Fourteen dogs with naturally occurring hypercortisolism (HC) were prospectively enrolled. Results of catecholamines and metanephrines:creatinine ratios before trilostane therapy of five dogs had been previously reported as part of a former study . Nine dogs were male (4 castrated) and five dogs female (4 spayed). Breeds included Dachshund (1), Giant Schnauzer (1), Nova Scotia Duck Tolling Retriever (1), Parson Jack Russell Terrier (1), Petit Bleu de Gascogne (1), Tibetan Terrier (1), Yorkshire Terrier (1), and 7 mixed-breed dogs. Age ranged between 6 and 14 years (median 8.5; standard error of mean (SEM): 0.6) and body weight between 7 and 58 kg (median 18.8; SEM: 3.7). Criteria for inclusion were the presence of clinical signs consistent with HC (e.g. polyuria, polydipsia, polyphagia, panting, skin signs, weakness, abdominal enlargement), a low-dose dexamethasone suppression test (LDDS test) or ACTH stimulation test results compatible with HC and the owner’s agreement to treat and regularly re-evaluate the dog over at least a 6-month period [9, 11]. Pituitary-dependent hypercortisolism (PDH) was diagnosed in all dogs by means of a normal or increased concentration of endogenous ACTH, bilateral symmetrical appearance of the adrenal gland determined by ultrasonography and/or demonstration of pituitary enlargement by computed tomography.
Twenty-five client-owned dogs were enrolled as controls. These dogs had been part of previous studies [9, 10, 12]. Eleven dogs were male (6 castrated) and 14 female (12 spayed), and breeds included Australian Shepherd (1), Berger Blanc Suisse (1), Bernese Mountain Dogs (2), Border Collie (1), Golden Retriever (2), Gordon Setters (2), Labrador Retriever (2), Nova Scotia Duck Tolling Retriever (1), Rhodesian Ridgeback (1), Siberian Huskies (2), Standard Poodle (1), Tervueren (1), and eight mixed-breed dogs. Age ranged between 2 and 15 years (median 7; SEM: 0.6) and body weight between 10.4 and 59.2 kg (median 26; SEM: 2.3). The dogs were considered as healthy, based on detailed information provided by their owners and the results of a physical examination, CBC, serum biochemistry profile, and urinalysis. The inclusion of the dogs in the study was approved by the veterinary office of the canton of Zurich and was in accordance with the guidelines and directives established by the Animal Welfare Act of Switzerland (TVB 199/2004).
Sample collecting and processing
All urine samples (healthy and sick dogs) used for analysis of catecholamines, metanephrines and creatinine were taken in hospital, either by free catch or by cystocentesis. In dogs with HC the first sample was taken in the morning during the initial work up for HC and once during trilostane therapy (at least 6 months after start of trilostane therapy). During trilostane therapy the urine was collected 2-3 h after the dose of trilostane in the morning. Urine collection and processing was done as reported previously [9, 12, 13]. Briefly, 10 ml of urine were placed in a silicone-coated tube containing 270-280 μL of 20% hydrochloric acid (HCl). Urinary pH was measured using pH indicator strips (range of pH 1 – 6) and HCl was added to achieve a pH ≤ 2 if needed. Samples were light protected and stored at −20 °C until analysis.
Measurement of Catecholamines and Metanephrines
Urine samples were analysed at the Institute of Clinical Chemistry, University Hospital Zurich, Zurich, Switzerland as previously described [9, 12, 13]. Urinary epinephrine, norepinephrine, total metanephrine and total normetanephrine were quantified by High Pressure Liquid Chromatography with amperometric detection as separate compounds. The terms “catecholamines” or “metanephrines” (plural form) includes epinephrine and norepinephrine or normetanephrine and metanephrine, respectively. The results were expressed as a ratio to urinary creatinine concentrations and will be listed in the following sections as: norepinephrine:creatinine, epinephrine:creatinine, normetanephrine:creatinine and metanephrine:creatinine.
Measurement of cortisol and endogenous ACTH
For the ACTH stimulation test, blood samples were taken before, and 60 min after, intravenous injection of 5 μg/kg synthetic tetracosactide (Synacthen®, Novartis Pharma Schweiz AG, Bern, Switzerland). Cortisol concentrations were measured by chemiluminescence assay (DPC Immulite® 1000, Siemens Schweiz AG, Zurich, Switzerland). The intra-assay coefficients of variation were 10% and 6.3% at cortisol levels of 74.5 and 521 nmol/L, respectively. The sensitivity of the assay was 5.5 nmol/L. Endogenous ACTH before ACTH stimulation was determined by a chemiluminescence assay (DPC Immulite® 1000, Siemens Schweiz AG, Zurich, Switzerland) previously validated for dogs [14, 15]. Blood was collected into chilled EDTA-coated tubes placed on ice and centrifuged at 4 °C. Cortisol and endogenous ACTH measurements were performed in house twice a week; plasma was stored either at −20 °C (cortisol) or at −80 °C (ACTH) until assayed.
The prospective study was performed between May 2006 and October 2014 at our hospital. The initial dose of trilostane for dogs with HC was 1-2 mg/kg q 24 h (in the morning). ACTH stimulation tests were performed prior to trilostane treatment (except for 1 dog) and at regular intervals thereafter (2, 4, 8, 12 and 16 weeks). The test was performed 2-3 h after the daily dose of trilostane according to the previously described treatment protocol . The treatment goal was to achieve a post-ACTH cortisol concentration of 41-138 nmol/L. The dose of trilostane was adjusted in dogs with post-ACTH serum cortisol >138 nmol/L or <41 nmol/L and clinical signs suggestive of hypercortisolism (polyuria, polydipsia, polyphagia) or hypocortisolism (reduced appetite, vomiting or nausea, soft feces), respectively.
Urine samples for determination of catecholamines and metanephrines were collected at diagnosis of HC and at least 6 months after starting trilostane therapy (ensuring that all dogs were clinically controlled for at least 2 months). At the re-evaluation during trilostane therapy, dogs had to be clinically controlled (polyuria, polydipsia and panting decreased, weakness and agility increased), owners had to be satisfied with the treatment outcome and post-ACTH cortisol concentrations had to be <138 nmol/L. Median time for urinary sample collection during trilostane therapy was 11 months (range: 6-40 months) after diagnosis.
Data were analyzed with non-parametric statistical methods (GraphPad Prism6, Graph Pad Software, San Diego, CA, USA, SPSS 22.0 for Windows, SPSS Inc., Chicago, IL USA). Ranges and median values are reported. The Wilcoxon signed rank test was used for comparisons between different time points, and the Mann-Whitney U-Test for comparisons between different groups. Linear correlations were calculated by Spearman’s non-parametric correlation. Values of p < 0.05 were considered significant.
Preliminary reference intervals for the urinary catecholamines and metanephrines were generated by the nonparametric method of percentile estimates with confidence intervals to determine the central 95th percentile interval (i.e. 2.5 through 97.5th percentile range) for results from clinically normal dogs . Reference intervals thus determined were: epinephrine: creatinine = 1-18; norepinephrine: creatinine = 1-19; metanephrine: creatinine = 12-255; normetanephrine: creatinine = 14-123.