Introduction
Naturally occurring hypoadrenocorticism (HA), commonly termed Addison’s disease, is a life-threatening endocrine disorder that requires an accurate and timely diagnosis to ensure successful outcomes.1 Hypoadrenocorticism in dogs is usually a result of primary adrenal gland failure, which is presumably due to immune-mediated destruction of the adrenal cortices.2,3 Most affected dogs are deficient in both cortisol and aldosterone, and these hormonal deficiencies can result in a multitude of clinical, hematologic, and biochemical abnormalities.4–6 Some dogs will present in hypovolemic shock with life-threatening electrolyte and acid-base disturbances, whereas other affected dogs have an insidious presentation in which chronic waxing and waning gastrointestinal signs and lethargy are often reported.4,7 The spectrum of clinical features of HA overlap with the clinical features of cardiovascular, urinary, and gastrointestinal diseases.1,4,8 As such, diagnostic testing for HA is frequently pursued in veterinary medicine, especially in emergency and referral centers.4
The ACTH stimulation test, which is performed by measuring serum or plasma cortisol concentrations before and 1 hour after IV administration of synthetic ACTH, is the gold standard for HA diagnosis in dogs.1 The ACTH stimulation test is accurate, and equivocal results are uncommon.9 However, the synthetic ACTH preparations used in testing are expensive and not readily available to some practitioners.1,10 These concerns have led to investigations of alternative diagnostic methods including basal cortisol concentrations, cortisol-to-ACTH ratios, aldosterone-to-renin ratios, urine electrolytes, a machine learning algorithm, and various integrations of routine hematologic and serum biochemistry results, among others.9–19 Most alternative tests have been either unreliable for HA diagnosis or met with other limitations, including difficulties with sample processing or assay availability.10–19 One commonly used alternative test is measurement of serum or plasma basal cortisol concentrations, which are highly sensitive for HA diagnosis.9,13,16 Basal cortisol concentrations are used to screen dogs for HA, especially in cases with a low to moderate index of suspicion for disease.1,13 However, cortisol secretion in dogs is episodic, and the reported specificities of basal cortisol concentrations at traditionally utilized cutpoints are not acceptable for confirming a diagnosis of HA.13,16,20
A test capable of assessing cortisol concentrations over a longer time period might offset the limitations of a single basal cortisol measurement. The urine cortisol-to-creatinine ratio (UCCR) provides an indirect assessment of circulating cortisol concentrations over a period of time and is less likely to be affected by rapid fluctuations in cortisol secretion.21 The UCCR is a screening test for hyperadrenocorticism in dogs, but it could have value for HA diagnosis as well.22–24 The UCCR only requires a single urine specimen, is less expensive than ACTH stimulation testing, and is a widely available test.25,26 The purpose of our study was to evaluate the UCCR for HA diagnosis. We also sought to determine diagnostic performance when urine cortisol concentrations for the UCCR were determined by either radioimmunoassay (RIA) or chemiluminescent immunoassay (CLIA), as these methodologies are often used in research settings and commercial diagnostic laboratories. We hypothesized that the UCCR would be a useful alternative test for HA diagnosis.
Materials and Methods
Study design
Dogs undergoing testing for HA with measurement of a basal serum cortisol concentration or an ACTH stimulation test were identified for potential inclusion in a prospective study evaluating the UCCR as a diagnostic test for HA. There were no requirements based on age, breed, or sex. Dogs in which cortisol measurements were obtained for assessment of possible hyperadrenocorticism were not eligible for inclusion nor were dogs that had been previously treated for hyperadrenocorticism. Dogs that had received exogenous glucocorticoids or azole antifungals in the 4 weeks prior to evaluation also were excluded.27–29 Urine specimens that were collected as part of the normal clinical evaluation in the 24 hours preceding serum cortisol measurements were used for UCCR determinations. If urine had not been obtained during this time period or if insufficient urine volume was available, then a urine specimen was obtained at the time of blood collection for serum cortisol measurements. Urine samples were collected from participating dogs by cystocentesis or natural voiding based on patient factors and clinician preference. The ACTH stimulation tests were performed by measuring serum cortisol concentrations immediately before and 1 hour after IV administration of 5 µg/kg of synthetic ACTH (Cosyntropin). Additional diagnostic testing and treatments were at the discretion of attending clinicians and were independent of study participation.
Case classification
Dogs were diagnosed with HA if both pre- and post-ACTH–stimulated serum cortisol concentrations were ≤ 55 nmol/L (2 µg/dL).1 A basal cortisol concentration > 55 nmol/L (2 µg/dL) or post-ACTH–stimulated cortisol concentrations > 138 nmol/L (5 µg/dL) were considered to be indicative of nonadrenal illness (NAI).9,13 Basal cortisol concentrations are not used for HA confirmation, but a basal cortisol concentration > 55 nmol/L (2 µg/dL) can be used to exclude a diagnosis of HA because it has a negative predictive value ≥ 99.8% across a wide range of prevalences.9,13,16 Dogs with basal cortisol concentrations ≤ 55 nmol/L (2 µg/dL) that did not have a follow-up ACTH stimulation test or dogs that had post-ACTH–stimulated cortisol concentrations between 55 and 138 nmol/L (2 and 5 µg/dL) were considered to have equivocal results and excluded from analysis.9,13,16 This project was approved by the Michigan State University Institutional Animal Care and Use Committee, and informed consent was obtained from owners of participating dogs.
Laboratory methods
All laboratory tests were conducted at the Michigan State University Veterinary Diagnostic Laboratory, which is an American Association of Veterinary Laboratory Diagnosticians–accredited laboratory. Serum cortisol concentrations were measured with a commercially available competitive CLIA (Immulite 2000 Cortisol; Siemens Healthcare Diagnostics Ltd) that has been described in our laboratory and others.9,13,16 Urine creatinine concentrations were measured in urine supernatant (centrifugation at 1,700 X g for 10 minutes) by the Jaffe method using a commercially available clinical chemistry analyzer (Beckman AU680; Beckman Coulter Inc). Urine cortisol concentrations were quantified in unextracted urine by use of either a commercially available 125I RIA kit (ImmuChem Coated Tube Cortisol; MP Biomedicals LLC) or the same CLIA used for serum cortisol measurements. Descriptions of the RIA and CLIA methodologies for urine cortisol determinations are available online (Supplementary Appendix S1), and additional descriptions of the CLIA methodology can be found elsewhere.30 Urine cortisol concentrations were determined by both RIA and CLIA for each case when possible, but specimen volume and availability of kits and reagents affected whether both assays could be performed. The UCCR was calculated by dividing the urine cortisol concentration (nmol/L) by the urine creatinine concentration (mmol/L) without adjusting for the units of measure, with the result rounded to the nearest integer (eg, a urine cortisol concentration of 47 nmol/L and a urine creatinine concentration of 22 mmol/L would result in a UCCR of 2).
Data and statistical analysis
The urine cortisol and UCCR data did not follow a normal distribution as assessed by Shapiro-Wilk testing and box-plot analysis and were reported as median and interquartile (25th to 75th percentile) range (IQR). Potential differences in urine cortisol concentrations between the RIA and CLIA methods were evaluated using a Wilcoxon signed rank test. Spearman rank correlation coefficients (ρ) were calculated to evaluate the relationship between RIA urine cortisol measurements and CLIA urine cortisol measurements. Characteristics of HA and NAI groups were compared using the Mann-Whitney U test or Fisher exact test as appropriate. The UCCR data were compared between HA and NAI dogs for each method of analysis (RIA or CLIA) using a Mann-Whitney U test. Commercially available software was used to construct receiver operating characteristics (ROC) curves, which plot the sensitivity against (1 – specificity) for all values of the studied variable, and to determine area under the ROC curves for UCCR diagnosis of HA. Sensitivity, specificity, and test accuracy (95% CIs) of selected UCCR cutpoints for the diagnosis of HA were calculated. Positive and negative predictive values were estimated using 2 previously reported prevalences.9,13 For all these assessments of UCCR diagnostic performance, the results of basal serum cortisol concentrations or ACTH stimulation tests were used as the reference standard as described in the case classification section. Statistical analyses were performed using commercially available software (GraphPad Prism version 6.0; GraphPad Software Inc), and for all analyses, values of P ≤ 0.05 were considered significant.
Results
One hundred fifty-six dogs were screened for inclusion, 8 of which were excluded from analysis because of equivocal test results. A total of 148 dogs, including 41 HA dogs and 107 NAI dogs, were included in the study. Characteristics of the HA and NAI groups are summarized (Table 1); age, sex, and neuter status of HA and NAI groups were not significantly different, although body weight was higher in the HA dogs. Thirty-nine of 41 HA dogs had classic electrolyte abnormalities associated with aldosterone deficiency including hyponatremia or hyperkalemia or both, whereas 2 HA dogs had normal electrolyte concentrations at the time of HA diagnosis. The most common diseases identified in NAI dogs included primary gastrointestinal disease (n = 53), kidney disease (13), esophageal disease (9), acute pancreatitis (9), and hepatobiliary disease (8); other disease processes were represented in ≤ 2 cases.
Characteristics of the dogs with hypoadrenocorticism (HA; n = 41) and nonadrenal illness (NAI; 107) included in the study.
| Characteristic | HA | NAI | P value |
|---|---|---|---|
| Median (IQR) age (y) | 4 (2–6) | 5 (2–9) | 0.095 |
| No. of males/females | 22/19 | 53/54 | 0.715 |
| No. of SI/neutered | 3/38 | 18/89 | 0.190 |
| Median (IQR) weight (kg) | 25.7 (17.4–33.4) | 19.6 (8.9–33) | 0.044 |
IQR = Interquartile (25th to 75th percentile) range. SI = Sexually intact.
Urine cortisol RIA versus CLIA
Of the 148 study dogs, 114 had urine cortisol concentrations determined by RIA, 109 had urine cortisol concentrations determined by CLIA, and 75 dogs had urine cortisol concentrations determined by both RIA and CLIA. Urine cortisol concentrations were strongly and positively correlated (ρ = 0.97; P < 0.001) in the 75 dogs in which both RIA and CLIA measurements had been performed, but urine cortisol concentrations were significantly higher when measured by CLIA as compared to RIA (Figure 1).
Scatterplots of urine cortisol concentrations in the 75 dogs in which these values were determined by both radioimmunoassay (RIA) and chemiluminescent immunoassay (CLIA) methods. The horizontal line within each plot represents the median. Urine cortisol concentrations as determined by CLIA (median, 433 nmol/L; interquartile range, 98 to > 1,380 nmol/L were higher than when determined by RIA (median, 85 nmol/L; interquartile range, 5 to 476 nmol/L). *P < 0.001.
Citation: Journal of the American Veterinary Medical Association 260, 9; 10.2460/javma.21.12.0538
UCCR for HA diagnosis
The UCCRs were significantly higher in NAI dogs (median, 22; IQR, 7 to 47.3) than in HA dogs (0, 0 to 0) when urine cortisol was determined by RIA (P < 0.001). The UCCRs also were significantly higher in NAI dogs (median, 71; IQR, 30 to 127) than in HA dogs (1; 0 to 1.3) when urine cortisol was determined by CLIA (P < 0.001). The area under the ROC curve demonstrating the performance of the UCCR for HA diagnosis was 0.99 (95% CI, 0.98% to 1.00%) when urine cortisol was measured by RIA and 1.0 (95% CI, 1.0 to 1.0) when urine cortisol was measured by CLIA (Figure 2). The most accurate cutpoint identified on the RIA ROC curve was a UCCR of ≤ 2, which was 97.2% sensitive (95% CI, 85.5% to 99.9%) and 93.6% specific (95% CI, 85.7% to 97.9%) for HA diagnosis. The most accurate cutpoint identified on the CLIA ROC curve was a UCCR of ≤ 10, which was 100% sensitive (95% CI, 84.6% to 100%) and 100% specific (95% CI, 95.9% to 100%). The sensitivity, specificity, and accuracy of these and additional selected cutpoints were tabulated (Table 2), and positive and negative predictive values were summarized (Table 3). A UCCR > 4 as determined by RIA or a UCCR > 10 as determined by CLIA both had negative predictive values of 100% when assuming a 3% prevalence of HA.
Receiver operating characteristic curves (dogs with hypoadrenocorticism vs dogs with nonadrenal illness) of the urine cortisol-to-creatinine ratio (UCCR) for the diagnosis of hypoadrenocorticism when urine cortisol concentrations were measured by RIA in 114 dogs (A) and CLIA in 109 dogs (B). The thin diagonal line displayed in each panel represents an uninformative test, wherein the area under the curve is 0.50. The area under the receiver operating characteristics curves for the RIA UCCR and CLIA UCCR determinations, which are represented by the thicker lines in each panel, were 0.99 (95% CI, 0.98 to 1.00) and 1.0 (95% CI, 1.0 to 1.0), respectively. See Figure 1 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 260, 9; 10.2460/javma.21.12.0538
Sensitivities (%), specificities (%), and accuracies (%) of selected urine cortisol-to-creatinine ratio (UCCR) cutpoints for the diagnosis of HA by means of radioimmunoassay (RIA) or chemiluminescent immunoassay (CLIA).
| Assay | Cutpoint | Sensitivity (95% CI) | Specificity (95% CI) | Accuracy (95% CI) |
|---|---|---|---|---|
| RIA | ≤ 0 | 86.1 (70.5–95.3) | 100 (95.4–100) | 95.6 (90.1–98.6) |
| ≤ 1 | 91.7 (77.5–98.3) | 96.2 (89.2–99.2) | 94.7 (88.9–98.0) | |
| ≤ 2 | 97.2 (85.5–99.9) | 93.6 (85.7–97.9) | 94.7 (88.9–98.0) | |
| ≤ 4 | 100 (90.3–100) | 82.1 (71.7–89.8) | 87.7 (80.3–93.1) | |
| ≤ 10 | 100 (90.3–100) | 69.2 (57.8–79.2) | 79.0 (70.3–86.0) | |
| CLIA | ≤ 1 | 77.3 (54.6–92.2) | 100 (95.9–100) | 95.4 (89.6–98.5) |
| ≤ 6 | 95.5 (77.2–99.9) | 100 (95.9–100) | 99.1 (95.0–100) | |
| ≤10 | 100 (84.6–100) | 100 (95.9–100) | 100 (96.7–100) | |
| ≤ 13 | 100 (84.6–100) | 98.9 (93.8–100) | 99.1 (95.0–100) | |
| ≤ 18 | 100 (84.6–100) | 90.8 (82.7–96.0) | 92.7 (86.1–96.8) |
The performance metrics for the UCCR when urine cortisol was measured by RIA were calculated using data from 36 dogs with HA and 78 dogs with NAI. The performance metrics for the UCCR when urine cortisol was measured by CLIA were calculated using data from 22 dogs with HA and 87 dogs with NAI.
See Table 1 for remainder of key.
Positive and negative predictive values (%) of selected UCCR cutpoints for the diagnosis of HA at 2 prevalences of the disease.
| 3% prevalence | 21% prevalence | ||||
|---|---|---|---|---|---|
| Assay | Cutpoint | PPV | NPV | PPV | NPV |
| RIA | ≤ 0 | 100 | 99.6 | 100 | 96.4 |
| ≤ 1 | 42.4 | 99.7 | 86.4 | 97.8 | |
| ≤ 2 | 31.9 | 99.9 | 80.1 | 99.2 | |
| ≤ 4 | 14.7 | 100 | 59.7 | 100 | |
| ≤ 10 | 9.1 | 100 | 46.4 | 100 | |
| CLIA | ≤ 1 | 100 | 99.3 | 100 | 94.3 |
| ≤ 6 | 100 | 99.9 | 100 | 98.8 | |
| ≤ 10 | 100 | 100 | 100 | 100 | |
| ≤ 13 | 72.9 | 100 | 95.9 | 100 | |
| ≤ 18 | 25.2 | 100 | 74.3 | 100 | |
Although not included in statistical analyses, 8 dogs had equivocal HA testing results. All 8 dogs had baseline serum cortisol concentrations ≤ 55 nmol/L (2 µg/dL). Six of these dogs did not have follow-up ACTH stimulation testing performed; the remaining 2 dogs had post-ACTH–stimulated cortisol concentrations between 55 and 138 nmol/L (2 and 5 µg/dL). The UCCR was determined by CLIA in all 8 dogs, and the median value was 18 (range, 5 to 56). One dog with a UCCR of 5 had a baseline cortisol concentration of 41 nmol/L and was azotemic, hyponatremic, and hyperkalemic. This dog underwent 2 ACTH stimulation tests 2 weeks apart; post-ACTH–stimulated cortisol concentrations were equivocal both times (66 and 69 nmol/L, respectively). Another dog with a UCCR of 6 had an undetectable baseline cortisol concentration (< 5.5 nmol/L) and an equivocal post-ACTH–stimulated cortisol concentration (103 nmol/L). This dog had an acute onset of gastrointestinal signs that responded to supportive care. The other 6 dogs with equivocal disease status had UCCR values > 13. Five of these dogs had gastrointestinal signs; the other dog not only had lower urinary tract signs but also eosinophilia and lymphocytosis noted on hematologic evaluation.
Discussion
The UCCR has been used for decades to screen dogs for hyperadrenocorticism because it is inexpensive, widely available through commercial diagnostic laboratories, and highly sensitive for detecting excess circulating cortisol concentrations.24–26 Results of the present study suggested that the UCCR could also have value as a diagnostic test for HA in dogs. The UCCR discriminated between HA and NAI in nearly all cases in this study population. The areas under the ROC curves were excellent for both RIA and CLIA methodologies, and several cutpoints were highly sensitive, specific, and accurate for HA diagnosis.
The current standard for HA diagnosis in dogs is the ACTH stimulation test, but testing expense and inconsistent availability of synthetic ACTH preparations have prompted multiple investigations of alternative diagnostic tests in recent years.9–18 Only measurements of baseline cortisol concentrations, and to a lesser extent the cortisol-to-ACTH ratio, have been used with any regularity in clinical practice.9,10,13,16,31 The lack of specificity of baseline cortisol concentrations for HA diagnosis at a clinically utilized cutpoint limits use of this test to screening purposes.9,13,16 The cortisol-to-ACTH ratio is both sensitive and specific.10 However, some overlap exists between HA and NAI dogs, and few commercial laboratories offer endogenous ACTH measurements.31 The performance characteristics of the UCCR for HA diagnosis established in our study were encouraging, but these results should not be interpreted as an implication that the UCCR is equivalent to the ACTH stimulation test. Hypoadrenocorticism is a life-threatening disease, treatment is life-long, and medication costs are substantial; these factors must be considered when determining the utility of a test for HA.1,5,7
The overall diagnostic performance of the UCCR for HA diagnosis when urine cortisol concentrations were measured by RIA or CLIA was similar. However, some overlap in UCCR values was observed between HA and NAI dogs when urine cortisol was measured by RIA, whereas no overlap in UCCR values was observed with the CLIA methodology. Although these appeared to be minor differences, any overlap limits clinical usefulness given the consequences of a misdiagnosis. The specificity of the RIA UCCR of 0 was 100%, but this dropped to 93.6% at a cutpoint of ≤ 2. More importantly, the positive predictive value drops from 100% to somewhere between 30% and 80% depending on HA prevalence. Perhaps the primary clinical value of the RIA UCCR would be disease screening as sensitivity and negative predictive values remain near 100% across a range of values. Conversely, the CLIA UCCR appeared to better discriminate between HA and NAI populations. The CLIA UCCR cutpoint of ≤ 10 was 100% accurate for HA diagnosis, which suggests that the CLIA UCCR might have clinical value beyond solely screening for disease. A larger study sample would be needed to increase the confidence of this finding, and a safer interim approach would be to interpret UCCR values near this cutpoint as equivocal results. Over 95% of HA dogs had a CLIA UCCR < 6, and over 98% of NAI dogs had a CLIA UCCR > 13. As such, the vast majority of cases would still be accurately classified if values between 6 and 13 were considered inconclusive. Even if the CLIA UCCR was not used for confirmatory purposes, it would still be an excellent screening test. This is in agreement with previous suggestions that the UCCR may be superior to measurements of baseline cortisol concentrations to screen dogs for HA.23
The differences in urine cortisol concentrations between methodologies were another notable finding. Many commercial diagnostic laboratories have transitioned to a CLIA for cortisol measurements in recent years, and a validation study of the commonly used CLIA methodology for unextracted canine urine was recently published.30,32 Paired RIA and CLIA measurements were strongly correlated in our study, but results obtained with CLIA were significantly higher than those with RIA. This contrasts with serum cortisol measurements, which are similar between RIA and CLIA methods, yet is in agreement with previous research in which immunoassays had higher results than other methods when determining urine cortisol concentrations.9,33 Urine cortisol concentrations were determined in unextracted urine in our study, whereas some laboratories perform extraction steps using organic solvents to minimize the influence of cortisol metabolites.33,34 Perhaps the CLIA values would have been closer to the RIA values if extracted urine were used, but it is unclear whether extraction actually improves diagnostic performance.33 The analytical specificity of the anti-cortisol antibodies used in an assay also influences the degree of cross-reactivity with conjugated corticosteroid metabolites and is likely responsible for some of these differences as well as differences among immunoassays.34 The anti-cortisol antibody used in the CLIA assay was recently changed by the manufacturer, which indicates that the results reported herein should not be broadly applied to any laboratory using CLIA methodologies.30 Some differences in cortisol measures can even occur among laboratories when using the same assays and reagents.35 As such, it is important that both methodology of cortisol measurement and laboratory-specific reference intervals and cutpoints are considered when interpreting UCCR results.
Urine specimens were collected in the hospital for UCCR determinations in all study dogs, which is different than the recommended approach of collecting urine samples at home when screening for hyperadrenocorticism.22,24 At-home collection lessens patient stress and false-positive UCCR results when screening for hyperadrenocorticism.36 One potential study limitation was that possible effects of at-home collection on UCCR accuracy when testing for HA are not known. The collection methods in the present study would likely mimic those in clinical practice, in which dogs undergoing HA testing are often evaluated on an urgent or emergent basis. Any added physiologic stress associated with in-hospital urine collection might be beneficial by further magnifying the differences between HA and NAI dogs. Another potential study limitation was that the disease status of several study dogs could not be determined. This could have affected sensitivity and specificity calculations. Both HA and NAI likely were represented in these 8 dogs based on the range of CLIA UCCR values, but follow-up information was unavailable. This limitation provides further support for interpreting CLIA UCCR values near the cutpoint of 10 as inconclusive results. Finally, a larger sample size would have strengthened study conclusions, and additional investigations that include greater numbers of dogs with HA and NAI are warranted.
In summary, we documented that the UCCR is a useful test to aid in the diagnosis of HA in dogs. At a minimum, the UCCR was an extremely sensitive test that could be used to screen dogs for HA. The high specificities also suggested that the UCCR could be used to confirm an HA diagnosis in some cases when ACTH stimulation testing is not possible, but larger studies are needed to corroborate this finding. Clinicians must be cognizant of the methodology of cortisol measurement and laboratory-specific reference intervals when interpreting results.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org
Acknowledgments
Funded by the Michigan State University College of Veterinary Medicine Trinket Fund.
The Michigan State University Veterinary Diagnostic Laboratory offers commercial laboratory services including serum and urine cortisol measurements. The authors declare that there were no additional potential conflicts of interest.
The authors thank Drs. Linda Okonkowski, Alysha Vincent, and Casey Dropkin for assistance with sample collection and processing, and Judy Eastman, Caitlyn Beets, and Enass Bassiouny for technical support.
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