It is suggested that the reader be familiar with the general features of canine hyperadrenocorticism and the normal regulation of adrenal corticosteroid levels before proceding with the following discussion
The diagnosis of Canine Hyperadrenocorticism is not straight forward. General bloodwork and urinalysis results, though they may be suggestive, are not specific for the diagnosis of hyperadrenocorticism (aka "Cushing's Syndrome").The plasma or urine cortisol (or related corticosteroid) measurements are the most non-specific tests that, at best , are only suggestive for the presence of canine hyperadrenocorticism. Initially, therefore, more direct modalities for making the initial diagnosis should employ provocative testing and imaging studies.HOWEVER, having said that, It should be noted that many other illnesses...non-adrenal in nature... and even stress will interfere with the interpretation of most of the commonly used methods.
Finally, not all dogs with Cushing's Syndrome read the text books...so it is often the case that dogs with this disease will test (falsely) negative. In these instances, it is necessary to re-interpret the results of several tests and imaging methods as a unit, combine this with the patient's clinical signs and then rely upon the clinical judgement of the veterinarian in making the defintive diagnosis.
Once an unequivocal diagnosis is obtained via initial provocative testing, other assays and imaging methods are used to ascertain the type of hyperadrenocorticism (i.e. PDH or ADH...see introduction for explanation of these and other basic information about adrenal gland activity) . This becomes important because the recommended treatments for each differ.
General Screening Tests:
Principle: Excess circulating corticosteroids (cortisol) spill into urine. The concentration of urine cortisol is a reflection of the production and circulation of adrenal gland steroid synthesis/release.
Method: This technique requires the collection of 24 hours worth of urine. For a human, this is probably not a terrible inconvenience. For a dog....it is quite impractical.
Interpretation::Since this is not performed anymore in veterinary medicine, the discussion of interpreting this test is deferred.
Urine Cortisol:Creatinine Ratio
Principle: Is similar to measuring urine cortisol, but more practical to perform. Urine cortisol concentration is "normalized"...compared to a standard urine concentration of creatinine, which is a metabolic toxin normally cleared by the kidneys. By comparing cortisol in urine relative to the urine creatinine level, the need to collect whopping volumes of urine over a 24 hour period is gone, and only a single urine sample is required.
Method: A single sample of urine is obtained and laboratory analyses of urine cortisol and creatinine are performed.
Interpretation: A low or "normal" (physiologic) concentration is interpreted as negative for hyperadrenocorticism. An excessive concentration of cortisol does not prove the presence of hyperadrenocorticism. In one study, 76% of dogs that did not have adrenal gland disease had an elevated urine cortisol:creatinine ratio.
Principle: In theory, the presence of excessive levels of plasma cortisol is considered indicative of oversecretion by the adrenal gland.
Method: A sample of blood is obtained and plasma is analyzed for its concentration of cortisol, which is compared to "normal" baseline cortisol concentrations.
Interpretation: Many dogs with hyperadrenocorticism have normal baseline cortisol levels when sampled randomly (there is fluctuation over a 24 hour period). Also, elevation of serum baseline cortisol levels can be seen with non-adrenal illnesses. This test is not sensitive and is not specific.
Provocative Tests for Hyperadrenocorticism:
Anyway, the principles of provocative testing involves a little understanding about the stimulation of normal adrenal activity by the pituitary and hypothalamus and the inhibition of these centers by elevated serum corticosteroid levels. For review of these phenomena, please refer to an earlier discussion. The goals of these initial tests are to determine with certainty 1) whether the animal has canine hyperadrenocorticism and 2) if it does, whether it is PDH (Pituitary-Dependent Hyperadrenocorticism) or ADH (Adrenal-Dependent Hyperadrenocorticism). The preferred provocative tests to diagnose hyperadrenocorticism are the: ACTH Stimulation Testand the Low Dose Dexamethasone Suppression Test (LDDST). Only the former provocative test is useful to diagnose Iatrogenic Cushing's Disease. Other tests (e.g High Dose Dexamethasone Suppression Test are help in distinguishing whether Cushing's disease is due to PDH or ADH. In addition to the ACTH Stimulation Test, measuring the concentration of endogenous blood ACTH may be helpful in the diagnosis of iatrogenic disease. The most common tests are described below.
ACTH Stimulation Test
Principle:As in nature, stimulation of the adrenal gland results in production and release of cortisol (see footnote) from the adrenal gland into the blood. In the presence of hyperadrenocorticism, the stimulation is exaggerated...the level of serum cortisol resulting from administration of ACTH to the patient is markedly elevated (in the case of hyPOadrenocorticism...not covered here... there is no response to ACTH). This (not the Low Dose Dexamethasone Suppression Test)` is the prefered screening test for dogs concurrently receiving phenobarbital for control of seizures.
Method: Synthetic (Cortrosyn® )ACTH is injected , 250 µg intramuscularly (or more recently, intravenously at 5 µg/kg) after obtaining a baseline cortisol* level (see footnote re: sex hormone levels); then 1 hour later, another blood sample is obtained and the cortisol* level is ascertained again.
This test does not distinguish PDH from ADH
Some dogs with non-adrenal illness will show an exaggerated response to ACTH stimulation
PDH: Approximately 85% of dogs with this form of Cushing's Syndrome will show exaggerated response to stimulation with exogenous ACTH.
ADH: Dogs with ADH...about 60% will show an exaggerated response; 40% will have a normal response, though when retested, some of these may show an exaggerated response
Iatrogenic: Dogs with signs of Cushing's Disease due to excessive or prolongued administration of corticosteroid medications will have little or NO stimulation when challenged by exogenous ACTH. This is one of the few methods to ascertain whether a dog actually has Iatrogenic Cushing's Disease
Note: In some animals with signs of hyperadrenocorticism, post-stimulation cortisol levels are not increased but one or more sex hormones (which can cause similar signs as cortisol when present in excess) may be increased. The hormones most likely to increase are: progesterone and/or 17-hydroxyprogesterone and/or androstenedione. Some are now advocating the measurement of all of these sex steroid hormones levels before and after stimulation with ACTH (if results of standard stimulation of cortisol levels are inconclusive or negative).
(Reference: JAVMA 226 (February 2005 ): pp556)
Low Dose Dexamethasone Suppression Test (LDDST)
Principle: As explained elsewhere, normal control of adrenocortical levels is effected by hormonal signals from the hypothalamus (secreting CRH) and pituitary gland (secreting ACTH) at the base of the brain. Normally, when blood corticosteroid levels are adequate, these then act to suppress the further secretion of CRH and ACTH respectively, and blood cortisol levels drop. This assay is designed to examine for the presence and magnitude of this response (i.e. whether there is a decrease in plasma cortisol, and if so by how much and for how long) due to elevated blood concentrations of an exogenously delivered corticosteroid...in this case dexamethasone. Dexamethasone is a potent corticosteroid that does not interfere with assays for plasma cortisol. Hence is well suited to provide the signal that, in normal dogs, induces a persistently decreased (< 1.0µg/dl for at least 8 hours) level of circulating cortisol.
A low concentration of dexamethasone is injected intravenously after obtaining blood to ascertain baseline cortisol levels. Samples of blood are then obtained 4 hr and 8 hr after administering the drug.
In the absence of any suppression, a diagnosis of Cushing's Disease can be made, but it is not possible to distinguish ADH from PDH. A High Dose Dexamethasone Suppression Test may be able to distinguish the two after the diagnosis of Cushing's Disease is made. False positive test occurs in dogs receiving phenobarbital for control of seizure. In these dogs, the ACTH Stimulation Test should be used.
PDH: Since animals with PDH have abnormal levels of circulating ACTH stimulating the adrenal glands and resulting in abnormal levels of cortisol, logic would dictate that the pituitary in these cases is resistant to the inhibitory ("feedback inhibition") effects of excessive corticosteroids. In fact, these pituitary tumor which secrete ACTH in excess are variably sensitive to low doses of circulating dexamethasone. Hence it is possible to see either no suppression (20% of dogs with PDH) or some suppression of cortisol levels... but only for a short time (2-4 hours). In cases where significant suppression is noted early, by 8 hours, circulating cortisol levels are again elevated to >1.4µg/dl . However...if the blood cortisol level at 8 hours is >1.0µg/dl but still < 1.4µg/dl, the result is considered ambiguous...and non-diagnostic.
The reason for the rise in cortisol at 8 hours in PDH dogs that suppress cortisol levels earlier is that dogs with Cushing's Syndrome clear (i.e. metabolize and/or excrete in urine or bile) circulating dexamethasone in less than 8 hours (whereas normal dogs require 12 or more hours to clear dexamethasone).
ADH: Adrenal tumors are autonomous...meaning that they synthesize cortisol independent of any other signals or events. Hence, no decrease in cortisol levels is detected at any time after administration of dexamethasone.
Iatrogenic: Iatrogenic Cushing's Syndrome can not be diagnosed using this procedure, since blood cortisol levels are already suppressed by the presence of exogenously administered corticosteroid preparations.
High Dose Dexamethasone Suppression Test
Principle:The test is primarily useful in distinguishing PDH from ADH once a diagnosis of hyperadrenocorticism is obtained via one of the previously discussed provocative tests. Keep in mind that in some instances the low dose dexamethasone suppression test is both diagnostic for hyperadrenocorticism and can distinguish PDH from ADH . However, in a few percent of PDH animals, no suppression is obtained, and it is not possible to ascertain the type of hyperadrenocorticism. In those instances of PDH where no suppression was observed with the low dose dexamethasone suppression test, suppression may be observed when very high doses of dexamethasone are used. In contrast, ADH animals should not suppress at any dose of dexamethasone, since these are autonomous adrenal tumors.
Method: A high concentration of dexamethasone in injected after obtaining blood for a baseline cortisol measurement. Blood is obtained at 4 hours and 8 hours after dexamethasone administration and cortisol levels are again ascertained.
Interpretation: Suppression has occured if the test meets one of these four criteria: 1) 4 hr cortisol < 1.4 µg/dl, 2) 4hr cortisol < 50% of baseline 3) 8hr cortisol < 50% of baseline 4) 8hr cortisol < 1.4µg/dl. The absence of suppression is interpreted as "inconclusive".
PDH: Approximately 70-75% of these animals will meet one of the above-defined criteria for suppression. Thus, one quarter to ~1onw third will a show false negative test. It is believed that some of these false negatives may result from tumors of a certain region (pars intermedia) of the pituitary gland...that is inherently more resistent to feedback inhibition by circulating corticosteroids. Assuming a positve Low Dose Dexamethasone test for Cushings Disease, if the cortisol is not suppressed with the higher dexamethasone dose, there is a 50% chance the dog has PDH or ADH.
ADH: As mentioned earlier, these are autonomous tumors unaffected by any of the usual regulatory pathways. However, in any animal, there is a periodic/diurnal fluctuation in the cortisol level which may also occur in these animals. Hence, it is theoretically possible to see some "suppression"....which is really not supression at all...but a chance sampling at a time when the cortisol level is lower than at the time previously sampled. Hence, though rare, it is possible to obtain results not entirely consistent with ADH though ADH: is present. In an ideal world, a dog with ADH will show now suppression from baseline (but...see PDH, above).
Non Provocative Tests
Principle: By definition, PDH means that the pituitary is over secreting ACTH..which is what is overstimulating the adrenal glands to secrete excessive quantities of adrenal cortical hormones, e.g. cortisol. In contrast, adrenal tumors (ADH) secrete adrenal hormones independent of control by the pituitary gland. The excessive quantities of cortisol produced by an adrenal tumor is expected to inhibit further production of ACTH by the pituitary gland (see discusion of feedback inhibition). Measurement of circulating [ACTH] should therefore distinguish between ADH (absence of circulating [ACTH] ) and PDH (excessive [ACTH])
Method: A single blood sample is collected, plasma is separated, and stored frozen (or with special preservative prior to assay for [ACTH]. Since ACTH is labile (unstable), failure to obtain and properly preserve sample will lead to erroneous results and misdiagnosis (the absence of [ACTH] will look like ADH.
PDH: As mentioned above, these animal should have high [ACTH]. In fact, 85%-90% will have normal to high [ACTH], BUT....10%-15% of dogs with PDH will have low or non-diagnostic [ACTH]. The latter may be due to random variation in ACTH secretion or, rarely, aberrant ACTH molecules manufactured by pituitary tumors that are not measured via standard [ACTH] assays. Also, there is a "gray zone" value of [ACTH] which is specific neither for PDH nor ADH.
ADH: Approximately 74% of these animals will have undetectable quantities of [ACTH], BUT 26% will have normal or non-diagnostic [ACTH] levels. These may result from instances where both ADH and PDH are concurrently present or may occur due to random sample variation in [cortisol] or [ACTH] or to the "gray zone" problem discussed above..
Abdominal Radiographs: May be helpful in determining whether hyperadrenocorticism (already diagnosed via another method) is ADH or PDH.
Principle: Adrenal masses may cause certain radiological changes usually related to just one of the two glands (sometimes bilateral adrenal tumors occur, though that is considered rare).
Method: Standard radiographic techniques is employed to assess the abdominal structures, especially the adrenal gland and nearby structures, such as kidney and liver.
PDH: Increased size of both adrenal glands, under the stimulation of excessive ACTH is expected. These may or may not be visualized with plain radiographs.
ADH: Usually one adrenal gland is enlarged (the one with the tumor). The tumorous mass(es) may be detected via its effects on the normal radiological positioning of other abdominal structures (called a "mass effect"). Mineral deposits in the abnormal adrenal gland may also be detected this way. Such mineralization is rare (but possible) in normal adrenal glands.
Abdominal Ultrasound: May also be helpful in determining whether hyperadrenocorticism (already diagnosed via another method) is ADH or PDH. Other abnormal adrenal anomalies may also be visualized even in the absence of clinical signs of hyperadrenocorticism. These include an adrenal cyst, a non-functioning adrenal tumor (tumor that does not cause clinical signs of hyperadrenocorticism because it does not secrete excessive quantities of corticosteroids), a pheochromocytoma (this is an entirely different type of adrenal tumor that secrets excessive quantities of "adrenaline-like" neurotransmitter molecules with potential life-threatening consequences!). Ultrasonography is currently limited though because the adrenal glands are often difficult to visualize. The normal size (small), location near other organs...where gas in the stomach or intestines, or excessive abdominal fat or mineralized or enlarged kidney(s) or liver or lymph nodes can obscure visualization.. are factors to reckon with. An ultrasonographer experienced in adenal ultrasonography is a definite plus. In one study, approximately 14% of ADH animals showed an enlarged adrenal. In PDH animals, because there is overlap in normal adrenal size and adrenal size observed with some affected animals, it is sometimes difficult to utilize this (size) criterion for diagnosis (maximum sensitivity was 77% for left adrenal; as low as 18% for the right adrenal).. Additionally, the ultrasonographic appearance of affected (hyperplastic or tumorogenic) adrenals may be similar to normal adrenals. Should clearly abnormal, bilaterally enlarged adrenal glands be seen (and hyperadrenocorticism is previously diagnosed), it is still possible to misdiagnose PDH when, in fact, a bilateral (rare) ADH is present.
Computed Tomography (CT): When applied to the abdomen, this technique is somewhat more sensitive than ultrasound, but some of the same limitations apply also. For example, adrenal enlargement is not always clearly apparent, compared to normal and it is not possible to distinguish benign and malignant adenal masses based upon size, shape or response to the contrast agent.
This technique can be employed to examine the pituitary gland at the base of the brain. The presence of a pituitary mass is consistent with PDH. But small pituitary masses may be missed. However, when concurrent neurological abnormalities are noted (in addition to the clinical signs of Cushing's Syndrome), masses are invariably large enough to be identified.
The ability to see small tumors is enhanced using Dynamic CT, however. This technique is useful ifhyphophysectomy is being considered for treatment...in order to precisely locate the abnormal pituitary gland tissue(s) (see treatment options.)
Magnetic Resonance Imaging (MRI): This technique is useful when a pituitary mass is present and it's size is not discernable via other imaging techniques. Approximately one half of PDH dogs without any concurrent neurological signs have masses detectable by MRI. The size and appearance can be serially evaluated and, if it appears that the mass is changing and may be threatening future neurological complications, a surgical treatment option may then be considered (even if clinical signs of Cushing's Syndrome are resolved medically) (see treatment options.)
The author wishes to acknowledge the following sources of information for this page:
Feldman & Neslon:Canine and Feline Endocrinology and Reproduction 2nd ed WB Saunders, 1996.
The author's personal notes obtained from numerous journals, presentations by endocrine specialists at professional meetings and participation in the Veterinary Information Network (VIN)
Behrend and Kemppainen:Diagnosis of Canine HyperadrenocorticismThe Veterinary Clinics of North America, Small Animal Practice, September, 2001, pp985
Axlund, Behrend and Winkler: Surgical Treatment of Canine Hyperadrenocorticism Compendium, May 2003 25:pp334
Hill, Kate E etal: Secretion of Sex Hormones in Dogs with Adrenal Gland Dysfunction J. American Veterinary Medical Association February 15th 2005 pp556