Association of four differently processed diets with plasma and urine advanced glycation end products and serum soluble receptor for advanced glycation end products concentration in healthy dogs.

Advanced glycation end products (AGEs), formed via the Maillard reaction (MR) during processing of foods, have been implicated in inflammatory and degenerative diseases in human beings. Cellular damage is primarily caused by AGE binding with the receptor for AGEs (RAGE) on cell membranes. An isoform of RAGE, soluble RAGE (sRAGE), acts as a decoy receptor binding circulating AGEs preventing cellular activation. Pet food manufacturing involves processing methods similar to human food processing that may increase dietary AGEs (dAGEs). We hypothesized that diet, plasma and urine AGEs, and serum sRAGE concentrations would differ between thermally processed diets. This study examined the association of four differently processed diets: ultra-processed canned wet food (WF); ultra-processed dry food (DF); moderately processed air-dried food (ADF) and minimally processed mildly cooked food (MF) on total plasma levels of the AGEs, carboxymethyllysine (CML), carboxyethyllysine (CEL), methylglyoxal hydroimidazolone-1, glyoxal hydroimidazolone-1, argpyrimidine, urine CML, CEL and lysinoalanine, and serum sRAGE concentration. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used to measure AGEs. sRAGE concentration was measured using a commercial canine-specific enzyme-linked immunosorbent assay kit. Total dAGEs (mg/100 kcal as fed) were higher in WF than in other diets. Plasma total AGEs (nM/50 µL) were significantly higher with WF, with no difference found between DF, ADF, and MF; however, ADF was significantly higher than MF. Urine CML (nmol AGEs/mmol creatinine) was significantly higher with DF than with WF and MF. There were no significant differences in total urine AGEs or serum sRAGE concentration between diets. In conclusion, different methods of processing pet foods are associated with varied quantities of AGEs influencing total plasma AGE concentration in healthy dogs. Serum sRAGE concentration did not vary across diets but differences in total AGE/sRAGE ratio were observed between MF and WF and, ADF and DF.

Pharmacokinetics and diuretic effect of furosemide after single intravenous, oral tablet, and newly developed oral disintegrating film administration in healthy beagle dogs.

BACKGROUND: Furosemide, a diuretic that acts on the loop of Henle, is commonly used to treat congestive heart failure in veterinary medicine. Some owners have difficulty in administering oral tablet medication to animal patients, which leads to noncompliance, especially during long-term administration. Oral disintegrating film (ODF) has the advantages of easy administration via a non-invasive route, rapid dissolution, and low suffocating risk. The objective of this study was to research the pharmacokinetic (PK) profiles and diuretic effect of furosemide after intravenous (IV), orally uncoated tablet (OUT), and newly developed ODF administration in healthy beagle dogs. In this study, a furosemide-loaded ODF (FS-ODF) formulation was developed and five beagle dogs were administered a single dose (2 mg/kg) of furosemide via each route using a cross-over design. RESULTS: The most suitable film-forming agent was sodium alginate; thus, this was used to develop an ODF for easy drug administration. No significant differences were detected in the PK profiles between OUT and FS-ODF. In the blood profiles, the concentration of total protein was significantly increased compared to the baseline (0 h), whereas no significant difference was detected in the concentration of creatinine and hematocrit compared to the baseline. FS-ODF resulted in a similar hourly urinary output to OUT during the initial 2 h after administration. The urine specific gravity was significantly decreased compared to the baseline in each group. The peak times of urine electrolyte (sodium and chloride) excretion per hour were 1 h (IV), 2 h (OUT), and 2 h (FS-ODF). CONCLUSIONS: These results suggest that the PK/PD of furosemide after administration of newly developed FS-ODF are similar to those of OUT in healthy dogs. Therefore, the ODF formulation has the benefits of ease and convenience, which would be helpful to owners of companion animals, such as small dogs (< 10 kg), for the management of congestive heart failure.

Evaluating extraction methods to study canine urine microbiota.

The urinary microbiota is the collection of microbes present in urine that may play a role in host health. Studies of urine microbiota have traditionally relied upon culturing methods aimed at identifying pathogens. However, recent culture-free sequencing studies of the urine microbiota have determined that a diverse array of microbes is present in health and disease. To study these microbes and their potential role in diseases like bladder cancer or interstitial cystitis, consistent extraction and detection of bacterial DNA from urine is critical. However, urine is a low biomass substrate, requiring sensitive methods to capture DNA and making the risk of contamination high. To address this challenge, we collected urine samples from ten healthy dogs and extracted DNA from each sample using five different commercially available extraction methods. Extraction methods were compared based on total and bacterial DNA concentrations and bacterial community composition and diversity assessed through 16S rRNA gene sequencing. Significant differences in the urinary microbiota were observed by dog and sex but not extraction method. The Bacteremia Kit yielded the highest total DNA concentrations (Kruskal-Wallis, p = 0.165, not significant) and the highest bacterial DNA concentrations (Kruskal-Wallis, p = 0.044). Bacteremia also extracted bacterial DNA from the greatest number of samples. Taken together, these results suggest that the Bacteremia kit is an effective option for studying the urine microbiota. This work lays the foundation to study the urine microbiome in a wide range of urogenital diseases in dogs and other species.

Analytical and physiological validation of an enzyme immunoassay to measure oxytocin in dog, wolf, and human urine samples.

Oxytocin (OT) promotes pro-sociality, bonding, and cooperation in a variety of species. Measuring oxytocin metabolite (OTM) concentrations in urine or saliva provides intriguing opportunities to study human and animal behaviour with minimal disturbance. However, a thorough validation of analytical methods and an assessment of the physiological significance of these measures are essential. We conducted an analytical validation of a commercial Enzyme Immunoassay (EIA; Arbor OT assay kit) to measure OTM concentrations in dog, wolf, and human urine samples. To test the assay's ability to detect changes in OTM concentrations, we administered oxytocin intranasally to 14 dogs. Assay performance with regard to parallelism was acceptable. Assay accuracy and extraction efficiency for dog and wolf samples were comparable to a previously validated assay (Enzo OT assay kit) but variation was smaller for human samples. Binding sensitivity and antibody specificity were better in the Arbor assay. Average OTM concentrations were more than twice as high as in comparable samples measured with the Enzo assay, highlighting a lack of comparability of absolute values between different assays. Changes in OTM concentrations after intranasal treatment were detected reliably. The Arbor assay met requirements of a "fit-for-purpose" validation with improvement of several parameters compared to the Enzo assay.

Reference intervals for electrophoretograms obtained by capillary electrophoresis of dialyzed urine from healthy dogs.

Electrophoresis of urine to evaluate protein fractions in dogs with proteinuria to differentiate glomerular from tubular damage has increased in recent years; however, capillary electrophoresis (CE) of urine has not been reported in a study of > 40 healthy animals, to our knowledge. We aimed to establish reference intervals (RIs) for the urine protein fractions obtained by CE of urine from healthy dogs. We obtained urine samples from 123 clinically healthy dogs of both sexes between December 2016 and April 2019; urine was frozen until CE was performed. The electrophoretic patterns obtained were divided into 5 protein fractions, and RIs were established in percentages and absolute values using nonparametric methods. RIs were obtained for the fractions (F) as follows: 5.5 to 56.2% for F1, 3.2 to 16.5% for F2, 3.5 to 16.2% for F3, 17.8 to 69.8% for F4, and 5.1 to 23.9% for F5. These RIs obtained by CE might be useful clinically as a basis for comparison with pathologic samples. Age was a statistically significant factor for F2 (p = 0.01) and F3 (p = 0.02), and sex was a statistically significant factor for F1 (p = 0.03).

Comparação entre os resultados da urinálise e dosagens bioquímicas urinárias de amostras de urina coletadas pelas técnicas de cistocentese guiada por ultrassom e sondagem uretral em cães / Comparison between the results of urinalysis and urinary biochemical tests of urine samples collected by techniques ultrasound-guided cystocentesisand urethral catheterization in dogs

Objetivou-se no presente estudo comparar as técnicas de coleta de urina via sondagem uretral e cistocentese guiada por ultrassom, afim de verificar se o método de coleta pode influir nos resultados laboratoriais. Foram utilizados 12 cães machos, sem histórico de enfermidades, dos quais coletou-se cinco mililitros (mL) de urina via sondagem uretral e cinco mL via cistocentese guiada por ultrassom, ambas no mesmo momento. Posteriormente foi realizada a análise física (cor, odor, densidade, turbidez), química (urobilinogênio, glicose, corpos cetônicos, bilirrubina, proteína, nitrito, pH, sangue e leucócitos) e sedimentoscopia (avaliação de 10 campos de luz, objetiva de 40x). Cilindros urinários, cristais, corpúsculos gordurosos, espermatozoides, bactérias e células vesicais foram classificados qualitativamente como: ausentes (0), discretos (1), moderados (2) e intensos (3). Hemácias, leucócitos, e células de descamação foram quantificadas a partir da média dos campos analisados. As análises bioquímicas de microalbuminúria, creatinina e proteína total urinárias foram realizadas a partir do sobrenadante urinário, removido das amostras após centrifugação, e utilizados kits reagentes, conforme recomendação do fabricante, sendo a leitura em espectrofotômetro. Em todos os testes realizados os valores de p encontrados foram superiores 0,05 (p>0,05), excluindo-se a possibilidade de haver diferenças significativas dos resultados laboratoriais obtidos pelas duas formas de coleta.

The objective of this study was to compare two techniques of urine collection, urethral catheterization and ultrasound-guided cystocentesis, in order to verify if the collection method may influence the laboratory results. Twelve male dogs were used, with no history of diseases, of which five milliliters (mL) of urine were collected by urethral catheterization and five mL by both at the same time. Subsequently, the samples underwent physical analysis (color, smell, density andturbidity), chemical analysis (urobilinogen, glucose, ketone bodies, bilirubin, protein, nitrite, pH, blood and leukocytes) and sedimentoscopy (evaluation of 10 light fields, 40x objective). Urinary casts, fatty corpuscles, spermatozoa, bacteria and bladder epithelial cells were classified qualitatively as absent (0), discrete (1), moderate (2) and intense (3). Red blood cells, leukocytes and desquamation cells were quantified from the mean of the analyzed fields. The urine supernatants were obtained after centrifugation and were used for biochemical analyzes of microalbuminuria, urinary protein and creatinine. The reagent kits were used as recommended by the manufacturer and the samples were read by spectrophotometry. All tests presented p values higher than 0,05 (p>0,05), excluding the possibility of significant differences between the laboratory results of both forms of urine collection.

Interobserver reliability of canine urine specific gravity assessed by analog or digital refractometers.

Refractometry is utilized routinely to evaluate canine urine specific gravity (USG) in veterinary clinical settings. We aimed to determine if the magnitude of interobserver reliability when assessing canine USG via refractometry could impact clinical judgment. USG was determined in 38 dogs by 3 registered veterinary technicians (RVTs) using both an optical analog refractometer and a digital refractometer. Summary statistics were reported, interobserver reliability was assessed via intraclass correlation coefficient (ICC) analysis through a 2-way mixed-effects model, and agreement between RVT pairs was compared through Bland-Altman plots. The median analog refractometer USG measurement was 1.018 (range: 1.004-1.040) and for the digital refractometer was 1.0176 (1.0035-1.0357). The analog refractometer average measure ICC was 0.995 (95% CI: 0.992, 0.997; p < 0.001). The digital refractometer average measure ICC was 0.999 (95% CI: 0.999, 1.000; p < 0.001). Strong agreement between all pairs of RVTs was seen via Bland-Altman plots for both analog and digital refractometers, with 95% CIs spanning no more than 0.002 in either the positive or negative direction for all pairings. The interobserver variability in canine USG measurements by RVTs was trivial and did not impact clinical judgment and decision-making.

Comparative pharmacokinetic profile of cimicoxib in dogs and cats after IV administration.

Cimicoxib is a selective COX-2 inhibitor (coxib) registered for the treatment of pain and inflammation in dogs. Pharmacokinetics of some coxibs have been described in dogs and cats. In cats, total body clearance values are lower and terminal half-lives of the coxibs are longer than those in dogs. The aim of this work was to evaluate if this is also the case for cimicoxib. For this purpose, blood pharmacokinetics and urinary excretion after IV administration were compared between these species. The in vitro metabolism of cimicoxib was also evaluated using canine and feline microsomes. In canine and feline microsomes, the formation rate of demethyl-cimicoxib, a phase 1 metabolite, was decreased in presence of quinidine, a specific human cytochrome P450 (CYP)2D6 inhibitor. IC50 values were 1.6 µM and 0.056 µM with canine and feline microsomes, respectively. As quinidine was about 30 times more potent in feline microsomes, the affinity of cimicoxib to the enzyme was considered to be about 30 times lower than that in canine microsomes. Total body clearance (ClB) of cimicoxib, was 0.50 L/h kg in dogs and 0.14 L/h kg in cats (P < 0.01) and terminal half-life, T½λz, was 1.92 and 5.25 h, respectively (P < 0.01). Dose eliminated in urine was 12.2% in dogs and 3.12% in cats (P < 0.01). Conjugated demethyl-cimicoxib represented 93.7% of this amount in dogs and 67.5% in cats. Thus cimicoxib, like other veterinary coxibs, was eliminated more slowly in cats. Both CYP2D15 (the canine ortholog of CYP2D6) and UDP-glucuronyltransferase enzyme systems have reduced ability to produce metabolites of cimicoxib in cats.

Desempenho de diferentes marcas de tiras reagentes relativo à densidade e à análise química da urina de cães e gatos / Performance of different brands of reagent strips for density and chemical analysis of dog and cat urine

Objetivou-se verificar a compatibilidade entre diferentes marcas de tiras reagentes para urinálise, tanto de uso veterinário, como de uso humano, e confrontar os parâmetros semiquantitativos desse instrumento com métodos quantitativos. Para isso, foram analisadas 77 amostras frescas de urina de cães e gatos e testados 04 modelos de tiras reagentes. Quanto à densidade urinária, houve correlação razoável entre os métodos quantitativo e semiquantitativo naquelas amostras com pH ácido, mas não naquelas com pH neutro ou alcalino. Quanto à concentração proteica, houve similaridade de 53,3% a 83,3% entre as marcas testadas e quando comparadas com a análise fotométrica houve uma correlação razoável (rs = 0,69752 a 0,75074). Em ponto de corte de 15mg/dL de proteína, a sensibilidade da tira reagente foi 82,5% e 100% para urina canina e felina, respectivamente. No tocante à hematúria, houve divergência razoável entre a sedimentoscopia e as diferentes marcas de tiras reativas. Quanto à piúria, há uma baixa sensibilidade das tiras em relação às amostras caninas com muitos resultados falso-negativos (33% a 75%), enquanto em amostras felinas a sensibilidade foi de 100%. Assim, independente da marca, as tiras reagentes devem servir apenas como teste rápido de triagem, sendo mais apropriado o uso de métodos quantitativos na avaliação clínica do paciente a partir da urinálise.

The aim was to verify the compatibility between different brands of urinary dipsticks, for both human and veterinary use, and to compare the semiquantitative parameters of this instrument with quantitative methods. For this, 77 fresh samples of urine from dogs and cats were analyzed e and 04 models of reagent strips were tested. Regarding urinary density, a reasonable correlation was observed between the quantitative and semiquantitative methods in those samples with acidic pH, which did not occur in those with neutral or alkaline pH. Regarding the protein concentration, there was similarity from 53.3% to 83.3% between the brands and in the comparative analysis between the control strip and the photometric analysis, there was a reasonable correlation (rs = 0.69752 to 0.75074). In cut-off point of 15mg/dL protein, the sensitivity of the reagent strip was 82.5% and 100% for canine and feline urine, respectively. Regarding hematuria, there was a reasonable divergence of results between sedimentation and tested dipsticks. As for pyuria, there is a low sensitivity of the strips in relation to canine samples with many false negative results (33% to 75%), while in feline samples the sensitivity was 100%. Thus, regardless of the brands, the reagent strips should serve only as a rapid screening test, while the use of quantitative methods in the clinical evaluation of the patient from urinalysis is more appropriate.

Stability of canine urine samples under different storage conditions.

The stability of canine urine samples is essential when the samples cannot be analyzed immediately. The objective of this study was to investigate the stability of canine urine samples at room temperature and under refrigerated conditions. Samples from 20 dogs were collected, divided, and stored at 4°C and 20°C. The samples were examined up to 48 h after collection for specific gravity, pH, protein, bilirubin, glucose, ketones, and sediment and at 4 h and 24 h for bacterial growth. Specific gravity and all chemistry parameters were stable for a minimum of 48 h in 90% of samples. The sediment was stable, apart from crystals. The bacterial growth of 3 bacterial species tested in vitro, as well as the clinical samples, was mostly constant over 24 h at the refrigerated temperature. In urine samples stored at room temperature, the total number of aerobic growing bacteria was increasing. The results of our study showed that routinely measured parameters were stable in unpreserved urine for a minimum of 4 h and up to 48 h in most cases. If it is not possible to culture urine immediately, it is recommended that urine samples be stored at 4°C for a period of up to 24 h.

La stabilité des échantillons d'urine canins est essentielle lorsque les échantillons ne peuvent être analysés immédiatement. L'objectif de la présente étude était d'examiner la stabilité d'échantillons d'urine canins à température ambiante et réfrigérés. Des échantillons provenant de 20 chiens furent prélevés, divisés et entreposés à 4 °C et 20 °C. Les échantillons furent examinés jusqu'à 48 h après le prélèvement pour la gravité spécifique, le pH, les protéines, la bilirubine, le glucose, les cétones et le sédiment, et à 4 h et 24 h pour la croissance bactérienne. La gravité spécifique et tous les paramètres chimiques étaient stables pour un minimum de 48 h dans 90 % des échantillons. Le sédiment était stable, sauf pour les cristaux. La croissance bactérienne de trois espèces bactériennes testée in vitro, ainsi que dans les échantillons cliniques, était généralement constante sur une période de 24 h à la température de réfrigération. Dans les échantillons d'urine entreposés à la température ambiante, le nombre total de bactérie aérobie augmentait. Les résultats de notre étude démontrent que les paramètres mesurés de routine sont stables dans de l'urine sans agent de préservation pour un minimum de 4 h et jusqu'à 48 h dans la majorité des cas. S'il n'est pas possible de mettre l'urine en culture immédiatement, il est recommandé que les échantillons d'urine soient entreposés à 4 °C pour une période allant jusqu'à 24 h.(Traduit par Docteur Serge Messier).

Urine creatinine concentration and urine protein-to-creatinine ratios in healthy nonracing Greyhounds.

BACKGROUND: Serum creatinine concentrations are higher in Greyhounds when compared with nonsighthound breeds. Greyhounds might also have higher urine creatinine concentrations compared with other breeds, which could affect urine protein-to-creatinine ratio (UPC) references. OBJECTIVES: We aimed to determine the UPC reference intervals (RIs) in healthy nonracing Greyhounds and compare this with UPC values in a group of healthy nonsighthounds and with the current International Renal Interest Society (IRIS) guidelines. METHODS: The study used an observational cross-sectional design, involving clinically healthy, nonracing Greyhounds (n = 98) and nonsighthound dogs of similar weight, age, and sex (n = 24). Packed cell volumes, total solids, urine protein concentrations, serum and urine creatinine concentrations, urine specific gravity (USG) measurements, and UPCs were determined. Linear regression was used to compare urine creatinine and urine protein concentrations, relative to the USG measurements, between Greyhound and nonsighthound groups. Greyhound UPC RIs were determined using nonparametric methods and compared with UPC values in nonsighthounds and current IRIS guidelines. RESULTS: Mean urine creatinine concentrations, adjusted for USGs, were approximately 22% higher in Greyhounds compared with nonsighthounds (P = .002). Mean urine protein concentration (P = .46) and UPC (P = .1) were not significantly different between Greyhounds and nonsighthounds. The upper limit of the Greyhound UPC RI was 0.20 or 0.42, depending on whether strict or moderate exclusion criteria were applied, respectively. CONCLUSIONS: Greyhounds have higher urine creatinine concentrations than nonsighthounds. Although the suggested RI for UPCs in Greyhounds is slightly lower than the cut-offs recommended in generic canine IRIS guidelines, this difference is not likely to be clinically significant.

Infecção de trato urinário inferior por Citrobacter sp em cadela com hipoadrenocorticismo / Citrobacter sp lower urinary tract infection in a bitch with hypoadrenocorticism

Citrobacter sp. é um microrganismo frequentemente encontrado em vagina de cadelas, mas corresponde a menos de 3% do total de microrganismos isolados em urocultura. O hipoadrenocorticismo (HA) é uma doença endócrina incomum e que leva a poliúria e hipostenúria. O objetivo deste trabalho é relatar o caso de uma fêmea da espécie canina, da raça Teckel, 11 anos, atendida com queixa de urina de odor alterado há vários dias, além de vômito, diarreia e inapetência há três dias, e poliúria e polidipsia há 2 meses, com histórico de tratamento anterior com mitotano para hiperadrenocorticismo. O diagnóstico foi de ITU por Citrobactersp., além de HA iatrogênico. Como destaques nos exames, relação sódio:potássio de 22,6 foi observada, sugestiva de HA, que foi confirmado por teste de estimulação com ACTH, além de urina inicialmente hipostenúrica (densidade 1,006), o que pode ser atribuído ao washout medular renal devido à hiponatremia causada pelo hipoadrenocorticismo. O tratamento foi feito com amoxicilina associada a ácido clavulânico por 20 dias, além da terapia específica para HA com prednisona e fludrocortisona, que foi continuado. A paciente recuperou-se bem, a densidade urinária aumentou após início do tratamento para HA e não houve recidiva da ITU em período de acompanhamento de 8 meses. Acredita-se que a baixa densidade urinária causada pelo HA tenha sido fator essencial para a ocorrência da ITU por Citrobacter.

Citrobacter sp. is a normal constituent of bitches' vagina, but it is related to less than 3% of total isolated microorganism in uroculture. Hypoadrenocorticism (HA) is an endocrine disease uncommonly diagnosed that leads to polyuria and hypostenuria. The aim of this work is report the case of a Teckel bitch, 11 years old, attended with complaint of altered urinary smell for several days, besides vomiting, diarrhea and inappetence for three days, and polyuria and polydispsia for two months, with previous mitotane treatment for hyperadrenocorticism. The diagnosis was Citrobacter UTI, besides iatrogenic HA. The most important exam results include sodium:potassium ratio of 22.6, suggestive of HA, that was confirmed by ACTH stimulation test. Besides, hypostenuria (urinary specific gravity ­ USG - of 1.006) was noted, attributed to renal medular washout due to hyponatremia seen in HA. Therapy comprised amoxicillin associated with clavulanic acid for 20 days, in addition to specific therapy for AH with prednisone and fludrocortisone. The patient recovery well, USG increased after the beginning of the treatment for HA and there was no UTI reinfection for a 8 month period. It is believed that low USG due to HA was essential for the occurrence of Citrobacter UTI.

Increasing dietary sodium chloride promotes urine dilution and decreases struvite and calcium oxalate relative supersaturation in healthy dogs and cats.

Urolithiasis is highly prevalent in dogs and cats, with struvite and calcium oxalate being most commonly diagnosed. Some commercial diets aimed at reducing the risk of urolithiasis are based on inclusion of sodium chloride (NaCl) in an attempt to dilute the urine and the risk of crystallization, but more information on the effect of differing levels of sodium inclusion is needed. The objective of this study was to compare the short-term effect of four diets differing only in NaCl content (base diet with 0.3% sodium and diets with added NaCl to achieve 0.7, 1.0 and 1.3% sodium as fed) on urinary ion concentrations and relative supersaturation (RSS) of struvite and calcium oxalate in dogs and cats. In both species, there was a significant increase in water intake and urine volume as dietary NaCl increased. Urine sodium concentration increased with increasing dietary NaCl. The highest sodium diet increased urinary calcium excretion in dogs only, while decreasing urinary calcium concentration. Calcium oxalate RSS and struvite RSS both significantly decreased, with the lowest RSS values reported on the highest sodium diet in both dogs and cats (p < .001). These results suggest that an increase in dietary NaCl decreases RSS values in both dogs and cats. Despite an increase in urinary calcium excretion in dogs, urinary calcium concentration and calcium oxalate RSS were lower on high sodium diets due to urine dilution. Long-term studies are needed to confirm the relationship between RSS and stone occurrence and recurrence.

Detection of allopurinol and oxypurinol in canine urine by HPLC/MS-MS: Focus on veterinary clinical pharmacokinetics.

Allopurinol is the most commonly used drug for the treatment of hyperuricemia in people, and in view of the risks of fatal hypersensitivity in patients with renal dysfunction, doses based on the glomerular filtration rate are proposed. In veterinary medicine, allopurinol is used in the treatment of canine leishmaniasis (CanL) caused by Leishmania infantum owing to the drug action of inhibiting the parasite's RNA synthesis. However, renal dysfunction frequently ensues from disease progression in dogs. The purpose of the present study was to standardize and validate a sensitive high-performance liquid chromatography-mass spectrometric (HPLC-MS/MS) method to determine the concentration of allopurinol and its active metabolite oxypurinol in canine urine for clinical pharmacokinetic investigation. Urine samples of eleven (11) dogs with naturally occurring CanL and in the maintenance phase of the treatment with alopurinol were used. For the chromatographic analysis of urine, the mobile phase consisted of a solution of 0.1 % formic acid (88 %) in 10 mM ammonium acetate. Separation of allopurinol and oxypurinol occurred in a flow of 0.8 mL/min on a C8 reverse phase column 5 µm, and acyclovir was the internal standard. The HPLC-MS/MS method was validated by reaching the limits of detection and quantification, reproducibility and linearity. The lower limit of quantification achieved by the method was 10 µg/mL for both allopurinol and oxypurinol. Calibration curves were prepared in blank urine added with allopurinol at concentrations of 10-1000 µg/mL, and oxypurinol at 10-200 µg/mL. Coefficients of variation of less than 15 % between intracurrent and intercurrent accuracy values were observed for both allopurinol and oxypurinol. Urine test samples remained stable after being subjected to freeze-thaw cycles and remaining at room temperature for 4 h. The method proved to be adequate to quantify allopurinol and oxypurinol in urine samples from dogs under treatment.

Cortisol urinary metabolites in dogs with hypercortisolism, congestive heart failure, and healthy dogs: pilot investigation.

Nonadrenal diseases (NAD), including congestive heart failure (CHF), can affect the conversion of cortisone to cortisol favoring the production of cortisol's urinary downstream metabolites 5α/5ß-tetrahydrocortisol (THF) relative to tetrahydrocortisone (THE). We hypothesized that healthy dogs would have lower urinary levels of cortisol, cortisone, THF, and THE than dogs with hypercortisolism (HC) or CHF, and the latter would have higher urinary levels of THF and lower THE than dogs with HC. Four, 9, and 8 dogs with HC, CHF, and normal health, respectively, were included in a pilot prospective cross-sectional study. A single morning voided urine sample was analyzed for urinary cortisol metabolites by liquid chromatography-mass spectrometry. The percentages of conjugated urinary metabolites were significantly higher in dogs with CHF than in healthy dogs (p = 0.001), and not different in HC dogs (p = 0.07). Log-transformed urine cortisol metabolites-to-creatinine ratios in healthy dogs were significantly lower than the 2 other groups (p < 0.001). The urinary free THE:THF ratio was significantly higher (p < 0.001) than the urinary total and conjugated THE:THF ratios. Health status did not affect the total, conjugated, and free THE:THF ratios (p = 0.61). Additional studies are needed to investigate differences in cortisol metabolites between dogs with HC and NAD to accurately discriminate between the groups.

Influence of preanalytic and analytic variables in canine and feline urine specific gravity measurement by refractometer.

Urine specific gravity (USG), which is usually measured by refractometry, is an important indicator of renal concentrating ability. Few studies have evaluated refractometers with separate scales for canine and feline urine. Variables such as protein content or storage time may influence the USG. We compared the effects of measuring USG with a refractometer with single or separate scales for canine and feline urine, investigated inter- and intra-observer variability, and measured agreement between whole urine and supernatant. We evaluated the correlation between USG and osmolality, the influence of urinary protein on USG and osmolality, and the impact of storage time up to 6 mo. We examined 252 canine and 126 feline samples. Bland-Altman analysis revealed higher USG values of the single-scale refractometer than the dual-scale refractometer, with a mean difference (bias) of < 0.001 for canine and 0.003 for feline specimens. Inter- and intra-observer variability were acceptable. Good agreement was shown between USG of whole urine and supernatant. Correlations between USG and osmolality were excellent (0.98-0.99, p < 0.001). Proteinuria up to 1 g/L had no major impact on USG or osmolality. Storage time had no significant effect on USG. The difference between the refractometers is clinically irrelevant, and the use of a refractometer with separate feline and canine scales is unnecessary. Whole urine and supernatant stored up to 6 mo can both be used for USG measurement. The influence of proteinuria <1 g/L on USG and osmolality is negligible.

Investigation of interference from synthetic colloids on the performance of a canine neutrophil gelatinase-associated lipocalin immunoassay.

BACKGROUND: Synthetic colloid solutions, administered by rapid infusion to volume-depleted dogs, might be present in high concentrations in subsequent urine samples. The potential for these solutions to affect the performance of ELISA measurements due to sample matrix effects when studying kidney injury biomarkers requires investigation. OBJECTIVE: We aimed to investigate two different synthetic colloid solutions, 4% succinylated bovine gelatin (GEL) and 6% hydroxyethyl starch (HES), for potential interferences with a commercially available canine neutrophil gelatinase-associated lipocalin (NGAL) ELISA. METHODS: Assay interference was assessed by measuring the linearity of NGAL concentrations measured using a canine NGAL ELISA after serial dilution of a canine pooled urine sample with an assay diluent, GEL, or HES. RESULTS: NGAL recovery from urine specimens containing up to 75% HES and up to 62.5% GEL was within acceptable limits (80%-120%). NGAL recovery from the urine specimen containing 75% GEL was poor (76%). Linear regression analysis demonstrated excellent linearity under dilution when a canine urine sample was diluted with the assay diluent, GEL, or HES. CONCLUSIONS: The presence of large amounts (>62.5%) of GEL in canine urine samples could cause negative interference in the performance of the NGAL ELISA investigated.

Evaluation of the accuracy of urine analyzers in dogs and cats.

The accuracy of urine analyzers used for dogs and cats has remained uncertain. This study examines the agreement between results of urine analysis obtained using two devices marketed for animals and for humans and the results of quantitative biochemical analysis. The degrees of concordance for bilirubin and ketones in the same category were ~80%, but for pH these were only ~60% in dogs and cats. Degrees of concordance for protein and the UP/C ratio clearly differed between the devices for animals and humans. We found that values for bilirubin and ketones obtained using urine analyzers may be reliable, but pH is unlikely to be accurate enough to be clinically useful for dogs and cats.

Variability among four refractometers for the measurement of urine specific gravity and comparison with urine osmolality in dogs.

BACKGROUND: Refractometry is often used in clinical veterinary medicine to estimate urine concentration. Variability among commonly-used refractometers has not been critically evaluated. OBJECTIVE: This study aimed to evaluate the variability of urine specific gravity (USG) among four refractometers and compare results of USG measurements with those of urine osmolality (Uosm ), the gold standard for determining urine concentrations. METHODS: USG was determined in 100 dogs using three optical refractometers, the American Optical Abbe refractometer 10450, Reichert TS 400 refractometer, and Heska Veterinary refractometer 2737-E02, and one digital refractometer, the Misco Palm Abbe Digital refractometer #PA203. Results were compared between each pair of refractometers and between each refractometer and urine osmolality determined by freezing point depression using a Multi-Osmette 2430E osmometer. Results were analyzed with Bland-Altman plots and Passing-Bablok regression analysis. RESULTS: The Reichert, Heska, and Misco refractometers provide USG measurements that can be used interchangeably based on small differences, consistently less than 0.002, between the USG measurements. The AO refractometer measured USG values with much larger differences, which were deemed clinically significant by the study parameters. None of the refractometers were able to accurately predict Uosm or vice versa within a clinically acceptable range. CONCLUSIONS: Varying degrees of differences were seen in the USG measurements among the different refractometers. These differences were refractometer-dependent, and the results from one instrument could affect clinical decisions.

Variability of first morning urine specific gravity in 103 healthy dogs.

BACKGROUND: Urine specific gravity (USG) is an integral part of the urinalysis and a key component of many clinical decisions, and fluctuations in USG have the potential to impact case management. OBJECTIVES: To determine the intraindividual variability of first morning USG results in healthy dogs. ANIMALS: One hundred three healthy client-owned dogs. METHODS: Dogs were deemed healthy based on clinical history and physical examination findings. Repeated USG measurements were performed over the course of 2 weeks. Three urine samples were collected each week for a total of 6 samples per dog. Sample collection was distributed evenly throughout the week. Urine samples were acquired immediately upon waking and before any ingestion of liquids, food, or exertion of physical activity in the dogs. All measurements were made using the same Misco digital refractometer. RESULTS: Intraindividual USG was variable over the course of the study. The mean difference between the minimum and maximum USG for each dog was 0.015 (SD, 0.007). The within-week difference between the minimum and maximum USG was less than over the complete 2-week study (0.009 [SD 0.006] for week 1 and 0.010 [SD 0.007] for week 2). The mean coefficient of variance across all 6 time points was 15.4% (SD 8.97%). CONCLUSIONS AND CLINICAL IMPORTANCE: Clinically important variation occurs in USG in healthy animals and might impact clinical decision-making when diagnostic cutoff points are utilized. Clinicians should be aware of inherent variability in this clinical variable when analyzing results.