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Naturally occurring hypoadrenocorticism is charac-terized by a deficiency of glucocorticoids with or

without mineralocorticoid deficiency. Some of the most common clinical signs include lethargy, vomiting, diar-rhea, weakness, tremors, collapse, polyuria, and poly-dipsia.1 Because of these vague clinical signs involving many different organ systems, hypoadrenocorticism may be mistaken for other disease processes such as re-nal failure, gastrointestinal tract disease, or neurologic disease. This is of particular concern because hypoadre-nocorticism can quickly become life threatening if not recognized and treated appropriately. Therefore, adre-nal gland function testing for hypoadrenocorticism is commonly performed.

The gold standard for the diagnosis of hypoadreno-corticism is the ACTH stimulation test.2 However, there have recently been problems with this test, including high cost and intermittent availability of the drugs used.3,4 Several ACTH products approved for use in hu-

Use of basal serum or plasma cortisol concentrations to rule out a diagnosis

of hypoadrenocorticism in dogs: 123 cases (2000–2005)

Elizabeth M. Lennon, bs; Tonya E. Boyle, dvm; Rae Grace Hutchins, bs; Arit Friedenthal, bs; Maria T. Correa, phd; Sally A. Bissett, bvsc, mvsc, dacvim; Lorra S. Moses; Mark G. Papich, dvm, ms, dacvcp; Adam J. Birkenheuer, dvm, phd, dacvim

From the Departments of Clinical Sciences (Lennon, Boyle, Hutchins, Friedenthal, Bissett, Moses, Birkenheuer), Molecular and Biomedical Sciences (Papich), and Population Health and Pathobiology (Correa), College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.

Presented in part at the 2006 American College of Veterinary Internal Medicine Forum, Louisville, June 2006.

Address correspondence to Dr. Birkenheuer.

Objective—To determine whether basal serum or plasma cortisol concentration can be used as a screening test to rule out hypoadrenocorticism in dogs.Design—Retrospective case-control study.Animals—110 dogs with nonadrenal gland illnesses and 13 dogs with hypoadrenocorticism.Procedures—Sensitivity and specificity of basal serum or plasma cortisol concentrations of either ≤ 1 µg/dL or ≤ 2 µg/dL to detect dogs with hypoadrenocorticism were estimated by use of the ACTH stimulation test as the gold standard. Results—Basal cortisol concentrations of ≤ 1 µg/dL had excellent sensitivity (100%) and specificity (98.2%) for detecting dogs with hypoadrenocorticism. For basal cortisol concen-trations of ≤ 2 µg/dL, sensitivity was 100% but specificity was 78.2%.Conclusions and Clinical Relevance—On the basis of sensitivity and specificity, basal serum or plasma cortisol concentrations had high negative predictive values over a wide range of prevalence rates and can be used to rule out a diagnosis of hypoadrenocorticism. Dogs with basal cortisol concentrations > 2 µg/dL that are not receiving corticosteroids, mitotane, or ketoconazole are highly unlikely to have hypoadrenocorticism. However, if the basal cortisol concentration is ≤ 2 µg/dL, little to no information regarding adrenal gland function can be obtained and an ACTH stimulation test should be performed. (J Am Vet Med Assoc 2007;231:413–416)

mans have recently been withdrawn from the market in the United States. The only remaining FDA-approved product is a synthetic hormone, cosyntropin, which has become quite expensive.4,5 Compounded ACTH products are available from compounding pharmacies but compounded drugs are not regulated by the FDA, and these products may vary among pharmacies with respect to potency, stability, purity, quality, and duration of activity.6 Even compounded products are expensive.

It has been the dogma in veterinary medicine that basal cortisol concentrations are not clinically useful as a diagnostic test. This conclusion has largely been drawn from results of research indicating that cortisol is secreted episodically and concentrations may periodi-cally decrease to less than the reference limit or even become undetectable in clinically normal dogs.7,8 Simi-larly, dogs with hyperadrenocorticism may have basal cortisol concentrations within the reference range.9,10 Conversely, evidence from the literature indicates that dogs with hypoadrenocorticism typically have very low basal cortisol concentrations.2,11-20 On the basis of those reports, we speculated that basal cortisol concen-trations could be used as a screening test to rule out hypoadrenocorticism in dogs. The purpose of the study reported here was to estimate the sensitivity and speci-ficity of basal cortisol concentrations for detection of hypoadrenocorticism in dogs.

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Criteria for Selection of Cases

Dogs with a diagnosis of hypoadrenocorticism made between November 2000 and December 2005 were iden-tified by examining the results of all ACTH stimulation tests conducted during that period. Dogs were excluded if mitotane was administered at any time, short-acting corticosteroids were administered within 1 week, long-acting corticosteroids were administered within 3 weeks, ketoconazole was administered within 24 hours preceding the ACTH stimulation test, or corticosteroids or ketocon-azole were administered at an unspecified time prior to the ACTH stimulation test. Dogs were also excluded if the medical record stated that the ACTH stimulation test was performed to diagnose hyperadrenocorticism or if a dexa-methasone suppression test or urine cortisol-to-creatinine ratio was performed at any time.

Procedures

Medical records were reviewed, and the following variables were recorded: signalment; ACTH stimula-tion test results; and, when available, serum sodium, potassium, cholesterol, and urea nitrogen concentra-tions. Only laboratory data obtained within 7 days of the ACTH stimulation test were recorded. Plasma or serum cortisol concentrations were analyzed by use of a competitive chemiluminescence immunoassaya that has been validated for measuring canine cortisol with good precision, linearity, and recovery.21 All calibrations and quality-control sample evaluations specified by the manufacturer were performed prior to each analysis.

The chemiluminescence assay used to measure corti-sol concentration had an analytic sensitivity of 0.2 µg/dL and an intra-assay and interassay precision of ≤ 10%. The assay was linear, with a recovery of at least 100%. The assay was highly specific, with only 8.6% cross-reactivity with corticosterone, but no interference from other steroidal hormones (eg, aldosterone, cortisone, estriol, and proges-terone). The assay was not affected by bilirubin, lipemia, hemolysis, or anticoagulants (ie, heparin and EDTA).

Sensitivity and specificity of basal cortisol concen-trations of ≤ 1 µg/dL and ≤ 2 µg/dL to detect dogs with hypoadrenocorticism were estimated by comparison with results of the gold standard ACTH stimulation test. On the basis of these sensitivities and specificities, predictive values were estimated assuming disease prevalence of 0.5% and 15% in a population of 10,000 dogs. The pro-portion of dogs screened for hypoadrenocorticism on the basis of clinical suspicion that actually have the disease was estimated as 15%. This estimate was based on evalu-ation of 558 dogs in which ACTH stimulation tests were performed at the authors’ institution between November 2000 and December 2005.

Results

One hundred twenty-three dogs met the inclusion criteria for this study, including 13 dogs with hypo- adrenocorticism and 110 control dogs. Six of the 13 dogs with hypoadrenocorticism were defined as atypi-cal in that they did not have serum electrolyte abnor-malities classically identified in hypoadrenocorticism. Selected biochemical values (sodium, potassium, so-dium-potassium ratios, BUN, and cholesterol) from the cases and controls were tabulated (Table 1).

All dogs with hypoadrenocorticism had basal cortisol concentrations ≤ 1 µg/dL; in contrast, 1.8% (2/110) of the dogs with nonadrenal gland illnesses had cortisol concen-trations ≤ 1 µg/dL (Figure 1). Likewise, all dogs with hypo-adrenocorticism had basal cortisol concentrations ≤ 2 µg/dL, compared with 21.8% (24/110) of the dogs with non-adrenal gland illnesses. Sensitivity and specificity of a basal cortisol concentration ≤ 1 µg/dL for detecting dogs with hypoadrenocorticism were 100% and 98.2%, respectively. When a cortisol concentration of ≤ 2 µg/dL was evaluated, sensitivity was 100% but specificity was 78.2%.

Assuming a sensitivity of 100%, specificity of 78.2%, and a disease prevalence of 0.5%, the positive and negative predictive values were 2.3% and 100%, re-spectively. Assuming a disease prevalence of 15%, the positive and negative predictive values were 45% and 100%, respectively.

Discussion

Results of this study indicated that a basal cortisol con-centration can be used to rule out hypoadrenocorticism

Figure 1—Basal serum or plasma cortisol concentrations in 13 dogs with hypoadrenocorticism and 110 dogs with nonadrenal gland illnesses. Samples with no detectable cortisol (< 1.0 µg/dL) were plotted with a value of 0.

Sodium Potassium BUN Cholesterol (referencerange, (referencerange, (referencerange, (referencerange,Disease 147–154mEq/L) 3.9–5.2mEq/L) Na+:K+ratio 8–27mg/dL) 138–317mg/dL)

Hypoadrenocorticism n = 13 n = 13 n = 13 n = 11 n = 13 142 (124–151) 4.8 (3.4–5.8) 30 (23–42) 48.6 (14–146) 179 (76–450)

Nonadrenal gland illness n = 99 n = 99 n = 99 n = 106 n = 97 149 (134–187) 4.8 (3.2–8.2) 31 (16–43) 21 (3–126) 276 (107–786)

Table 1—Selected serum biochemical values (mean [range]) in dogs with hypoadrenocorticism or nonadrenal gland illnesses.

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in dogs that are not receiving corticosteroids, mitotane, or ketoconazole with excellent negative predictive value. The potential for severe or fatal consequences if the diag-nosis of hypoadrenocorticism is missed makes the routine use of a screening basal cortisol concentration important in any dog in which hypoadrenocorticism is a differential diagnosis. The lower cost of a basal cortisol measurement (presently $40.00 at the authors’ institution), compared with a complete ACTH stimulation test (presently $195 for a 20-kg [44-lb] dog), allows clinicians to screen more dogs routinely for hypoadrenocorticism.

In the study reported here, 6 of 13 dogs with hy-poadrenocorticism did not have alterations in serum sodium or potassium concentrations. This was in part because of the study design in which dogs that had re-ceived short- or long-acting glucocorticoids prior to hav-ing an ACTH stimulation test performed were excluded. These dogs were excluded because the glucocorticoids likely suppressed either the basal cortisol concentration or the ability to secrete cortisol in response to exogenous ACTH. This ultimately resulted in the exclusion of 6 dogs with hypoadrenocorticism, 5 of which had electro-lyte abnormalities typical of hypoadrenocorticism. Even if those dogs had been included, 37% of dogs would have been deemed atypical with regard to clinical signs. This high proportion of dogs with atypical hypoadrenocorti-cism was also likely a result of the nature of the casel-oad, which consisted almost exclusively of dogs receiv-ing secondary and tertiary care (the authors’ hospital is a referral-only institution). In addition, many private veterinary hospitals have the ability to rapidly measure serum electrolytes and many clinicians suspect the con-dition in dogs with typical electrolyte abnormalities, so presumably many dogs with hypoadrenocorticism are recognized at primary and secondary care facilities and are not referred. Many dogs that are referred are treated with fluids containing high concentrations of sodium and low concentrations of potassium, which might mask the typical electrolyte abnormalities.

Some potential limitations of this study include the small number of dogs with hypoadrenocorticism and our inability to determine in every control case wheth-er the clinician was performing the ACTH stimulation test to detect hypoadrenocorticism or hyperadreno-corticism. It was possible to ascertain from the medi-cal records that in 50% (55/110) of the control cases, suspected hypoadrenocorticism was the reason that the ACTH stimulation test was performed. The specificity of the cutoff values of ≤ 1 µg/dL and ≤ 2 µg/dL for these cases alone was 98% and 74.6%, respectively.

To compensate for the small number of cases with hypoadrenocorticism, a review of the literature report-ing basal cortisol concentrations in dogs with hypoad-renocorticism was performed. Because of the manner in which the data were presented, it was not possible to determine the exact number of dogs with basal cortisol concentrations > 1 µg/dL; however, it was possible to ascertain the number of dogs with basal cortisol con-centrations > 2 µg/dL. In 11 published studies,2,10-19 only 1.1% (6/561) of dogs with hypoadrenocorticism had basal cortisol concentrations > 2 µg/dL. These data strongly support the conclusions of the present study. These data, combined with results of the study reported

here, suggest that the sensitivity of basal cortisol con-centrations > 2 µg/dL to rule out hypoadrenocorticism is quite high (99%), but larger studies should be per-formed to confirm these findings.

Because hypoadrenocorticism is considered a rare disease and the prevalence in 1 study22 was approxi-mately 5/1,000 dogs, the positive predictive value of a basal cortisol concentration to diagnose hypoadreno-corticism is likely to be poor if the test was used as a screening test in apparently healthy dogs. Despite this, the negative predictive value should remain excellent over a wide range of prevalence rates. If basal cortisol concentration measurements were used to screen our control population of dogs that did not have hypoadre-nocorticism by use of a cutoff value of > 2 µg/dL, only 24 of 110 dogs would have required an ACTH stimula-tion test; the remainder would have been spared fur-ther testing and their owners spared the substantial additional cost. We recommend the use of this test to screen sick dogs in which there is a suspicion of hypo-adrenocorticism and an ACTH stimulation test is be-ing considered. Because of the relatively poor positive predictive value and because hypoadrenocorticism re-quires lifelong treatment and monitoring, a diagnosis of hypoadrenocorticism should always be confirmed with an ACTH stimulation test. Interpretation of the basal cortisol concentration should always be made in light of clinical signs, and if hypoadrenocorticism remains a top differential diagnosis despite a basal cortisol con-centration > 2 µg/dL, an ACTH stimulation test should be performed because in rare cases basal cortisol con-centration may be > 2 µg/dL.

Methods of cortisol measurement vary among labo-ratories. Therefore, the cutoff value to rule out hypoad-renocorticism may also vary but is likely to be close to 2 µg/dL. Further studies comparing cortisol measure-ments among laboratories would be useful.

a. IMMULITE Cortisol, Diagnostic Products Corp, Los Angeles, Calif.

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