The acute effects of a protein-rich meal on the urinary corticoid:creatinine ratio in healthy dogs.

The objective of this prospective crossover study was to investigate the effects of a high-protein diet on canine urinary corticoid-to-creatinine ratio (UCCR). The hypothesis was that meal-induced hypercortisolism is, as has been shown in humans, a predictable and consistent finding in healthy dogs. Eight clinic-owned beagles were randomly assigned to one of two groups. The allocation to the groups defined the sequence of a protein-enriched meal (meal A) or no meal on the first and second days, whereas on the third day all dogs again received an identical meal (meal B) to test reproducibility. Urinary corticoids were measured using a solid-phase, competitive CLIA on unextracted urine. Contrary to our expectations, consistent incremental responses of the UCCR were not observed (meal A vs. no meal [anova]: absolute increase, F = 2.546, p = 0.162; relative increase, F = 4.084, p = 0.09; AUC(UCCR) , F = 0.279, p = 0.616). Nevertheless, the robust increases in two dogs above 60% of baseline suggest that the collection of urine prior to feeding likely increases the specificity of the UCCR to discriminate between dogs with and without hypercortisolism.

Diagnostic efficacy of the leukogram and the chemiluminometric ACTH measurement to diagnose canine hypoadrenocorticism.

BACKGROUND AND OBJECTIVE: The gold standard in the diagnosis of canine hypoadrenocorticism (HA) is the adrenocorticotropin (ACTH) response test. As synthetic ACTH (tetracosactide [Synacthen®]) is currently not available in the European Union, the evaluation of other diagnostic tests seemed warranted. The diagnostic efficacy of electrolytes, the leukogram and endogenous ACTH concentrations to diagnose HA was investigated. MATERIAL AND METHODS: The medical records of 145 dogs with clinical signs suspect for spontaneous HA were included in a retrospective study. HA was diagnosed (n = 38) or ruled out (n = 84) by using an ACTH response test. In 23 patients HA was excluded by basal cortisol measurement. The diagnostic performance of various variables was assessed based on receiver operating characteristic (ROC) curves and by calculating differential positive rates. A decision tree (IBM SPSS Decision Trees 20, IBM Corporation) was constructed with the variables neutrophil to lymphocyte ratio (N/LR) and sodium to potassium ratio (Na/KR) to illustrate the diagnostic efficacy of the respective test results. RESULTS: The best single variables to diagnose HA were the endogenous ACTH concentration (area under the ROC curve [ROC AUC] 0.97; cutoff > 50 pmol/l: sensitivity 96%, specificity 100%) and the Na/KR (ROC AUC 0.905; cutoff ≤ 22: sensitivity 92%, specificity 91%). The diagnostic performance of various variables of the leukogram was poor to moderate (ROC AUC 0.625-0.828). 68% of dogs with HA had a Na/KR ≤ 22 and a N/LR ≤ 2.3, a combination not observed in dogs with non-adrenal diseases. CONCLUSION AND CLINICAL RELEVANCE: As secondary HA is very rare, endogenous ACTH measurement is a very good alternative to the traditional ACTH response test. Data also suggest that the combination of a Na/KR ≤ 22 and a N/LR ≤ 2.3 is highly specific and can be used to rule in HA.

[Buccal glucose measurements in dogs using safety lancets for blood sampling]. / Bukkale Glukosemessung beim Hund unter Probengewinnung mit Sicherheitslanzetten.

OBJECTIVE: Glucose measurements at the pinna or paws are an important part of the management of diabetic dogs. Despite extensive rubbing or warming of the testing site, an adequate blood sample cannot always be obtained. Therefore, the objective of this study was to evaluate the lip as an alternative sampling site with respect to practicability and tolerance by the dogs and reliability of the measured values. MATERIAL AND METHODS: Nine clinic-owned male-castrated healthy beagles were included in this prospective study. Glucose measurements were performed using a glucometer developed for veterinary use, before and 15 minutes after oral application of glucose syrup. Capillary blood was collected using two commercially available safety lancets (Wellion Safety lancet [23 gauge] and Wellion Safety lancet special [blade 0.8 mm]) at the buccal mucosa of the upper lip and at the pinna. Measured values were compared with those obtained from venous plasma (reference method). RESULTS: Buccal glucose measurements were easy to perform and were well tolerated by the dogs without obvious pain reactions. There was no difference between the glucose concentrations from the lip samples and those determined using the reference method (p = 0.793, F < 1). The glucose application had no effect on the observed differences (reference value minus glucometer value; p = 0.63, F < 1). However, in one dog, a clinically problematic discrepancy of 3.6 mmol/l was observed. The special lancet caused noticeable secondary bleeding in two dogs. CONCLUSION AND CLINICAL RELEVANCE: The results of this study indicate that the buccal mucosa is a convenient and reliable alternative sampling site for glucose measurements in dogs. Problematic deviations from reference values are possible after oral glucose application and the use of blade-like needles is not encouraged.

Configuration of antibodies for assay of urinary cortisol in dogs influences analytic specificity.

Whether the variation in the reported urinary corticoid-to-creatinine ratio in dogs is affected by the application of 2 commonly applied anticortisol antibodies was investigated. Free-catch morning urine samples of 50 healthy dogs were analyzed in duplicate with the use of 2 different polyclonal antibodies (antibody A and B) raised in different rabbits. Antibody A was raised against cortisol-3-carboxymethyl-oxime and antibody B against cortisol-21-hemisuccinate linked to BSA. Enzyme immunoassays were applied by using corresponding biotinylated labels. To examine possible cross-reactions with conjugated and nonconjugated cortisol metabolites, EIA measurements were performed with urine samples both before (directly assayed) and after diethyl-ether extraction, as well as after reversed-phase HPLC. Although the results correlated (P < 0.001), urinary corticoid concentrations and accordingly the urinary corticoid-to-creatinine ratios were 8 times higher when using antibody A than when using antibody B (mean ± SD corticoid concentrations, 223 ± 131 vs 29 ± 12 nmol/L; P < 0.001). Irrespective of the antibody used, extraction significantly decreased measured corticoid concentrations (antibody A, 158 ± 120 nmol/L; antibody B, 15 ± 8 nmol/L; P < 0.001), but the decrease was conspicuous when antibody A was used. Antibody A cross-reacted significantly with polar (eg, conjugated) metabolites, clearly depicted in the chromatogram by 3 additional peaks in earlier fractions well separated from cortisol. In contrast the assay that used antibody B was specific, showing only 1 major peak in the fractions eluting authentic cortisol. In summary, the study indicates that the configuration of the antibody considerably influences the analytic specificity of cortisol assays and underlines the pivotal importance of assay validation for each species and sample material.

Hyperthyroidism in dogs caused by consumption of thyroid-containing head meat.

Two female spayed dogs belonging to the same owner were admitted for further examinations because of clinical signs and laboratory values compatible with hyperthyroidism. Sonography of the ventral aspect of the neck revealed small thyroid glands in both dogs. The hypothesis that the dogs suffered from alimentary hyperthyroidism caused by feeding head meat containing thyroid gland tissue was confirmed by consultation of the slaughtering plant, determination of iodine concentrations in deep-frozen samples and hormone measurements in 5 other dogs receiving head meat from the same supplier. After changing the diet, thyroxine concentrations declined and clinical signs were no longer observed.

Point-of-care ß-hydroxybutyrate measurement for the diagnosis of feline diabetic ketoacidaemia.

OBJECTIVES: To evaluate accuracy and precision of a hand-held ketone meter measuring ß-hydroxybutyrate and to determine its diagnostic performance to rule out ketoacidaemia in diabetic cats. METHODS: The ketone meter was validated by calculating within-day precision at different ß-hydroxybutyrate concentrations and by comparison with a laboratory method. To determine its diagnostic performance to diagnose ketoacidaemia, 217 sets of data (venous blood gas analysis and ß-hydroxybutyrate measurements) were retrospectively analysed. Sensitivities and specificities were calculated with the help of receiver-operating characteristic curves. RESULTS: The ketone meter reliably detected ß-hydroxybutyrate at concentrations >0·1 mmol/L and reproducibility was acceptable. Measurements highly correlated with laboratory results (r=0·97; P<0·001), but a significant negative bias was found at high concentrations. A ß-hydroxybutyrate concentration of >2·55 mmol/L had a sensitivity of 94% and a specificity of 68% for diagnosing ketoacidaemia. Many cats with high ß-hydroxybutyrate concentrations and normal blood pH had an elevated chloride gap suggestive of superimposed hypochloraemic metabolic alkalosis. CLINICAL SIGNIFICANCE: The commercially available point-of-care ketone meter Precision Xtra is a valid tool to measure ß-hydroxybutyrate in diabetic cats. Concentration <2·55 mmol/L enable ketoacidaemia to be excluded and should lead to redirection of differential diagnoses.

The influence of trilostane on steroid hormone metabolism in canine adrenal glands and corpora lutea-an in vitro study.

Trilostane is widely used to treat hyperadrenocorticism in dogs. Trilostane competitively inhibits the enzyme 3-beta hydroxysteroid dehydrogenase (3ß-HSD), which converts pregnenolone (P5) to progesterone (P4) and dehydroepiandrosterone (DHEA) to androstendione (A4). Although trilostane is frequently used in dogs, the molecular mechanism underlying its effect on canine steroid hormone biosynthesis is still an enigma. Multiple enzymes of 3ß-HSD have been found in humans, rats and mice and their presence might explain the contradictory results of studies on the effectiveness of trilostane. We therefore investigated the influence of trilostane on steroid hormone metabolism in dogs by means of an in vitro model. Canine adrenal glands from freshly euthanized dogs and corpora lutea (CL) were incubated with increasing doses of trilostane. Tritiated P5 or DHEA were used as substrates. The resulting radioactive metabolites were extracted, separated by thin layer chromatography and visualized by autoradiography. A wide variety of radioactive metabolites were formed in the adrenal glands and in the CL, indicating high metabolic activity in both tissues. In the adrenal cortex, trilostane influences the P5 metabolism in a dose- and time-dependent manner, while DHEA metabolism and metabolism of both hormones in the CL were unaffected. The results indicate for the first time that there might be more than one enzyme of 3ß-HSD present in dogs and that trilostane selectively inhibits P5 conversion to P4 only in the adrenal gland.

[Diagnostic efficacy of plasma ACTH-measurement by a chemiluminometric assay in canine hyperadrenocorticism]. / Klinische Anwendung einer chemiluminometrischen ACTH-Bestimmung beim caninen Hyperadrenokortizismus.

This retrospective study was performed to investigate the diagnostic efficacy of the chemiluminometric ACTH-measurement to differentiate between pituitary and adrenal dependent hyperadrenocorticism (HAC) in dogs. 49 dogs with pituitary HAC, 10 dogs with adrenal HAC and 1 dog with a combination of both pathologies were included. Dogs with HAC like symptoms, where HAC had been ruled out, served as controls (n = 18). All dogs with adrenal HAC, as well as 9 dogs with pituitary HAC had an ACTH concentration below the detection limit of 2.2 pmol/l (10 pg/dl) plasma. Using 2.2 pmol/l as a cut-off the sensitivity and specificity to diagnose pituitary HAC was 0.82 (95 % CI 0.686 - 0.914) and 1 (95 % CI 0.692 - 1), respectively. With the help of the chemiluminometric assay, a correct classification was possible in 85 % of patients with HAC. As an ACTH-concentration below the detection limit was found in dogs with adrenal as well as pituitary HAC, additional discriminatory tests are necessary in these cases.

Sudden death in a dog with lymphoplasmacytic hypophysitis.

A 10-year-old dog with a history of progressive anorexia and weight loss died suddenly despite treatment. Histopathological examination revealed severe follicular lymphoplasmacytic adenohypophysitis and atrophy of the zona fasciculata and zona reticularis of the adrenal cortex. It is likely that lack of production of adrenocorticotropic hormone and cortisol was the cause of death of this dog.

Tailored reference limits for urine corticoid:creatinine ratio in dogs to answer distinct clinical questions.

To establish reference intervals for the urinary corticoid:creatinine ratio (UCCR) determined by chemiluminometric immunoassay, UCCR was measured by this method in 50 healthy dogs. To assess the diagnostic performance of different cut-off levels, the UCCR of 66 dogs with hyperadrenocorticism and 87 dogs with diseases mimicking hyperadrenocorticism were used to construct a receiver operating characteristic (ROC) curve. The upper reference limit derived from morning samples in healthy dogs was 30.81 × 10(-6). The area under the ROC curve was 0.94. The diagnostic cut-off with the highest negative likelihood ratio was 26.5 × 10(-6) (sensitivity 1, specificity 0.54), whereas the cut-off with the highest positive likelihood ratio was 161.2 × 10(-6) (specificity 0.988, sensitivity 0.515). The application of these two different diagnostic cut-offs eliminated the necessity to perform additional tests in 53 per cent of the patient population.

Ketone measurements using dipstick methodology in cats with diabetes mellitus.

OBJECTIVES: To compare the results of urine and plasma ketone dip test in a group of diabetic cats with possible ketosis or ketoacidosis, using laboratory plasma beta-hydroxybutyrate measurements as the gold standard. METHODS: According to clinical examinations, plasma beta-hydroxybutyrate measurements and venous blood gas analysis, 54 cats with diabetes mellitus were classified as non-ketotic (n=3), ketotic (n=40) or ketoacidotic (n=11). Plasma and urine acetoacetate concentrations were determined using urine reagent strips. RESULTS: Although there was a significant positive correlation between blood and urine ketone measurements (r=0.695, P<0.001), the results differed significantly (Z=-3.494, P<0.001). Using the differential positive rates, the best cut-off value to detect cats with ketoacidosis was 1.5 mmol/l for urine and 4 mmol/l for plasma. The sensitivity/specificity was 82/95 per cent for urine and 100/88 per cent for plasma, respectively. CLINICAL SIGNIFICANCE: The urine and plasma ketone dip tests have a different diagnostic accuracy, and results have to be interpreted differently. Because of its high sensitivity, the plasma ketone dip test performs better than the urine ketone dip test to identify cats with impending or established ketoacidosis.

The desmopressin stimulation test in dogs with Cushing's syndrome.

Desmopressin is a synthetic analogue of the hypothalamic peptide vasopressin and binds to specific pituitary vasopressin (V3) receptors. The V3-receptor is overexpressed in pituitary corticotrope tumors and the injection of desmopressin induces a marked ACTH and cortisol release in human patients with pituitary- (PDH), but not adrenal tumor (AT) dependent hyperadrenocorticism. In this prospective study, we investigated the effects of desmopressin on serum cortisol levels in 80 dogs suspected of Cushing's syndrome. The aim was to find a sensitive and specific test to exclude AT. According to standard tests the dogs were divided into 3 groups (group 1=other disease, n=27; group 2=PDH, n=46; group 3=AT, n=7). Desmopressin was injected as an i.v. bolus of 4microg and serial blood samples were collected before and after 30, 60 and 90min. Desmopressin significantly stimulated cortisol release in dogs with PDH (median 51%, range -24 to 563%; p<0.0001), whereas no increase was seen in dogs with AT (median -12%, range -44 to 5%; p=0.063) and in controls (median +7%, range -36 to 196%; p=0.131). Using a cut off value of 10% increase over baseline, it was possible to exclude AT in 75% of patients. The results of this study suggest that the desmopressin test could be a useful tool in differentiating pituitary from adrenal dependent Cushing's syndromes. Additional dogs with adrenocortical tumor must be tested in order to recommend its use in clinical practice.

Simultaneous canine distemper virus, canine adenovirus type 2, and Mycoplasma cynos infection in a dog with pneumonia.

The present case is the first description of a triple infection with canine distemper virus (CDV), canine adenovirus (CAV) type 2, and Mycoplasma cynos in a dog. The 5-month-old female Miniature Pinscher was euthanized because of dyspnea, croaking lung sounds, weight loss, and lymphopenia. Pathologic examination revealed a fibrinous necrotizing pneumonia with large amphophilic intranuclear and acidophilic intracytoplasmatic inclusion bodies in different lung cells. Immunohistochemically, CDV antigen was present in lung and many other organs. In situ hybridization for detection of CAV nucleic acid showed positive signals in the lung only. Polymerase chain reaction of lung tissue and consecutive sequencing of the amplification product identified CAV type 2. Bacteriologic examination of lung tissue yielded large amounts of M cynos. This infection was confirmed by immunohistochemistry detecting abundant positive signals in the lung tissue.