Vol. 22, No.
2 February 2000 V
CE Refereed Peer Review
FOCAL POINT
★ Several tests can be used to
distinguish pituitary-dependent
hyperadrenocorticism from
adrenal tumor, but results of
such tests can be nondiagnostic
in some animals.
KEY FACTS
■ Measurement of corticotropin
(ACTH) requires special sample
handling.
■ Results of high-dose
dexamethasone suppression
testing are never diagnostic
of adrenal tumor.
■ Results of the ACTH assay are
nondiagnostic approximately
25% of the time.
■ Metyrapone suppression
testing may have advantages
over high-dose dexamethasone
suppression testing or ACTH
assay.
C O N T I N U I N G E D U C AT I O N S E R I E S
Successfully complete the quizzes for each CE article in this series and
receive a certificate indicating completion of a course of study in Diagnostic
Testing for Hyperadrenocorticism. This is the third of three articles.
Differentiating
Tests to Evaluate
Hyperadrenocorticism
in Dogs and Cats
University of Pennsylvania
Carole A. Zerbe, DVM, PhD
ABSTRACT: Tests to distinguish pituitary-dependent hyperadrenocorticism (PDH) from
adrenal tumor (AT) are based on the fact that animals with PDH have high plasma concentra-
tions of corticotropin (ACTH) and a raised threshold for negative feedback, whereas those with
AT have low ACTH and a normal negative feedback pathway that is already maximally activat-
ed by glucocorticoids secreted by the AT. Animals with PDH should have suppression of plas-
ma cortisol after dexamethasone administration; however, some dogs with PDH fail to have
suppression, and thus lack of suppression is nondiagnostic. Results of the ACTH assay can be
diagnostic for either AT or PDH, but test results are nondiagnostic in approximately 25% of
cases. Metyrapone suppression testing and corticotropin-releasing hormone stimulation test-
ing may be useful differentiating tests but cannot yet be routinely recommended.
W
hen a definitive diagnosis of hyperadrenocorticism (HAC) has been
made via screening tests, a determination of which form of HAC (i.e.,
pituitary-dependent HAC [PDH] or adrenal tumor [AT]) the animal
has must be made. Tests to distinguish AT from PDH are called differentiating tests
and are critical in making a prognosis and selecting therapy. For example, there is a
50% chance that a dog with AT has benign adenoma,1,2 a disease that can be cured
by unilateral adrenalectomy. A dog with adenocarcinoma may best be treated by
chemotherapy (mitotane), but the dosage would need to be much higher than that
needed to treat a dog with PDH. In general, prognosis is best for unilateral adrenal
adenoma, good for PDH, and guarded for adrenal adenocarcinoma.
SAMPLE HANDLING
Hormone assays for differentiating tests include measurement of cortisol, 11-des-
oxycortisol (11-DOC; a precursor for cortisol), and corticotropin (ACTH). Corti-
sol and 11-DOC are steroid hormones that can be measured in serum or plasma.
Small Animal/Exotics Compendium February 2000

For measurement of cortisol, adrenal axis associated with

NEUROAMINES
EDTA-treated plasma samples each disorder. Dogs and cats
do not need to be cooled; serum with PDH have bilateral ad-
Hypothalamus
samples should be shipped in renal hyperplasia, which is
an insulated container with Excessive CRH? caused by excessive ACTH
cool packs.3 Eleven-DOC can secretion. Glucocorticoid neg-
be measured in serum or plas- Anterior ative feedback continues to op-
ma, and all samples should be Raised erate, albeit at a greater thresh-
threshold pituitary
shipped using cool packs. old than is normal. Because
for negative
ACTH is measured in plasma; feedback Excessive ACTH glucocorticoid negative feed-
it is a very labile protein hor- Bilateral back is in operation in animals
mone, and special handling is adrenal with AT, ACTH concentra-
required.4 Ideally, blood should hyperplasia tions are low and the normal
be collected in EDTA (purple- contralateral adrenal cortex
topped) tubes to which apro- EXCESSIVE CORTISOL atrophies. Most differentiating
tinin has been added. Apro- Figure 1—Alterations of the hypothalamic–pituitary– tests are therefore designed to
tinin is a protease inhibitor adrenal axis in animals with pituitary-dependent hypera- take advantage of the fact that
that retards the breakdown of drenocorticism. Note the increased corticotropin (ACTH) pituitary disease is often associ-
ACTH in plasma.5 The sample concentrations and the raised threshold to negative feedback. ated with high plasma ACTH
should be centrifuged immedi- (CRH = corticotropin-releasing hormone) concentrations and a raised
ately, and the plasma collected threshold for glucocorticoid
into plastic tubes. The sample negative feedback; animals
should be stored frozen rather NEUROAMINES with AT tend to have low con-
than at 4˚C and mailed within centrations of ACTH and a
a day. The container should be Hypothalamus normal glucocorticoid negative
shipped with frozen refrigerant feedback (Figures 1 and 2).
packs using next-day service. Low CRH Examples of differentiating
If aprotinin is not used, tests include the ACTH assay,
ACTH is more labile and sam- Anterior high-dose dexamethasone sup-
ple-handling procedures be- Normal pituitary pression (HDDS) test, corti-
come even more critical. Blood negative cotropin-releasing hormone
should be collected into cold feedback Low ACTH (CRH) stimulation test, and
EDTA tubes placed on ice and metyrapone suppression test
centrifuged; plasma should Adrenal (see Differentiating Tests). It is
tumor
then be immediately collected important to note that results
into cold plastic tubes and of some screening tests, namely
EXCESSIVE CORTISOL
frozen. Samples should remain the low-dose dexamethasone
frozen until the assay is per- Figure 2—Alterations of the hypothalamic–pituitary– suppression test and combined
formed and thus should be adrenal axis in animals with adrenal tumor. Note the de- dexamethasone suppression/
shipped on dry ice using creased corticotropin (ACTH) concentrations and normal ACTH stimulation test, may
overnight service. It is recom- negative feedback pathway. (CRH = corticotropin-releas- be diagnostic for PDH. Conse-
mended that the reference lab- ing hormone) quently, in addition to accu-
oratory be consulted before rately identifying HAC in 58%
collecting and shipping samples; some laboratories will to 92% of dogs,6–9 these screening tests also may differenti-
supply blood collection tubes with aprotinin added. ate PDH from AT in approximately 33% to 61% of dogs
Normal values vary among laboratories; therefore, with HAC.9–11 This article discusses all differentiating tests
the normal range of the laboratory that analyzes the (which can be used in cats and dogs unless otherwise not-
sample should be used. Normal ranges provided in this ed) in terms of use, rationale, protocol, normal values, in-
article are only approximate values. terpretation, advantages, and disadvantages.

DIFFERENTIATING TESTS High-Dose Dexamethasone Suppression Test

To distinguish PDH from AT, one must understand Rationale
the abnormalities of the hypothalamic–pituitary– A high dose of dexamethasone suppresses cortisol

APROTININ ■ BILATERAL ADRENAL HYPERPLASIA ■ GLUCOCORTICOID NEGATIVE FEEDBACK

Compendium February 2000 Small Animal/Exotics

concentrations in dogs and cats with preted as confirmation of AT.10,11,14 Ad-

PDH but not in those with AT. This
Differentiating Tests
ditional testing (e.g., ACTH assay,
is because animals with AT already Most Common metyrapone suppression, or CRH stim-
have maximum activation of the neg- ■ High-dose dexamethasone ulation) or nonhormonal tests (e.g.,
ative feedback pathway with conse- suppression test imaging studies) are required.15
quent low or undetectable concentra-
■ Corticotropin assay
tions of ACTH; further suppression Comments
of ACTH is unlikely. In contrast, the This test is easy to perform, and no
high dose of dexamethasone will sup- Less Common (not currently in use) special sample handling is required.
press ACTH and subsequently corti- ■ Corticotropin-releasing hormone Some dogs, however, may become ill
sol in most dogs with PDH. stimulation test after testing (especially with the 1.0
■ Metyrapone suppression test mg/kg dose), and results are never di-
Protocol agnostic for AT. HDDS test results
A pre-dexamethasone blood sample are diagnostic for PDH in 73% (154
is collected for baseline plasma cortisol Screening Tests That May Also of 210) of dogs.10,11,14 Although rare, 2
determination, dexamethasone (dogs, Serve as Differentiating Tests of 75 dogs with AT had test results
0.1 mg/kg; cats, 1.0 mg/kg) is adminis- ■ Low-dose dexamethasone consistent with PDH (i.e., false-posi-
tered intravenously (IV), and post-dexa- suppression test tive results); it should be noted, how-
methasone samples are collected 4 and ■ Combined dexamethasone ever, that results were borderline in
8 hours later.11,12 In dogs, a dexametha- these two cases.2,10,11,14
suppression/corticotropin
sone dose of 1.0 mg/kg may be used if
cortisol concentrations were not sup- stimulation test Corticotropin Assay
pressed in response to the 0.1 mg/kg Rationale
dose; this higher dose, however, should be used with cau- The ACTH assay measures endogenous ACTH con-
tion in dogs with diabetes mellitus or basal cortisol con- centrations, which tend to be high or high-normal in
centrations above 12 µg/dl.12 In contrast, the 1.0 mg/kg dogs and cats with PDH but low-normal to unde-
dose is recommended in cats (in this species, the 0.1 tectable in those with AT.
mg/kg dose is used to screen for HAC rather than to dif-
ferentiate between PDH and AT).13 Protocol
The low-dose dexamethasone suppression test typi- A blood sample is collected for determination of
cally refers to a test of dexamethasone suppression us- ACTH concentration. ACTH is a labile protein hor-
ing a dose of 0.01 to 0.015 mg/kg; in dogs, it is used to mone that degrades quickly with improper sample han-
screen for the presence of HAC. When higher doses of dling. See the Sample Handling section for specific in-
dexamethasone are used for testing, the test is referred structions.
to as the HDDS test and is used as a differentiating test
in dogs. This would be straightforward, except the dos- Interpretation and Normal Values
es of dexamethasone required for screening and differ- This test is generally considered to be the most accu-
entiating are higher in cats than those used in dogs. rate means of differentiating PDH from AT and can be
Consequently, terminology can be confusing (Table I). used to diagnose AT or PDH (unlike the HDDS test,
which cannot confirm the presence of AT).2,9,10,14,16 In
Interpretation
This test should be used only after a TABLE I
diagnosis of HAC has been confirmed. Comparison of Dexamethasone Suppression Testing in Cats and Dogs
A plasma cortisol concentration of less
Dexamethasone Dose
than 50% of the pretesting concentra-
Test (mg/kg) Species Purpose
tion or below the laboratory-estab-
lished value at either 4 or 8 hours after LDDS 0.01–0.015 Dogs Screen for HAC
dexamethasone administration is con- HDDS 0.1 Dogs Differentiate PDH from AT
sistent with PDH.9,11 Because approxi- HDDS 0.1 Cats Screen for HAC
mately 27% of dogs with PDH do not HDDS 1.0 Dogs and cats Differentiate PDH from AT
have cortisol suppression in response to AT = adrenal tumor; HAC = hyperadrenocorticism; HDDS = high-dose dexametha-
high doses of dexamethasone, failure to sone suppression; LDDS = low-dose dexamethasone suppression; PDH = pituitary de-
adequately suppress cannot be inter- pendent HAC.

DEXAMETHASONE DOSES ■ FAILURE TO SUPPRESS CORTISOL ■ FALSE-POSITIVE RESULTS

Small Animal/Exotics
dogs, plasma concentrations above
approximately 40 to 45 pg/ml
Usual Diagnostic Criteria to
(8.8 to 9.9 pmol/L) are consistent Interpret the Corticotropin Assay
with PDH, whereas concentra-
tions below 20 pg/ml (4.4 pmol/L) Value (pg/ml)
are consistent with AT. Concen- <20
trations that fall between these
ranges are nondiagnostic; either 20–40
the test should be repeated or ad- >40
ditional testing performed.
Based on the literature and us-
ing the diagnostic criteria provided
(Table II), results were diagnostic in only 75% (n = 127)
of animals evaluated.2,10,14,16 Responses were nondiagnos-
tic in 23% of dogs, and 4% of dogs would have been in-
correctly diagnosed as having PDH. If diagnostic crite-
ria are changed so that a range of 20 to 45 pg/ml
represents nondiagnostic samples, 29% (37 of 127) of
dogs had nondiagnostic responses, 71% had correct re-
sponses, and no dogs had incorrect responses. Further-
more, nondiagnostic results were obtained more fre-
quently in dogs with AT (42%) than in those with
PDH (15%). In one study, 12 of 41 dogs with adrenal
tumor (29%) still required additional investigation to
determine the cause of their HAC even after one to
three samples were obtained for ACTH assay.2 However,
multiple samples may allow differentiation even if the
first sample is nondiagnostic.
Less is known about ACTH concentrations in nor-
mal or affected cats.13,17 When a recent review was pub-
lished, the ACTH assay had been determined for only
25 cats with HAC.13 In those cases, the assay was diag-
nostic for AT in two of two cats with AT and for PDH
in 22 of 23 cats (96%) with PDH. 13 One cat with
PDH had a nondiagnostic result.
Comments
Because ACTH is a labile hormone, blood samples
for ACTH assay require special handling.4 Sample han-
dling, however, is somewhat less critical when aprotinin
is added to the collection tubes.5 Additionally, test re-
sults may be nondiagnostic even after multiple samples
have been assayed. This test does have the advantage of
requiring only a single blood sample, and results may
be diagnostic for AT or PDH.
Corticotropin-Releasing Hormone
Stimulation Test
Rationale
Corticotropin-releasing hormone stimulates release
of ACTH from the pituitary corticotroph and conse-
quently cortisol from the adrenal cortex. This would
occur in the presence of a pituitary tumor but not AT.
NONDIAGNOSTIC RESULTS ■ PITUITARY CORTICOTROPH ■ 11β-HYDROXYLASE
Compendium February 2000
TABLE II Protocol
A pretesting blood sample is
collected, CRH (1 µg/kg) is ad-
ministered, and samples are col-
Interpretation lected 15 (for ACTH measure-
Consistent with ment) and 30 (for cortisol
adrenal tumor measurement) minutes later.
Nondiagnostic
Consistent with pituitary- Interpretation and
dependent Normal Values
hyperadrenocorticism Normal values have not been
established. Both plasma cortisol
and ACTH concentrations should increase after CRH
challenge in dogs and cats with PDH but not in those
with AT.
Comments
Corticotropin-releasing hormone is not readily avail-
able commercially and has not been extensively investi-
gated.18–20 CRH stimulation testing is not recommend-
ed for general use at this time.
Metyrapone Suppression Testing
Rationale
Metyrapone decreases plasma cortisol concentrations
by inhibiting the enzyme 11β-hydroxylase, the final en-
zyme in the cortisol synthetic pathway. With metyra-
pone-induced 11β-hydroxylase inhibition, 11-DOC
accumulates in the presence of ACTH. Therefore, ex-
cessive ACTH concentrations found in dogs with PDH
would be expected to result in high plasma 11-DOC
and low plasma cortisol concentrations after adminis-
tration of metyrapone. This test has not been evaluated
for use in cats.
Protocol
A pre-metyrapone blood sample is collected, and four
doses of metyrapone (25 mg/kg orally) are adminis-
tered at 6-hour intervals. The second and final blood
sample is collected 24 hours after the first sample.
Interpretation and Normal Values
Like the ACTH assay and unlike the HDDS test, re-
sults of testing may be consistent with either AT or
PDH. Blood samples are assayed for both cortisol and
11-DOC. For results of testing to be valid, there must
be a decrease in plasma cortisol concentrations after ad-
ministration of metyrapone. If cortisol concentrations
fall and 11-DOC concentrations increase to 15 ng/ml
or higher, results are consistent with PDH. If 11-DOC
concentrations remain below 15 mg/dl, results are con-
sistent with AT.21 These recommendations may change
with greater experience.
Small Animal/Exotics Compendium February 2000

Approach to differentiation of canine hyperadrenocorticism

Pituitary/adrenal Imaging studies

function tests

ACTH assay HDDS test (dexamethasone Radiography or Computed Magnetic

dose: dogs, 0.1 or 1.0 ultrasonography tomography resonance
mg/kg; cats, 1.0 mg/kg) imaging

Low Borderline High Suppression No suppression

(<20 pg/ml)a (20–40 pg/ml)a (>40 pg/ml)a

AT Nondiagnostic PDH PDH or, rarely, AT or PDH

result false-positive
(PDH or AT) result

Repeat Perform HDDS Perform Perform ACTH Use higher dose Perform
ACTH assay test imaging assay (1.0 mg/kg) of imaging studies
studies dexamethasone
aNormal values may differ according to the laboratory used.

Figure 3—Algorithm to differentiate the form of canine hyperadrenocorticism. Although important, imaging studies do not assess
function and, like all data, should be cautiously interpreted. (ACTH = corticotropin; AT = adrenal tumor; HDDS = high-dose
dexamethasone suppression; PDH = pituitary-dependent hyperadrenocorticism)

Comments suppression test, when used in concert with the ACTH

This test has the advantages of quick outpatient test-
ing, results that are consistent with AT or PDH, and
easy sample handling. It has the disadvantages of re-
quiring owner compliance for drug administration and
two outpatient visits 24 hours apart. Also, the laborato-
ry must assay 11-DOC in addition to the more com-
mon steroid, cortisol. Although this test needs to be
further investigated before it can be strongly recom-
mended for use, it has better diagnostic accuracy, sensi-
tivity, and specificity than does the ACTH assay or
HDDS test.21
SUMMARY
Current choices for hormonal tests of differentiation
of HAC are the ACTH assay and HDDS test (Figure
3). Each has its own advantages and disadvantages. I
believe that the CRH stimulation test and metyrapone
assay, may be useful tests in the future.
REFERENCES
1. Kintzer PP, Peterson ME: Mitotane treatment of 23 dogs
with cortisol-secreting adrenocortical neoplasms. JAVMA 205:
54–61, 1994.
2. Reusch CE, Feldman EC: Canine hyperadrenocorticism due
to adrenocortical neoplasia. J Vet Intern Med 5:3–10, 1991.
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5. Kemppainen RJ, Clark TP, Peterson ME: Preservative effect
of aprotinin on canine plasma immunoreactive adrenocorti-
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362, 1994.
Compendium February 2000
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sone suppression/ACTH stimulation test in dogs with hy-
peradrenocorticism. JAVMA 189:1562–1566, 1986.
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Small Animal/Exotics
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About the Author
Dr. Zerbe is affiliated with the Department of Clinical Stud-
ies, School of Veterinary Medicine, University of Pennsyl-
vania, Philadelphia, Pennsylvania.