Vol. 22, No.
1 January 2000 V
CE Refereed Peer Review
FOCAL POINT
★Each test used to confirm
a diagnosis of hyperadreno-
corticism (HAC) evaluates the
hypothalamic–pituitary–adrenal
axis from a different perspective
and thus must be interpreted in
light of its sensitivity, specificity,
and other factors.
KEY FACTS
■ The corticotropin stimulation
test is the only screening test
that can be used in cats or dogs
with clinical signs of HAC and
concurrent or recent treatment
with glucocorticoids.
■ The low-dose dexamethasone
suppression test is not useful
in cats.
■ The urine cortisol:creatinine ratio
test is easy to perform but has
very low specificity.
■ The combined dexamethasone
suppression/corticotropin
stimulation test is a useful
screening test that may be able
to detect pituitary-dependent
HAC.
■ Test results in animals with
nonadrenal disease may be
consistent with HAC.
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 second of three articles.
Screening Tests
to Diagnose
Hyperadrenocorticism
in Cats and Dogs
University of Pennsylvania
Carole A. Zerbe, DVM, PhD
ABSTRACT: Several screening tests are available to confirm the presence of hyperadrenocorti-
cism (HAC), each of which evaluates a specific aspect of pituitary–adrenal function. The corti-
cotropin (ACTH) stimulation test assesses the capacity of the adrenal gland to secrete cortisol,
whereas the low-dose dexamethasone suppression (LDDS) test determines the integrity of the
negative feedback pathway. The combined dexamethasone suppression/ACTH stimulation test
evaluates both adrenocortical reserve and negative feedback. The urine cortisol:creatinine ratio
test measures urinary cortisol excretion relative to creatinine. In addition, the LDDS and com-
bined tests can also identify the pituitary-dependent form of HAC and thus may function as
differentiating tests.
T
wo questions should be addressed in the approach to diagnosing hyper-
adrenocorticism (HAC; sometimes referred to as Cushing’s syndrome):
Does the patient have HAC, and, if so, what form? Assessment of clinical
signs, history, complete blood count, urinalysis, and biochemical profile can
raise the suspicion that an animal has HAC, but only hormonal testing can con-
firm its presence. This article, the second in a series on the pathophysiology of
HAC and hormonal tests to evaluate the condition, focuses on tests used to con-
firm the presence of HAC (i.e., screening tests). Once a diagnosis of HAC is
confirmed, a determination of whether it is adrenal or pituitary dependent must
be made. Tests to distinguish an adrenal tumor (AT) from pituitary-dependent
HAC (PDH) are referred to as differentiating tests and will be the subject of the
final article in this series.
SAMPLE HANDLING
Evaluation of the hypothalamic–pituitary–adrenal (HPA) axis involves collecting
blood or urine to determine cortisol or corticotropin (ACTH) concentrations.
General recommendations regarding sample handling are provided in this article;
Small Animal/Exotics Compendium January 2000

practitioners are strongly advised to follow the specific

instructions provided by their reference laboratory.
Cortisol, a steroid hormone, can be measured in plas-
ma, serum, or urine.1,2 Plasma is generally preferred over
serum and should be collected in EDTA (purple-topped)
tubes.2 In serum, cortisol is temperature sensitive; samples
should be packed with frozen refrigerant packs and Recent, current, or possible history of steroid use
shipped to the laboratory via next-day or second-day de-
livery service.2 Urine samples should be centrifuged to re-
move debris and handled as described for serum samples.1 ACTH stimulation testa
Sample handling for plasma ACTH determination will
be discussed in the article on differentiating tests.

SCREENING TESTS Normal response Subnormal response

To confirm the presence of HAC, the HPA axis can
be evaluated via urine cortisol concentration, adreno-
cortical steroid–secreting capacity, or the integrity of
the negative feedback pathway. The number of tests Normal or HAC Iatrogenic CSb
available to diagnose HAC suggests that they are not all
100% effective; indeed, there is no single best screening
test for HAC in cats or dogs, although each has advan- Use a different
tages and disadvantages. For example, only the ACTH screening test and/or
stimulation test can distinguish between iatrogenic and retest if clinical signs
naturally occurring HAC and is therefore the only test persist and no other
diagnosis is obtained
that can evaluate the HPA axis in an animal that is cur-
rently receiving or has recently received steroid therapy;
other tests would give unreliable results. Conversely, in LDDS testc
addition to confirming the presence of HAC, the low-
dose dexamethasone suppression (LDDS) test or the
combined dexamethasone suppression/ACTH stimula- Lack of Lack of Adequate
tion test (combined test) can differentiate PDH from suppression suppression suppression
AT in approximately 33% to 61% of patients.3–5 with 4- or 8-hr with 4- or 8-hr
If a practitioner is reasonably certain (based on clini- result <50% result >50%
of baseline of baseline
cal signs and history) that HAC is present, the LDDS
or combined test may be the best and quickest method
to screen for and potentially differentiate the cause of HAC (PDH) or HAC (test cannot Normal or false-
HAC. If HAC is only a slight possibility, the urine cor- false-positive differentiate negative result
tisol:creatinine ratio (UCCR) test might be the fastest result between PDH
and least expensive, and thus the best, initial approach. and AT) or false-
Because false-positive and false-negative results may oc- positive result Consider ACTH
cur with any of these tests, findings should be interpret- stimulation
testing
Figure 1—In the approach to dogs with possible hyperadreno-
corticism (HAC), veterinarians must first consider whether the
patient has strong or weak evidence of HAC based on clinical aUsed to rule out iatrogenic disease and determine the extent of adrenal
signs, history, and laboratory (i.e., complete blood count, bio- cortical suppression, which helps to determine how quickly steroid therapy
chemical profile, and urinalysis) abnormalities. A history of can be tapered.
bIatrogenic CS is also referred to as iatrogenic HAC (technically incorrect) and
steroid use also affects initial testing choices. Once these deci-
iatrogenic hypoadrenocorticism.
sions have been made, tests can be chosen and interpreted as cIn addition to confirming a diagnosis of HAC, the LDDS and the combined
indicated. (ACTH = corticotropin; AT = adrenal tumor; CS = (i.e., combined dexamethasone suppression/ACTH stimulation test) tests also
Cushing’s syndrome; DEX = dexamethasone; HPA = hypotha- have the advantage of distinguishing PDH from AT in 40% to 60% of cases.
d The ACTH stimulation test has the advantage of requiring only two samples
lamic–pituitary–adrenal axis; LDDS = low-dose dexameth-
and 1 or 2 hours to complete.
asone suppression test; PDH = pituitary-dependent HAC;
UCCR = urine cortisol:creatine ratio)
Figure 1
 Compendium January 2000 Small Animal/Exotics

Approach to dogs with suspected HAC

Strong evidence of HAC Weak evidence of HAC

Pursue other Evaluate HPA ACTH UCCR test

diagnostics first axis at a later stimulation test
date if clinical
Exaggerated response signs persist
and other
diagnoses Normal High
cannot be ratio ratio
found
HAC or false-positive result
No evidence HAC or
of HAC or false- false-positive
Confirm HAC with negative result result
additional testing

Confirm
HAC with
additional
Combined testc testing
ACTH stimulation testd

Lack of Normal Normal Subnormal Exaggerated

Lack of Normal
suppression suppression to response response response
suppression to suppression to
to DEX, DEX,
DEX, normal DEX, normal
exaggerated exaggerated
response to response to
response response to Normal Iatrogenic HAC or false-
ACTH ACTH
to ACTH ACTH or HAC CS positive result

HAC (test cannot HAC or HAC (PDH) or Normal or Retest using Confirm HAC with
differentiate false-positive false-positive false-negative LDDS or additional testing
between PDH result result result combined testc
and AT) or false-
positive result

Retest in Reevaluate at
~1 mo a later time
or with the
LDDS test
Small Animal/Exotics
ed cautiously and in conjunction with history, physical
examination, and clinicopathologic testing. Multiple
tests are often required for accurate assessment of the
HPA axis.
The algorithms presented in Figures 1 and 2 provide
a reasonable approach to testing in dogs and cats with
clinical signs, history, and laboratory abnormalities that
are consistent with HAC, although obviously not every
clinical situation is addressed. Tests used to screen for
HAC in dogs and cats (see Screening Tests) are dis-
cussed in the following sections. Because values vary
among laboratories, the normal range of the laboratory
where the tests are performed should be used for inter-
pretation; normal ranges provided in this article are only
approximate values.
Corticotropin Stimulation Test
Use and Rationale
The ACTH stimulation test is used to screen for the
presence of HAC and hypoadrenocorticism and to dis-
tinguish between iatrogenic and spontaneous HAC in
cats and dogs. It can also test the efficacy of mitotane,
ketoconazole, or metyrapone therapy.
This is a test of adrenocortical reserve. ACTH stimu-
lates secretion of cortisol from the adrenal glands; the
amount secreted reflects the capacity of the adrenal cor-
tex to secrete cortisol. For example, in patients with
adrenal atrophy, ACTH stimulation would result in lit-
tle or no cortisol secretion, whereas those with adrenal
hyperplasia would have an exaggerated response.
Protocol
The ACTH stimulation test is quick, economic, and
easy to perform; it requires only 1 to 2 hours and two
(dogs) to three (cats) blood samples to complete, de-
pending on the specific preparation used (i.e., the natu-
ral pituitary gland extract [ACTH gel, which is no
longer commercially available] or synthetic ACTH [co-
syntropin]) and the patient’s species. After reconstitu-
tion, cosyntropin is stable for up to 4 months when refrig-
erated or up to 6 months when stored at –20˚C.6,7
Two protocols exist for dogs. 4,8–10 In one, a pre-
ACTH blood sample is collected for plasma cortisol de-
termination, 2.2 IU/kg ACTH gel is injected intramus-
cularly (IM), and a post-ACTH sample is collected 2
hours later. In the other protocol, a pre-ACTH blood
sample is collected, 5 µg/kg cosyntropin is injected in-
travenously (IV), and a post-ACTH sample is collected
1 hour later.11
Several protocols can be used in cats; regardless of the
method used, a pretesting blood sample must be col-
lected.12–15 After IM administration of 2.2 IU/kg ACTH
gel, post-ACTH blood samples are collected 1 and 2
ADRENOCORTICAL RESERVE ■ COSYNTROPIN ■ CORTISOL CONCENTRATIONS
Compendium January 2000
ACTH stimulation test
Subnormal Normal Exaggerated
response response response
Iatrogenic Normal or HAC or false-
Cushing’s HAC positive result
syndrome
Retest using Confirm with
DEX DEX suppression
suppression
aCombined dexamethasone suppression/ACTH stimulation test.
Figure 2—Approach to cats with suspected hyperadrenocorti-
cism (HAC) based on clinical signs, history, and laboratory
(i.e., complete blood count, biochemical profile, and urinalysis)
abnormalities. (ACTH = corticotropin; AT = adrenal tumor;
DEX = dexamethasone; PDH = pituitary-dependent HAC)
hours later. Alternatively, 0.125 mg cosyntropin (half a
vial) may be given IM or IV; two post-ACTH samples
are collected 30 and 60 minutes later when the IM
route is used, and one post-ACTH sample is collected
60 to 90 minutes later when the IV route is used. In
cats, IV cosyntropin is recommended because it pro-
vides higher circulating ACTH concentrations.13 Two
post-ACTH samples are necessary after administration
of ACTH gel or when cosyntropin is given IM: Ap-
proximately one third of cats will have peak cortisol
concentrations when the first post-ACTH sample is col-
lected, but concentrations may be much lower in the
second sample.
Normal Values
Because pre- and post-ACTH cortisol concentrations
differ between cats and dogs, it is important to use the
normal reference range for the species being tested.
Normal reference ranges for pre-ACTH cortisol con-
centrations are approximately 0.5 to 4 µg/dl (14 to 110
nmol/L) in dogs and 0.3 to 5.0 µg/dl (8.3 to 138
nmol/L) in cats. Normal reference ranges for post-
ACTH cortisol concentrations are approximately 8 to
20 µg/dl (220 to 552 nmol/L) in dogs and 5 to 15
µg/dl (138 to 414 nmol/L) in cats.
 Compendium January 2000 Small Animal/Exotics

Approach to cats with moderately to strongly suspected HAC

Combined testa DEX suppression test (0.1 mg/kg)

Lack of Lack of Normal Normal Adequate Lack of

suppression suppression to suppression to suppression to suppression suppression
to DEX, DEX, normal DEX, DEX, normal
exaggerated response to exaggerated response to
response to ACTH response to ACTH Normal or HAC or
ACTH ACTH false-negative false-positive
result result

HAC (test HAC (PDH) or Normal or

cannot HAC or false- Confirm with ACTH
positive result false-positive false-negative
differentiate result result stimulation test
between AT
and PDH)

Interpretation Mitotane reduces the number of cells producing corti-

Exaggerated response to ACTH stimulation is consis- sol (via adrenocortical destruction) and consequently
tent with HAC but may be seen in some dogs and cats the number of cells that can respond to ACTH stimu-
with chronic illnesses that do not directly involve the lation. The other drugs do not destroy adrenocortical
HPA axis (see the Adrenal Function Testing in Cases of tissue but rather block enzymes in the cortisol synthetic
Nonadrenal or Concurrent Illness section).16–19 Approx- pathway.24,26 The amount of reduction in ACTH-stim-
imately 80% to 95% of dogs with HAC will have an ulated cortisol secretion is related to the degree that the
exaggerated response to ACTH,4,5,19–22 including about pathway is blocked. Ideally, with therapy, both pre- and
85% of dogs with PDH and about 50% of dogs with post-ACTH cortisol concentrations should be within
AT.4,5,19–21 However, this test does not appear to be as the pretesting normal range.
sensitive for the diagnosis of HAC in cats; responsive- Although a normal response to ACTH may indicate a
ness to ACTH challenge was increased in only 51% of healthy HPA axis, approximately 5% to 20% of dogs
cats and borderline increased in 16% of cats.23 with HAC (pituitary or adrenal dependent) will also have
A reduced response to ACTH is consistent with normal responses.4,5,19–22 When this happens in a dog in
adrenocortical atrophy, such as that which occurs in which HAC is strongly suspected, the LDDS test should
Addison’s disease and iatrogenic Cushing’s syndrome. be considered. Alternative approaches include retesting
For example, if response to ACTH the animal at a later date or after med-
is reduced in a dog with suspected ically stabilizing it (Figure 1).
Cushing’s syndrome, a likely inter- Screening Tests
pretation is that the dog has Cush- Comments
ing’s syndrome caused by glucocor- ■ Corticotropin stimulation test Of the screening tests, ACTH stim-
ticoid administration that resulted ■ Low-dose dexamethasone ulation has the highest specificity
in atrophy of the adrenal cortex. suppression test (dogs only) (82% to 91%; Table I) 5,19,22 and
This iatrogenic Cushing’s syndrome ■ High-dose dexamethasone thus the fewest false-positive results
would appropriately be treated by suppression test (cats only) compared with the LDDS, com-
steroid withdrawal rather than with bined, or UCCR test. Some authors
■ Combined dexamethasone
mitotane therapy. have consequently recommended
Reduced response to ACTH also suppression/corticotropin ACTH stimulation as the best gen-
occurs in dogs that are receiving mi- stimulation test (combined test) eral test for diagnosis of HAC. 22
totane or ketoconazole and in cats ■ Urine cortisol:creatinine ratio test Conversely, ACTH stimulation is
that are receiving metyrapone.24–26 less sensitive than is either the LDDS

ADRENOCORTICAL ATROPHY ■ IATROGENIC CUSHING’S SYNDROME ■ MITOTANE

Small Animal/Exotics Compendium January 2000

TABLE I ferences can lead to confusion in the selection

and performance of dexamethasone suppression
Sensitivity, Specificity, and Diagnostic Accuracy
tests (Table II).
of Screening Tests in Dogs
Study Use and Rationale
The LDDS test is used to screen for the pres-
Test Parameter (%) 1a,5 2 19 3 22 4 31 5 34
ence of HAC in dogs; a low dose of dexametha-
LDDS Sensitivity 100 100 96 85 — sone suppresses adrenocortical cortisol secretion
Specificity 73 44 70 73 — in normal dogs but not in dogs with HAC. This
Diagnostic 92 58 89 83 — suppression is caused by activation of the cortisol
accuracy negative feedback pathway leading to decreased
ACTH Sensitivity 89b 80 95 — — ACTH and subsequent cortisol secretion. There
stimulation Specificity 82b 86 91 — — is also a raised threshold for glucocorticoid-in-
Diagnostic 87b 84 93 — — duced cortisol suppression.
accuracy
UCCR Sensitivity 50 75 — 99 100 Protocol
Specificity 100 24 — 77 22 Although easy to perform, the LDDS test re-
Diagnostic 59 37 — 91 76 quires 8 hours to complete and the 8-hour sam-
accuracy ple is the most critical for interpretation.5,27 A
Combinedc Sensitivity 93 — — — —
pretesting blood sample is collected to determine
Specificity 73 — — — — baseline plasma cortisol, 0.01 to 0.015 mg/kg
Diagnostic 90 — — — — dexamethasone is injected IV, and blood is col-
accuracy lected for testing 4 and 8 hours after administra-
tion. Diluting the dexamethasone with sterile
a
This table includes data not presented in the original abstract. saline (1:10) may allow more accurate dosing.
b
Values were determined from the ACTH stimulation portion of the com-
bined test.
Dexamethasone is available in polyethylene gly-
c
Combined dexamethasone suppression/corticotropin stimulation test. col (Azium®, Schering-Plough, Union, NJ) and
ACTH = corticotropin; LDDS = low-dose dexamethasone suppression; as dexamethasone sodium phosphate; either
UCCR = urine cortisol:creatinine ratio; — = parameter not investigated. preparation is useful, and dosing is based on the
concentration of the active ingredient. This
or combined test.5,19,22 In addition to sensitivity, speci- screening test is not useful in cats when the 0.01-mg/kg
ficity, and diagnostic accuracy, such factors as length of dose of dexamethasone is used but may be if a dose of
time to complete the protocol, questionable history of 0.1 mg/kg is used.14,28,29
steroid use, presence of concurrent illness, and cost
should be weighed when deciding which test to use. Normal Values and Interpretation
The ACTH stimulation test is the only screening test To interpret the LDDS test, the 8-hour post-dexa-
that can be used in dogs or cats with clinical signs of methasone sample should be evaluated first; in normal
HAC and current or recent treatment with glucocorti- dogs, cortisol values at this time should be below 1.0 to
coids and the only test that distinguishes between iatro- 1.4 µg/dl (28 to 39 nmol/L). Failure to suppress corti-
genic and spontaneous HAC. This test cannot be used sol at 8 hours is consistent with a diagnosis of HAC
to distinguish AT from PDH. and may represent an adrenal tumor or PDH. Alterna-
tively, such findings may represent a false-positive value.
Low-Dose Dexamethasone Suppression Test The 4-hour sample is evaluated only when 8-hour
The LDDS test typically refers to tests using a dexa- test results are consistent with HAC. Cortisol suppres-
methasone dose of 0.01 to 0.015 mg/kg; in dogs, it is sion to less than 50% of the baseline concentration or
used to screen for the presence of HAC. When higher below 1.0 to 1.4 µg/dl (28 to 39 nmol/L) at 4 hours
doses of dexamethasone are used, the test is referred to with an “escape” or rebound of suppression in the 8-
as a high-dose dexamethasone suppression (HDDS) test. hour sample is consistent with PDH. Alternatively, an
In dogs, the higher dose is used to determine whether 8-hour value above 1.0 to 1.4 µg/dl (28 to 39 nmol/L)
HAC is pituitary or adrenal dependent (i.e., it is used that is less than 50% decreased from baseline is also
as a differentiating test). In cats, however, the dexa- consistent with PDH. Therefore, in addition to the ac-
methasone doses required for screening and differenti- tual cortisol concentration, the pattern of plasma corti-
ating are higher than are those used in dogs. These dif- sol suppression is also important and may provide a di-

CORTISOL NEGATIVE FEEDBACK PATHWAY ■ CORTISOL SUPPRESSION ■ 8- VS 4-HOUR SAMPLE

Compendium January 2000 Small Animal/Exotics

agnosis of PDH. Test results TABLE II dogs. Increased plasma corti-

are never diagnostic for AT; Comparison of Dexamethasone Suppression sol concentrations associated
in other words, a lack of sup- Tests in Cats and Dogs with HAC increase urinary
pression is consistent with cortisol concentrations. Cre-
Dexamethasone
PDH and AT. atinine measurements are used
Test Dose (mg/kg) Species Purpose
Approximately 5% to 10% to adjust for urine dilution.
of dogs with HAC may have LDDS 0.01–0.015 Dogs Screen for HAC
cortisol suppression that is Protocol
similar to that in normal HDDS 0.1 Dogs Differentiate A single urine sample is
PDH from AT
dogs.4,20 When results of the submitted for cortisol and
LDDS test are normal but HDDS 0.1 Cats Screen for HAC creatinine measurements.31–35
clinical suspicion of HAC It has been recommended
remains high, the animal HDDS 1.0 Dogs Differentiate that urine samples be col-
should be retested in 1 to 2 and cats PDH from AT lected by the owners in the
months using the ACTH AT = adrenal tumor; HAC = hyperadrenocorticism; HDDS = animal’s home environment32
stimulation, combined, high-dose dexamethasone suppression; LDDS = low-dose dexa- to avoid the stress associated
and/or LDDS test. In addi- methasone suppression; PDH = pituitary-dependent hyper- with a hospital visit and its
tion, chronic nonadrenal ill- adrenocorticism. subsequent effect on urinary
ness or recent or concurrent steroid concentrations.32
therapy with such drugs as glucocorticoids and pheno-
barbital may be associated with a reduced suppressive Normal Values and Interpretation
effect of dexamethasone (false-positive value, see the Normal values for UCCR differ among laboratories. A
Adrenal Function Testing in Cases of Nonadrenal or high UCCR is consistent with HAC.31–35 However, ap-
Concurrent Illness section).16–19 Variations in dexam- proximately 75% to 80% of dogs with nonadrenal illness
ethasone metabolism among dogs may account for a have increased UCCRs, and thus test results should be
percentage of false-positive and -negative test results.30 interpreted cautiously and always confirmed with anoth-
er screening test. Experience with this test in cats is limit-
Comments ed.35 Because of the rarity of feline HAC, additional tests
The LDDS test is the most sensitive of the screening of the HPA axis are recommended in this species.
tests (85% to 100%; Table I),5,19,22,31 but specificity is
low (44% to 73%).5,19,22,31 Thus, although this test is Comments
good at detecting HAC, it may also falsely detect HAC The specificity of the UCCR test is very low, ranging
in dogs with nonadrenal illness. It is therefore impor- from 22% to 77% (Table I).5,19,31,34 Because of the high
tant to use this test only when there is a high index of sensitivity reported in some studies, it has been recom-
suspicion for HAC. Although this test is time consum- mended that this test be used for its negative predictive
ing to complete, a diagnosis of PDH can be deter- value (i.e., if the UCCR is normal, HAC is unlikely).
mined in about 40% to 60% of the cases, thus saving Thus it is a reasonable test to screen for HAC in dogs
time as well as the expense of additional testing.3–5 in which the suspicion of HAC is low or in those that
Although LDDS is an excellent screening test in cannot be hospitalized for testing; however, false-nega-
dogs, a certain percentage of normal cats apparently tive results (i.e., normal UCCR in dogs with HAC) oc-
have an escape of serum cortisol suppression at the 8- cur in as many as 8% to 40% of cases.5,19
29
hour sample. Thus this test may give false-positive re-
sults and is not suitable for use in cats. Instead, a dose Combined Dexamethasone
of 0.1 mg/kg of dexamethasone may be used to screen Suppression/Corticotropin Stimulation Test
cats for the presence of HAC. When the higher dose is Use and Rationale
utilized, the test’s sensitivity is approximately 75%.23 As The combined test principally screens for HAC in
is recommended for dogs, additional testing should be dogs and cats and may also distinguish PDH from
performed in cats in which test results are normal but adrenal-dependent disease in dogs (i.e., in dogs, the
clinical signs strongly suggest HAC. combined test is both a screening [ACTH stimulation]
and a differentiating [HDDS] test); in cats, it is a com-
Urine Cortisol:Creatinine Ratio Test bination of two screening tests. It allows for evaluation
Use and Rationale of adrenocortical reserve capacity as well as integrity of
The UCCR test is used to screen for HAC in cats and the negative feedback pathway in both species.

ESCAPE OF CORTISOL SUPPRESSION ■ DEXAMETHASONE DOSES ■ NEGATIVE PREDICTIVE VALUE

Small Animal/Exotics
Protocol
Several different protocols have been used.10,17,36–40 A
pre-dexamethasone blood sample is collected for plas-
ma cortisol determination, dexamethasone (0.1 mg/kg
IV) is administered, and a post-dexamethasone sample
is collected 4 hours later. Immediately after the post-
dexamethasone sample is collected, either ACTH gel
or cosyntropin is administered (see the Corticotropin
Stimulation Test Protocol section); a post-ACTH sam-
ple is collected 1 (if cosyntropin is used) or 2 (if ACTH
gel is used) hours later. When ACTH gel is used in
cats, samples are collected 1 and 2 hours after adminis-
tration. An alternative protocol calls for collection of
the post-dexamethasone sample at 2 rather than 4
hours. Either protocol is acceptable.
Normal Values
Normal values are the same as for the ACTH stimu-
lation or HDDS test alone (i.e., pretesting values are
about 0.5 to 4.0 µg/ml [14 to 110 nmol/L] in dogs and
0.3 to 5.0 µg/ml [8.3 to 138 nmol/L] in cats). Post-
dexamethasone values should be below 1.0 to 1.4
µg/ml (28 to 39 nmol/L); normal post-ACTH values
are 8 to 20 µg/ml (220 to 552 nmol/L) in dogs and 5
to 15 µg/ml (138 to 414 nmol/L) in cats.
Interested in
writing for
COMPENDIUM?
For small animal articles, please contact
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For exotics articles, please contact
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email britchie@vet.uga.edu).
Compendium January 2000
Interpretation
Although interpretation of the combined test is simi-
lar to that of the individual tests, the combined test
should be evaluated as a unit. The ACTH stimulation
portion of the test serves as a screening test with the
same use, advantages, and disadvantages as the ACTH
stimulation test used alone. The addition of the dexa-
methasone suppression component permits (1) identifi-
cation of animals with HAC that respond normally to
ACTH but fail to show normal cortisol suppression in
response to 0.1 mg/kg of dexamethasone, and (2) im-
mediate diagnosis of PDH in dogs that have an exag-
gerated response to ACTH and normal suppression fol-
lowing dexamethasone administration.
A normal response to ACTH in conjunction with
resistance to dexamethasone suppression is consistent
with HAC, and further testing is indicated for differ-
entiation. Experience with the combined test in cats is
limited.17,23,40,41 Cats with HAC are expected to have
an exaggerated response to ACTH and resistance to
dexamethasone suppression, although ACTH stimula-
tion is exaggerated in only approximately 50% of cats
tested.23 As with the other screening tests, equivocal or
normal results in animals with suspected HAC should
be followed by an LDDS (dogs) or HDDS (cats) test
or by retesting in 1 to 2 months if clinical signs persist
(Figures 1 and 2).
Comments
Recommendations regarding the use of the combined
test to diagnose and differentiate HAC in dogs are con-
flicting.10,36–39 One researcher stated that the combined
test should not be recommended in dogs 38,39 even
though he determined that the test accurately detected
HAC in 86%38 or 76%39 of dogs evaluated. In another
study, diagnostic accuracy (90%) was similar to that of
the LDDS test (Table I); sensitivity (93%) and speci-
ficity (73%) were also similar to the LDDS test.5 The
combined test has a few advantages over the LDDS
test: The combined test can be accomplished in 3 to 6
hours as opposed to 8 hours for the LDDS test, can
provide information about adrenocortical reserve as
well as integrity of the negative feedback pathway, and
allows PDH to be diagnosed in dogs with normal sup-
pression of cortisol after dexamethasone administration
but an exaggerated response to ACTH challenge.
Adrenal Function Testing in Cases
of Nonadrenal or Concurrent Illness
Results of adrenal function testing may be abnormal
in dogs and cats with nonadrenal illness; the stress of
illness can activate the HPA axis by stimulating ACTH
and/or cortisol hypersecretion or by altering cortisol
COMBINED TEST ■ CATS
Compendium January 2000
metabolism. Difficulties occur when nonadrenal illness
causes excessive urinary or plasma cortisol concentra-
tions, an exaggerated response to ACTH, or a lack of
suppression to dexamethasone in animals without HAC.
For example, results of adrenal function testing are ab-
normal in dogs and cats with chronic renal or liver dis-
ease, uncontrolled diabetes mellitus (DM), and other
diseases.16–19 Environmental factors may also affect test
results.32,42
Sensitivity, specificity, and diagnostic accuracy are
methods to assess a screening test’s ability to accurately
distinguish dogs with HAC from normal dogs or those
with nonadrenal illness. In this article, specificity refers
to the ability of tests of the HPA axis to correctly iden-
tify dogs that do not have HAC. The optimum speci-
ficity is 100%. Of the commonly used screening tests,
the ACTH stimulation test has the highest specificity
(82% to 91%) and thus has been suggested by one
group22 as the better test in screening for HAC in dogs
(Table I). Specificity is lower for the LDDS (44% to
73%) and combined (73%) tests. Reported specificity
for the UCCR test varies dramatically (22% to 77%).
For most tests, sensitivity varied greatly but was gen-
erally highest for the LDDS test (85% to 100%), the
combined test (93%), and the ACTH stimulation test
(80% to 95%). Sensitivity for the UCCR ranged from
50% to 100%, depending on the study. Diagnostic ac-
curacy was highest for the LDDS test, combined, and
ACTH stimulation tests; results of the UCCR test var-
ied greatly.
Testing dogs with nonadrenal illness for HAC should
be postponed if possible; if postponement is not possi-
ble, the ACTH stimulation test has been recommended
as being most appropriate.22 When the dexamethasone
suppression portion is added to the ACTH stimulation
test, diagnostic accuracy and sensitivity are improved
while specificity is lowered. Perhaps the combined test
would be a better choice for these patients, but further
investigation is necessary before such a recommenda-
tion can be made.
Some drugs, namely anticonvulsants and glucocorti-
coids, may also cause false-positive results. Glucocorti-
coids interfere with tests of the HPA axis in two ways:
by cross-reacting with the cortisol assay (cortisone, hy-
drocortisone, prednisone, and prednisolone) and/or by
activating the cortisol negative feedback pathway with
resultant adrenocortical atrophy. Veterinarians must re-
alize that topical, otic, or ophthalmic preparations are
readily absorbed and exert the same effect as do par-
enteral glucocorticoids.43–46 Phenobarbital administered
to normal or epileptic dogs on a short- (8 weeks) or long-
(12 months) term basis did not affect pre– or post–
ACTH-stimulated cortisol concentrations.47,48 Howev-
FALSE-POSITIVE RESULTS ■ GLUCOCORTICOIDS ■ CONCURRENT DISEASE
Small Animal/Exotics
er, one of five dogs had lack of cortisol suppression fol-
lowing a low dose of dexamethasone at 6 and 12
months after initiation of phenobarbital therapy.47
Hyperadrenocorticism can be complicated by such
concurrent diseases as DM, renal disease, urinary tract
infection, thromboembolism, pneumonia, dermatopa-
thy, and others. In addition, because patients with
HAC and concurrent illness are middle-aged to geri-
atric, diseases more common to older dogs and cats oc-
cur more frequently. Although dogs with DM have
been documented to have abnormal adrenal function
test results, well-regulated dogs had normal responses
to ACTH stimulation and dexamethasone suppres-
sion.16 This suggests that the presence of DM itself does
not alter test results but rather that the degree of regula-
tion is important. Furthermore, poorly regulated dogs
with DM apparently have abnormal results in the ab-
sence of HAC.18 It is recommended that testing be de-
layed when possible until patients are adequately regu-
lated. For example, a ketoacidotic diabetic patient
should be stabilized before its HPA axis is evaluated.
However, although uncommon, some severely ill ani-
mals with complications associated with HAC (e.g.,
sepsis, pneumonia, thromboembolism) would benefit
from rapid diagnosis and concurrent treatment of HAC
and its complications.
Whether to test or stabilize first may be a difficult de-
cision. For certain patients, such as a dog with a previ-
ous history and clinical signs consistent with HAC and
a well-known complication of HAC (e.g., pulmonary
thromboembolism) or a cat with DM and thin, fragile
skin that tears easily (with or without the presence of
infection or ketoacidosis), I would choose to evaluate
the HPA axis and stabilize the patient concurrently. In
these and other limited circumstances, waiting for the
patient to normalize may be detrimental, and some pa-
tients may not improve until cortisol concentrations
begin to decrease.
CONCLUSION
There are many tests to evaluate pituitary adrenal
function, but none is best for all dogs and cats. There-
fore veterinarians must understand which test to use
and why. Multiple tests of pituitary adrenal function
are often required to effectively evaluate the HPA axis.
In general, a diagnosis of HAC should be confirmed
with more than one test. Clinical experience with HPA
testing in cats is extremely limited, and thus multiple
testing and cautious interpretation are especially impor-
tant in this species.
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About the Author
Dr. Zerbe is affiliated with the Department of Clinical
Studies, School of Veterinary Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania.