Cortisol concentrations following stimulation of healthy and adrenzpathic dogs with two doses of tetracosactrin A prospective study was undertaken to compare intravenous

tetracosactrin at doses of 5 pg/kg and 250 pg for diagnosing

hyperadrenocorticism in dogs. Both healthy dogs and dogs with

pituitarydependent hyperadrenocorticism were evaluated with

the two doses of the drug, and serum cortisol concentrations were

compared at 60 minutes post-stimulation. Some of the dogs had

additional samples taken at 90 and l20 minutes. For four dogs with

hyperadrenocorticism, timed samples were also obtained at 150,

180 and 240 minutes post-injection. Cortisol concentrations 60

minutes after stimulation with either 5 pg/kg or 250 pg intravenous

tetracosactrin were similar for both healthy dogs and dogs with

hyperadrenocorticism. The lower dose can therefore be used for

diagnosing hyperadrenocorticism in dogs.

L. A. FRANK, R. C. DENOVO, A. c. KRAJE AND J. w. OLIVER*

J&al of Small Animal Practice (2000) 41,308-311

Departments of Small Animal Clinical Sciences and *Comparative Medicine, University of Tennessee, College of Veterinary Medicine, Knoxville, TN 3701, USA

I INTRODUCTlON

The adrenocorticotropic hormone (ACTH) stimulation test is commonly used to screen dogs suspected of having hyper- adrenocorticism and to monitor mitotane therapy in dogs with hyperadrenocorti- cism. In addition, it is the test of choice for the diagnosis of iatrogenic hypetadreno- corticism and hypoadrenocorticism. Tetracosactrin (Cortrosyn; Organon), a synthetic polypeptide (containing a 24 amino acid sequence), is one of the A C T H preparations available. I t is a single-use lyophilised product that requires reconstitution with saline. While ACTH is currently readily available, there is always a possibility of decreased availability of the product in the future. Because a dog with hyperadrenocorticism will have multiple A C T H stimulation tests performed, storing and re-using a portion of the vial would be useful to enable multiple doses to be taken from a single vial. T h e authors recently demonstrated that the bioactivity of tetracosactrin is maintained when stored frozen at -20°C in plastic syringes for six months (Frank and Oliver 1998).

Various protocols using tetracosactrin have been recommended for the A C T H

stimulation test in dogs. Some use the entire vial (250 pg intravenously [IV]) per dog (Feldman and others 1982, Feldman and Nelson 1996) because tetracosactrin is packaged as a single-use vial. While there is no contraindication to the use of 250 pg of tetracosactrin per dog, many believe this dose to be excessive and that maximum stimulation can be achieved using lower doses of A C T H (Eiler and Oliver 1980, Kaplan and others 1995, Kerl and others 1999). Studies in normal dogs comparing 1, 3 or 5 pg/kg tetracosactrin subcuta- neously (Kemppainen and others 1982) and 1, 5 or 10 pg/kg IV (Kerl and others 1999) found comparable stimulation of the adrenal cortex at all doses, although the response was more prolonged at the 5 and 10 pg/kg doses in the latter study. Similar cortisol concentrations at 60 minutes post- tetracosactrin stimulation were also achieved in normal dogs when the entire vial was used (250 pg IV) compared with 1 pg/kg IV; however, the entire vial resulted in a more sustained response (Kemppainen 1983).

The purpose of the present study was to validate the use of a 5 pglkg dose of tetra- cosactrin for diagnosing hyperadrenocorti- cism in dogs. Cortisol concentrazions were compared in normal and adrenopathic dogs after stimulation with either 5 pg/kg or 250 pg tetracosactrin.

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FIG 1. Mean rerum cortisol concentration 60 minuter after stirnulation wlth 5 ~ U k g or 250 pg tetracosactrin in 15 healthy dogs. Ban indicate standard deviation

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Table 1. Mean serum cortisol concentration (nmol/litre [+SD]) post-tetracosactrin stimulation in four dogs with hy peradrenocorticism

Dose Time (minutes) 60 90 120 150 180 240

Time Post-Tetracosactrin (minutes) 5 pg/kg 940.8 685.6 374.4 200.3 125.0 92.4 FIG 2. Mean serum cortisol concentration 60, 90 and 120 minutes after (431.8) (547.4) 228.4) (932.5) (78.9) (99.3) stimulation with 5 pg/kg or 250 pg tetracosactrin in six healthy dogs. 250 pg 893.1 939.2 746.3 489.7 239.2 103.2 Different letters indlcate significant dlfference (Pe0.05). Bars indicate (443.1) (531.1) (559.2) (466.0) (170.8) (86.9) standard deviation

MATERIALS AND METHODS

Normal dose Fifteen healthy, privately owned dogs were randomly assigned to receive either 5 pg/kg or 250 pg tetracosactrin IV at the start of the study Dogs were returned in one to two weeks, at which time the doses were reversed and administered a second time. All dogs weighed between 5.9 and 20.9 kg (mean 12.8 kg). Smaller dogs were chosen in order to maximise the difference between dosages. Dogs were excluded from the study if they had received any cortico- steroid preparation (systemic or topical) within six months before the start of the study. Dogs were not allowed to receive any steroid preparation while participating in the study. Serum was collected before and 60 minutes after tetracosactrin injec- tion. In addition, samples were obtained at 90 and 120 minutes from six of the dogs. Serum samples were stored at -70°C until assayed for cortisol concentration at the completion of the study.

Adrenopathic dogs Six dogs with confirmed pituitary- dependent hyperadrenocorticism, weighing between 4.8 and 18.6 kg (mean 11.6 kg), were tested with 5 pg/kg tetracosactrin IV, followed by a second test four to seven days later using 250 pg of the drug IV. Again, smaller dogs were selected in order to maximise the difference between the two dosages. Pituitary-dependent hyper- adrenocorticism was confirmed by the following tests: low dose dexamethasone suppression (LDDS) test in 5/6 dogs (all suppressed to less than 35.9 nmol/litre ar four hours, but increased by eight hour post-dexamethasone suppression), endog- enous ACTH concentration in all dogs (ACTH concentrations were greater than

8.8 pmol/litre in four dogs and between 4.4 and 8.8 pmol/litre in two dogs) and ultrasonography in all dogs (all adrenal glands were similar in size to the contra- lateral gland and were either enlarged or in the upper range of normal).

The ACTH stimulation test was carried out at least four days following the LDDS test. Blood was collected from the jugular vein before and 60 minutes after tetra- cosactrin administration. For four of the dogs, additional samples were taken at 90, 120, 150, 180 and 240 minutes. Serum was assayed for cortisol within 48 hours to better serve the patients‘ needs. In addi- tion, an aliquot from each dog was stored at -70°C until the serum was collected from the second stimulation test, at which time all samples from a given dog were assayed for cortisol. Analyses of serum cortisol concentrations were performed in the authors’ endocrinology laboratory using a solid-phase radioimmunoassay procedure (Coat-a-Count; Diagnostic Products Corporation [DPC], Los Angeles, CA) that has been validated for use in dogs.

Statistical analysis For each sampling time, cortisol concentra- tions after stimulation with the two doses of tetracosactrin were compared using the paired Student t-test (Sigmastat 1994). Val- ues were considered significant if P<0.05.

RESULTS

In healthy dogs, cortisol concentrations 60 minutes post- tetracosactrin stimulation were similar when comparing the two dosages (Fig 1) . In the six dogs in which multiple timed samples were obtained, cortisol concentrations at 60 and 90 min- utes post-stimulation were likewise similar

(Fig 2). In 4/6 dogs receiving the 250 pg dose, however, peak cortisol concentration was reached at 90 minutes instead of 60 minutes. Cortisol concentrations 120 minutes post-tetracosactrin were significantly higher with the 250 pg as compared with the 5 pg/kg dose. As expected, there was a wide variation

in cortisol concentrations post-stimulation among the dogs with hyperadrenocorti- cism. The 5 pg/kg dose of tetracosactrin resulted in a small but statistically greater cortisol response 60 minutes after stimula- tion as compared with the 250 pg dose (Fig 3). When multiple sampling times were compared in four of these dogs, the 250 pg dose of ACTH resulted in peak cortisol at 90 minutes, although the corti- sol concentration was similar to that obtained at 60 minutes with both doses of ACTH (Table 1; Fig 4 ) . The 250 pg dose resulted in a more sustained cortisol response at 120 and 150 minutes, how- ever, as compared with the 5 pg/kg dose.

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Tetracosactrin Dose FIG 3. Mean serum cortisol concentration 60 minutes after stimulation with 5 pg/kg or 250 pg tetracosactrin in six dogs with pituitarydependent hyperadrenocorticism. Different letters indicate significant difference (Pe0.05). Bars indicate standard deviation

JOURNAL OF SMALL ANIMAL PRACTICE V O L 41 JULY 2000 309 __

Four dogs with hyperadrenocorticism receiving 250 pg tetracosactrin and all six hypercorticoid dogs receiving 5 pg/kg tetracosactrin had cortisol concentrations greater than 552 nmol/litre (Fig 4). Only one of the two dogs with post-stimulation cortisol concentrations less than 552 nmol/ litre had multiple timed samples taken. For this dog, the cortisol concentration never increased above 552 nmol/litre with the 250 pg dose of tetracosactrin (Fig 4C).

DISCUSSION

The ACTH stimulation test assesses gluco- corticoid reserve. In dogs with pituitary- dependent hyperadrenocorticism, cortisol concentrations post-stimulation are greatly increased because of adrenal cortical hyper- plasia. The response of dogs with adreno- cortical tumours to ACTH is more variable and was not evaluated in this study.

The results from this study are in agree- ment with those of Kerl and others (1999) and indicate that the 5 pg/kg dose of tetra- cosactrin adequately stimulates the adrenal

glands in both healthy dogs and those witk hyperadrenocorticism. This finding is sup ported by the fact that occupation of only : small fraction of the total ACTH receptoi sites results in full stimulation of steroid0 genesis (Gill 1979). The use of a smaller, ye equally effective, dose of synthetic ACTE permits the remainder of the vial’s content: to be divided and frozen in plastic syringe: for future use. A previous study showec tetracosactrin to be stable, with bioactiviq retained when frozen in plastic syringes fo up to six months (Frank and Oliver 1998)

In the present study, peak cortisol wa attained at 90 minutes in healthy dog when 250 pg tetracosactrin was used, bu was reached at 60 minutes with thc 5 pg/kg dose. While the 5 pg/kg peak con centration was lower at 90 minutes thar that obtained using the 250 pg dose, thi difference was not statistically significant In previous studies, peak cortisol concen trations in healthy dogs were reachec 90 minutes after stimulation with 5 pg/ki (Kerl and others 1999), 10 pg/kg (Kerl an( others 1999) and 250 pg (Kemppaine~ 1983, Hansen and others 1994) dose

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Post-Tetracosactrin Stimulation Time (minutes) FIG 4. Serum cortisol concentration at multiple timed intervals after stimulation with 5 pg/kg or 250 pg tetracosactrin in four individual dogs with pituitarydependent hyperadrenocorticism

of tetracosactrin; however, the cortisol concentration reached at 60 minutes was not significantly different from that of the peak concentration (Kemppainen 1983, Hansen and others 1994, Kerl and others 1999). Only the 1 pg/kg dose resulted in a cortisol peak at 60 minutes (Kemppainen 1983, Kerl and others 1999).

The difference in time to peak cortisol concentration with the 5 pg/kg tetracosac- trin dose between the present study and that of Kerl and others ( 1 999) may be attributed to the maximum total dose of tetracosactrin administered in each study, reflecting a more sustained response with a greater dose of tetracosactrin. The average weight of healthy dogs in the present study was 12.8 kg (range 5.9 to 20.9 kg), whereas the mean body- weight of dogs in the study by Kerl and oth- ers (1999) was 21 kg (range 10 to 35 kg).

Reports in the literature support adequate stimulation with smaller doses of tetracosactrin in healthy dogs (Kemppainen 1983, Kerl and others 1999); however, there was concern that the maximum response might not occur with these doses in dogs with hyperplastic adrenal glands. Interestingly, all six dogs with hyperadrenocorticism in the present study showed a small but statistically greater response at 60 minutes with the 5 pg/kg dose of tetracosactrin than with the 250 pg dose. Although ACTH is more rapidly eliminated in dogs with pituitary- dependent hyperadrenocorticism (Greco and others 1998), no apparent change in efficacy was noted in these adrenopathic dogs. Unfortunately, the dogs with hyperadrenocorticism were not randomly assigned as in the ‘normal dog’ part of the study because they were being evaluated for inclusion in a concurrent study. The small, yet statistically significant, differ- ence in response with these dogs may be attributed to a greater glucocorticoid reserve before the first stimulation as compared with the second stimulation, although the reason for this is unknown.

Consistent with previous reports in the literature (Feldman and Nelson 1996, Van Liew and others 1997), the results of the

310 JOURNAL OF SMALL ANIMAL PRACTICF VOL 41 JULY 2000 -

ACTH stimulation test in the present study were not always diagnostic for hyperadrenocorticism. Interestingly, this was seen in only two dogs with the 250 pg dose of tetracosactrin and not in any of the dogs receiving the 5 pg/kg dose. The ACTH stimulation test has been reported to be abnormal in 80 to 85 per cent of dogs with pituitary-dependent hyperadreno- corticism (Feldman and Nelson 1996). Evaluating the 90 minute sample in the present study did not improve the diag- nostic ability of this test in the one dog in which multiple timed samples were taken.

Another application for using a lower dose of tetracosactrin might be in screen- ing dogs for hypoadrenocorticism. In humans, in addition to its use as a screen- ing test for hyperadrenocorticism, a low dose ACTH stimulation test may identify those steroid-treated patients with mild adrenal suppression at risk of developing steroid withdrawal symptoms (Dickstein and others 1991). In another study, in dogs treated with prednisone, the ACTH stimulation test (using 1 kg/kg tetracosac- trin) detected mild adrenocortical inhibi- tion (Kemppainen 1983).

porcine ACTH with synthetic ACTH in adrenal stimu

Conclusions Cortisol concentrations obtained after stimulation with 5 pg/kg tetracosactrin were similar at 60 minutes post-ACTH Veterinary Medical Association 211, 322-325

to those after stimulation with 250 pg of tetracosactrin, in both healthy dogs and those with hyperadrenocorticism. Although peak cortisol concentration was not achieved until 90 minutes using the higher dose, the 90 minute cortisol concentration was similar to the 60 minute sample result, and did not aid in the diag- nosis of hyperadrenocorticism. Therefore, 5 pg/kg tetracosactrin adequately stimu- lates the adrenal glands at 60 minutes post- stimulation in both normal dogs and those with hyperadrenocorticism.

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Acknowledgements The study was supported by the University of Tennessee College of Veterinary Medi- cine Companion Animal Fund.

References DICKSTEIN, G., SHECHNER, C., NICHOLSON, W. E., ROSNER, I.,

SHEN-ORR. Z., AOAWI. F. & LAHAV. M. (1991) Adreno- corticotropin stimulation test: effects of basal cor- tisol level, time of day, and suggested new sensitive low dose test. Journal of Clinical Endocrinology and Metabolism 72, 773-778

EILER, H. &OLIVER, J. (1980) Combined dexamethasone suppression and tetracosactrin (synthetic ACTH) stirnulation test in the dog: new approach to testing of adrenal gland function. American Journal of Veterinary Research 41, 1243-1246

FELDMAN. E. C. & NELSON, R. W. (1996) Hyperadreno- corticism. In: Canine and Feline Endocrinology and Reproduction, 2nd edn. W. B. Saunders, Philadel- phia. pp 187-265

FELDMAN, E. C., STABENFELOT, G. H., FARVER, T. B. & AODIEGO. L. A. (19821 Cornoarison of aaueous

lation tests of the female dog. American Journal of Veterinary Research 43, 522-524

FRANK, L. A. &OLIVER, J. W. (1998) Comparison of serum cortisol concentrations in clinically normal dogs after administration of freshly reconstituted versus recon- stituted and stored frozen tetracosactrin. Journal of the American Veterinary Medical Association 212, 1569-1571

GILL, G. N. (Ed) (1979) ACTH regulation of the adrenal cor- tex. In: Pharmacology of Adrenal Cortical Hormones, 2nd edn. Pergamon Press, New York. pp 35-66

GRECO, D. S., BEHREND, E. N., BROWN, S. A,. ROSYCHUK, R. A. W. & GROMAN. R. P. (1998) Pharmacokinetics of exogenous corticotropin in normal dogs, hospi- talized dogs with non adrenal illness and adreno- pathic dogs. Journal of Veterinary Pharmacology and Therapeutics 21. 369-374

HANSEN, B. L., KEMPPAINEN. R. J . & MACDONALD. J. M. (1994) Synthetic ACTH (tetracosactrin) stimulation tests in normal dogs: comparison of intravenous and intramuscular administration. Journal of the Ameri- can Animal Hospital Association 30, 38-41

KAPLAN, A. J.. PETERSON, M. E. & KEMPPAINEN, R. J . (1995) Effects of disease on the result of diagnostic tests for use in detecting hyperadrenocorticism in dogs. Journal of the American Vetennary Medical Associa- tion 207, 445-451

KEMPPAINEN, R. J. (1983) Use of a low dose synthetic ACTH challenge test in normal and prednisone-treated dogs. Research in Veterinary Science 35, 240-242

KEMPPAINEN, R. J., THOMPSON, F. N. & LORENL, M. D. (1982) Effects of dexamethasone infusion on the plasma cortisol response to tetracosactrin (synthetic ACTH) injection in normal dogs. Research in Veterinary Science 32, 181-183

KERL. M. E., PCIERSON, M. E.. WALLACE, M. S., MELIAN. C. & KEMPPAINEN, R. J. (1999) Evaluation of a low-dose syn- thetic adrenocorticotropic hormone stimulation test in clinically normal dogs and dogs with naturally devel- oping hyperadrenocorticism. Journal of the American Veterinary Medical Association 214, 1497-1501

SIGMASTAT (1994) User's Manual: Statistical Software. Jandel Scientific, San Rafael. CA 8 , 1-92

VAN LIEW. C. H., GRECO. D. S. & SALMAN. M. D. (1997) Comparison of results of adrenocorticotropic hormone stimulation and low-dose dexamethasone suppression tests with necropsy findings in dogs: 8 1 cases (1985-1995). Journal of the American

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