulation tests to monitor adrenocortical reserve. Post ACTH cortisol concentration should optimally be within the normal basal reference range (25 – 125 nmol/l).

Feline Hyperadrenocorticism

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Spontaneous hyperadrenocorticism is rare in the cat, but both PDH and AT have been reported. Iatrogenic hyperadrenocorticism is less common than in the dog, but may be associated with chronic depot glucocorticoid or progestagen therapy.

Clinical signs are similar to those in the dog (see Table 8). Insulin-resistant diabetes mellitus can be associated with hyperadrenocorticism or acromegaly.

ACTH Stimulation Test

Protocol:

• Obtain a basal serum sample
• Inject 125 µg Synacthen iv
• Collect further samples after 60 and 90 minutes

Interpretation: Adrenal responsiveness to ACTH is much less marked in the dog than in the cat. Post ACTH cortisol concentrations on either sample in excess of 324 nmol/l are suggestive of hyperadrenocorticism, levels in excess of 441 nmol/l being strongly suggestive.

Accuracy: 15-30% of cats with hyperdrenocorticism have normal ACTH responses (false negatives). Non-adrenal illness and stress have been associated with false positives. Interpretation must therefore be made in the light of the history and physical findings.

Low Dose Dexamethasone Suppression Test

Protocol:

• Obtain a baseline serum sample
• Inject 0.1 mg/kg (the same dosage as the canine high dose test)
• Sample at 4, 6 and 8 hours after injection

Interpretation: Consistent absence of suppression (>40 nmol/l) on all 3 samples is strongly suggestive of hyperadrenocorticism. Increased cortisol only at 8 hours is consistent with hyperadrenocorticism, but should not be the sole diagnostic criteria.

Accuracy: Low case numbers have limited evaluation of the sensitivity and specificity of this test, however it has been reported that cats without hyperadrenocorticism but with non-adrenal illness do suppress at this higher dose of dexamethasone. In another study, 3 of 4 cases histologically confirmed hyperadrenocorticism had abnormal responses on this test.

High Dose Dexamethasone Suppression Test

Single test protocol at 0.1 mg/kg dexamethasone is now used in cats although previously ultra-high dose tests (1mg/kg) were performed. At 0.1 mg/kg dose any post-dexamethasone cortisol <50% of basal value supports PDH once hyperadrenocorticism is confirmed.

Endogenous ACTH

Protocol: consult the submitting laboratory

Interpretation: normal or elevated levels (>45pg/ml) are consistent with PDH. AT may be associated with low normal or subnormal levels.

Suggested Reading

Feldman, E.C., Nelson R W 1996. Second Edition Canine and Feline Endocrinology and Reproduction, W.B. Saunders, Philadelphia

Nelson, R.W., Couto G G, 1998, Small Animal Internal Medicine Second Edition, Mosby Year Book, Missouri

Peterson, M.E., Endocrine and Metabolic Disorders in Current Veterinary Therapy XII, Bonagura (Ed) 1995, W.B. Saunders, Philadelphia

Bruyette, D.S., Endocrine and Metabolic Diseases in Consultations in Feline Internal Medicine Volume 2, 1994 August, J.R. (Ed)., W.B. Saunders, Philadelphia

Torrence, A.O.; Mooney, C.T., Manual of Small Animal Endocrinology 1998 Second Edition, BSAVA Publications, Cheltenham