Objective-To determine the effects of protease inhibitors and holding times and temperatures before processing on the stability of substance P in bovine blood samples. Samples-Blood samples obtained from a healthy 6-month-old calf. Procedures-Blood samples were dispensed into tubes containing exogenous substance P and 1 of 6 degradative enzyme inhibitor treatments: heparin, EDTA, EDTA with 1 of 2 concentrations of aprotinin, or EDTA with 1 of 2 concentrations of a commercially available protease inhibitor cocktail. Plasma was harvested immediately following collection or after 1, 3, 6, 12, or 24 hours of holding at ambient (20.3° to 25.4°C) or ice bath temperatures. Total substance P immunoreactivity was determined with an ELISA; concentrations of the substance P parent molecule, a metabolite composed of the 9 terminal amino acids, and a metabolite composed of the 5 terminal amino acids were determined with liquid chromatography–tandem mass spectrometry. Results-Regarding blood samples processed immediately, no significant differences in substance P concentrations or immunoreactivity were detected among enzyme inhibitor treatments. In blood samples processed at 1 hour of holding, substance P parent molecule concentration was significantly lower for ambient temperature versus ice bath temperature holding conditions; aprotinin was the most effective inhibitor of substance P degradation at the ice bath temperature. The ELISA substance P immunoreactivity was typically lower for blood samples with heparin versus samples with other inhibitors processed at 1 hour of holding in either temperature condition. Conclusions and Clinical Relevance-Results suggested that blood samples should be chilled and plasma harvested within 1 hour after collection to prevent substance P degradation.

Objective-To investigate age-related and regional differences in estimated metabolite concentrations in the brain of healthy dogs by means of magnetic resonance spectroscopy (MRS). Animals-15 healthy Beagles. Procedures-Dogs were grouped according to age as young (n = 5; all dogs were 2 months old), adult (5; mean age, 4.5 years), or geriatric (5; all dogs were 12 years old). Imaging was performed by use of a 1.5-T MRI system with T1- and T2-weighted spin-echo and fluid-attenuated inversion recovery sequences. Signal intensity measurements for N-acetyl aspartate, creatine, choline, and lactate-alanine (the spectroscopic peaks associated with alanine and lactate could not be reliably differentiated) were determined with MRS, and areas under the spectroscopic peaks (representing concentration estimates) were calculated. Ratios of these metabolite values were compared among age groups and among brain regions with regression analysis. Results-The choline-to-creatine ratio was significantly higher in young dogs, compared with other age groups. The N-acetyl aspartate-to-choline ratio was significantly lower in young dogs and geriatric dogs than in adult dogs. When all age groups were considered, the choline-to-creatine ratio was significantly higher and N-acetyl aspartate-to-choline ratio was significantly lower in the frontal lobe than in all other regions. The N-acetyl aspartate-to-creatine ratio was significantly lower in the cerebellum than in other regions. Conclusions and Clinical Relevance-Metabolite ratios varied significantly among age groups and brain regions in healthy dogs. Future studies should evaluate absolute concentration differences in a larger number of dogs and assess clinical applications in dogs with neurologic diseases.

A high rate of mortality, expense, and complications of immunosuppressive therapy in dogs underscores the need for optimization of drug dosing. The purpose of this study was to determine, using a flow-cytometric assay, the 50% T-cell inhibitory concentration (IC50) of dexamethasone, cyclosporine, and the active metabolites of azathioprine (6-mercaptopurine) and leflunomide (A77 1726) in canine lymphocytes stimulated with concanavalin A (Con A). Whole blood was collected from 5 privately owned, healthy dogs of various ages, genders, and breeds. Peripheral blood mononuclear cells, obtained by density-gradient separation, were cultured for 72 h with Con A, a fluorochrome-tagged cell proliferation dye, and various concentrations of dexamethasone (0.1, 1, 10, 100, 1000, and 10 000 μM), cyclosporine (0.2, 2, 10, 20, 30, 40, 80, and 200 ng/mL), 6-mercaptopurine (0.5, 2.5, 50, 100, 250, and 500 μM), and A77 1726 (1, 5, 10, 25, 50, and 200 μM). After incubation, the lymphocytes were labeled with propidium iodide and an antibody against canine CD5, a pan T-cell surface marker. Flow cytometry determined the percentage of live, proliferating T-lymphocytes incubated with or without immunosuppressants. The mean (± standard error) IC50 was 3460 ± 1900 μM for dexamethasone, 15.8 ± 2.3 ng/mL for cyclosporine, 1.3 ± 0.4 μM for 6-mercaptopurine, and 55.6 ± 22.0 μM for A77 1722. Inhibition of T-cell proliferation by the 4 immunosuppressants was demonstrated in a concentration-dependent manner, with variability between the dogs. These results represent the initial steps to tailor this assay for individual immunosuppressant protocols for dogs with immune-mediated disease.

Objective—To investigate clinical use of proton magnetic resonance spectroscopy (1H MRS) and to compare metabolic brain bioprofiles of dogs with and without hepatic encephalopathy. Animals—6 dogs with hepatic encephalopathy and 12 control dogs. Procedures—Conventional MRI and single-voxel 1H MRS were performed with a 3-T magnet. Images for routine MRI planes and sequences were obtained. Single-voxel 1H MRS was performed with a point-resolved sequence with a short echo time (35 milliseconds) and voxel of interest placement at the level of the basal ganglia. Metabolites of interest included the glutamine-glutamate complex (sum quantification of glutamate and glutamine), myoinositol, N-acetyl aspartate, total choline, and creatine. Data were analyzed with postprocessing fitting algorithm software, and metabolite concentration relative to water and ratios with creatine as the reference metabolite were calculated. Results—Compared with control dogs, dogs with hepatic encephalopathy had specific changes, which included significantly higher concentration relative to water of the glutamine-glutamate complex and significantly lower concentration of myoinositol. Choline and N-acetyl aspartate concentrations were also slightly lower in dogs with hepatic encephalopathy than in control dogs. No differences in creatine concentration were detected between groups. Conclusions and Clinical Relevance—1H MRS aided in the diagnosis of hepatic encephalopathy in dogs, and findings supported the assumption that ammonia is a neurotoxin that manifests via glutamine-glutamate complex derangements. Use of 1H MRS may provide clinically relevant information in patients with subclinical hepatic encephalopathy, equivocal results of bile acids tests, and equivocal ammonia concentrations or may be helpful in monitoring efficacy of medical management.

Objective- To compare pharmacokinetics after a single IM or SC injection of ceftiofur crystalline-free acid (CCFA) to bearded dragons (Pogona vitticeps). Animals- 8 adult male bearded dragons. Procedures- In a preliminary experiment, doses of 15 and 30 mg/kg, SC, were compared in 2 animals, and 30 mg/kg resulted in a more desirable pharmacokinetic profile. Then, in a randomized, complete crossover experimental design, each bearded dragon (n = 6) received a single dose of 30 mg of CCFA/kg IM or SC; the experiment was repeated after a 28-day washout period with the other route of administration. Blood samples were collected at 10 time points for 288 hours after injection. Plasma concentrations of ceftiofur and desfuroylceftiofur metabolites were measured via reverse-phase high-performance liquid chromatography. Data were analyzed with a noncompartmental model. Results-No adverse effects were observed. Plasma concentrations greater than a target minimum inhibitory concentration of 1 μg/mL were achieved by 4 hours after administration by both routes. Mean plasma concentrations remained > 1 μg/mL for > 288 hours for both routes of administration. Conclusions and Clinical Relevance-A single dose of CCFA (30 mg/kg) administered IM or SC to bearded dragons yielded plasma concentrations of ceftiofur and its metabolites > 1 μg/mL for > 288 hours. The SC route would be preferred because of less variability in plasma concentrations and greater ease of administration than the IM route. Future studies should include efficacy data as well as evaluation of the administration of multiple doses.

Objective- To determine the pharmacokinetics of hydromorphone hydrochloride after IV and IM administration in American kestrels (Falco sparverius). Animals-12 healthy adult American kestrels. Procedures- A single dose of hydromorphone (0.6 mg/kg) was administered IM (pectoral muscles) and IV (right jugular vein); the time between IM and IV administration experiments was 1 month. Blood samples were collected at 5 minutes, 1 hour, and 3 hours (n = 4 birds); 0.25, 1.5, and 9 hours (4); and 0.5, 2, and 6 hours (4) after drug administration. Results- Plasma hydromorphone concentrations were determined by means of liquid chromatography with mass spectrometry, and pharmacokinetic parameters were calculated with a noncompartmental model. Mean plasma hydromorphone concentration for each time was determined with naïve averaged pharmacokinetic analysis.Plasma hydromorphone concentrations were detectable in 2 and 3 birds at 6 hours after IM and IV administration, respectively, but not at 9 hours after administration. The fraction of the hydromorphone dose absorbed after IM administration was 0.75. The maximum observed plasma concentration was 112.1 ng/mL (5 minutes after administration). The terminal half-life was 1.25 and 1.26 hours after IV and IM administration, respectively. Conclusion and Clinical Relevance- Results indicated hydromorphone hydrochloride had high bioavailability and rapid elimination after IM administration, with a short terminal half-life, rapid plasma clearance, and large volume of distribution in American kestrels. Further studies regarding the effects of other doses, other administration routes, constantrate infusions, and slow release formulations on the pharmacokinetics of hydromorphone hydrochloride and its metabolites in American kestrels may be indicated..

Objective—To describe findings of 3.0-T multivoxel proton magnetic resonance spectroscopy (1H-MRS) in dogs with inflammatory and neoplastic intracranial disease and to determine the applicability of 1H-MRS for differentiating between inflammatory and neoplastic lesions and between meningiomas and gliomas. Animals—33 dogs with intracranial disease (19 neoplastic [10 meningioma, 7 glioma, and 2 other] and 14 inflammatory). Procedures—3.0-T multivoxel 1H-MRS was performed on neoplastic or inflammatory intracranial lesions identified with conventional MRI. N-acetylaspartate (NAA), choline, and creatine concentrations were obtained retrospectively, and metabolite ratios were calculated. Values were compared for metabolites separately, between lesion categories (neoplastic or inflammatory), and between neoplastic lesion types (meningioma or glioma) by means of discriminant analysis and 1-way ANOVA. Results—The NAA-to-choline ratio was 82.7% (62/75) accurate for differentiating neoplastic from inflammatory intracranial lesions. Adding the NAA-to-creatine ratio or choline-to-creatine ratio did not affect the accuracy of differentiation. Neoplastic lesions had lower NAA concentrations and higher choline concentrations than inflammatory lesions, resulting in a lower NAA-to-choline ratio, lower NAA-to-creatine ratio, and higher choline-to-creatine ratio for neoplasia relative to inflammation. No significant metabolite differences between meningiomas and gliomas were detected. Conclusions and Clinical Relevance—1H-MRS was effective for differentiating inflammatory lesions from neoplastic lesions. Metabolite alterations for 1H-MRS in neoplasia and inflammation in dogs were similar to changes described for humans. Use of 1H-MRS provided no additional information for differentiating between meningiomas and gliomas. Proton MRS may be a beneficial adjunct to conventional MRI in patients with high clinical suspicion of inflammatory or neoplastic intracranial lesions.