Development and validation of an LC-MS/MS method for the quantification of oral-sugar probes in plasma to test small intestinal permeability and absorptive capacity in the domestic cat (Felis catus).

A novel method for quantifying the concentration of lactulose, rhamnose, xylose, and 3-O-methylglucose (3-OMG) in cat plasma using liquid chromatography-mass spectrometry (LC-MS) was developed. Domestic male cats (n = 13) were orally dosed with a solution containing the four sugars to test the permeability and absorptive capacity of their intestinal barrier. Plasma samples were taken 3 h later and were prepared with acetonitrile (ACN), dried under N2, and reconstituted in 90 % ACN with 1 mM ammonium formate. Stable isotope labelled 13C standards for each analyte were used as internal standards. Chromatographic separation was conducted using a Phenomenex Luna NH2 column with a gradient elution system of deionized water and 90 % ACN with 1 mM ammonium formate at 300 µL/min for 13 min total analysis time. Recovery trials were conducted in triplicate over three days with RSD values (%) for each day ranging from 1.2 to 1.4 for lactulose, 5.4 - 6.0 for rhamnose, 3.3 - 5.5 for xylose, and 2.6 - 5.6 for 3-OMG. Inter-day variations for each analyte were not different (p > 0.05). Limit of detection and quantification were 0.2 and 0.7 µg/mL for lactulose, 0.8 and 2.4 µg/mL for rhamnose, 0.6 and 1.8 µg/mL for xylose, and 0.3 and 1.1 µg/mL for 3-OMG, respectively. Plasma sugar concentrations recovered from cats were above the limit of quantification and below the highest calibration standard, validating the use of this method to test intestinal permeability and absorptive capacity in cats.

Rapid Patient-Side Evaluation of Endothelial Glycocalyx Thickness in Healthy Sedated Cats Using GlycoCheck® Software.

The endothelial glycocalyx (EG) determines transvascular fluid fluxes, and influences inflammation, coagulation, and capillary blood flow. The GlycoCheck® software calculates EG thickness using sidestream dark field videomicroscopy recordings. This method has not been evaluated for use in cats. The aim of the present study was to evaluate the use of GlycoCheck® for estimating EG thickness in healthy cats, and to investigate the variability of EG thickness in this population. One hundred and one healthy research-purposed cats were included in the study. The cats were sedated, and a handheld videomicroscope, connected to GlycoCheck® software, was used to evaluate the sublingual microvasculature. The parameters measured included perfused boundary region (PBR, an indirect measurement of EG thickness) in vessels between 5 and 25 µm in diameter, valid vessel density, percentage red blood cell filling, and median red blood cell column width. Heart rate, respiratory rate, pulse oximetry and oscillometric blood pressure readings were also recorded. There were 35 neutered male cats, 11 intact males, 38 neutered females, and 17 intact females. The average age was 63 months (range, 11-160 months). Tolerance intervals for PBR (vessel diameter 5-25 µm) were 1.89-3.00 µm (95% CI, lower limit 1.76-2.04, upper limit 2.83-3.13 µm); for valid vessel density were 73.33-333.33 µm/mm2 (95% CI, lower limit 77.00-99.33, upper limit 312.67-350.33 µm/mm2); for percentage red blood cell filling were 59.85-85.07% (95% CI, lower limit 58.97-63.33, upper limit 83.07-88.20 %); and for median red blood cell column width were 5.63-8.59 µm (95% CI, lower limit 5.28-6.07, upper limit 8.14-9.51 µm). There was a negative association between median red blood cell column width and body weight (p = 0.007). The median red blood cell column was significantly wider in intact females when compared to spayed females (p = 0.033). The GlycoCheck® analysis was easily performed in healthy sedated cats. Clinical variables did not have an effect on the EG thickness. These results suggest that this technique could be valuable for evaluation of the EG and microvascular parameters in cats.

Effect of non-steroidal anti-inflammatory drugs on glomerular filtration rate and urinary N-acetyl-ß-D-glucosaminidase activity in cats after dental surgery.

OBJECTIVE: To compare the effects of meloxicam or carprofen on glomerular filtration rate (GFR), and to evaluate the effect of meloxicam on urinary N-acetyl-ß-D-glucosaminidase (NAG) activity, of cats after dental surgery. STUDY DESIGN: Randomized, blinded, controlled trial. ANIMALS: A total of 24 mixed breed cats. METHODS: Cats were randomly assigned to one of three groups (n = 8 per group): meloxicam (0.2 mg kg-1); carprofen (4 mg kg-1); or saline (2 mL). Acepromazine (0.04 mg kg-1) and buprenorphine (0.02 mg kg-1) were administered intramuscularly as preanaesthetic medication. Test drugs were injected subcutaneously at the time of preanaesthetic medication. Anaesthesia was induced with intravenous propofol and maintained with isoflurane in oxygen. Mean arterial blood pressure (MAP), respiratory rate (fR), heart rate (HR) and haemoglobin oxygen saturation values (SpO2) were recorded. All cats underwent ultrasonic dental scaling with polishing. Teeth extraction involved mucosal flap creation, removal of alveolar bone and flap closure. Plasma iohexol clearance (ICL), a measure of GFR, was estimated before and 24 hours after anaesthesia induction in all cats. Urinary NAG index was estimated in saline and meloxicam groups at the same time points as GFR. Between-group and -time point differences in GFR and NAG index were compared using mixed model analyses. Data are presented as mean ± standard deviation (p < 0.05). RESULTS: There was no significant difference in plasma ICL rate (range: from 1.22 ± 0.05 to 1.27 ± 0.04 mL kg minute-1) between groups or between time points. Urinary NAG index (range: from 1.0 ± 0.19 to 1.36 ± 0.29 Units gram-1) was not significantly different between meloxicam and saline groups. MAP, HR, fR and SpO2 did not differ significantly between groups. CONCLUSIONS AND CLINICAL RELEVANCE: Meloxicam and carprofen appeared to produce nonsignificant effects on GFR, and meloxicam did not affect the urinary NAG activity, of cats after dental surgery.

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.

Felis catus papillomavirus type 2 virus-like particle vaccine is safe and immunogenic but does not reduce FcaPV-2 viral loads in adult cats.

Felis catus papillomavirus type 2 (FcaPV-2) commonly infects the skin of domestic cats and has been associated with the development of skin cancer. In the present study, a FcaPV-2 virus-like particle (VLP) vaccine was produced and assessed for vaccine safety, immunogenicity, and impact on FcaPV-2 viral load. This is the first report of the use of a papillomavirus VLP vaccine in domestic cats. The FcaPV-2 VLP vaccine was given to ten adult cats that were naturally infected with FcaPV-2, and a further ten naturally infected cats were sham vaccinated as a control group. The rationale for vaccinating cats already infected with the virus was to induce neutralizing antibody titers that could prevent reinfection of new areas of skin and reduce the overall viral load, as has been demonstrated in other species. Reducing the overall FcaPV-2 viral load could reduce the risk for subsequent PV-associated cancer. The vaccine in this study was well-tolerated, as none of the cats developed any signs of local reaction or systemic illness. In the treatment group, the geometric mean anti-papillomavirus endpoint antibody titers increased significantly following vaccination from 606 (95% CI 192-1913) to 4223 (2023-8814), a 7.0-fold increase, although the individual antibody response varied depending on the level of pre-existing antibodies. Despite the immunogenicity of the vaccine, there was no significant change in FcaPV-2 viral load in the treatment group compared to the control group, over the 24 week follow-up period. A possible reason is that FcaPV-2 was already widespread in the basal skin layer of these adult cats and so preventing further cells from becoming infected had no impact on the overall viral load. Therefore, these results do not support the use of a FcaPV-2 VLP vaccine to reduce the risk for PV-associated cancer in cats in which FcaPV-2 infection is already well established. However, these results justify future studies in which the vaccine is administered to younger cats prior to FcaPV-2 infection becoming fully established.

Nonlinear mixed models of growth curves from domestic shorthair cats in a breeding colony, housed in a seasonal facility to predict obesity.

Obesity is highly prevalent in pet cats (Felis silvestris catus) and is associated with several comorbidities, yet very little is understood about the early life risk factors. The aim of this study was to develop a mathematical model that describes growth in cats, to determine which early life variables were associated with being overweight in adulthood and to identify when during life the overweight phenotype developed in a colony of cats. We used pedigree, birth and growth data of 212 colony cats that were fed ad libitum from weaning. A nonlinear mixed-effects model was fitted to the body weight data to describe growth curves. A subset of 146 cats that had reached 9 years of age was used to evaluate the body weight curves to 9 years, and candidate early life predictors of peak weight were evaluated using logistic regression. In contrast with other species, candidates such as maternal factors, birth weight and litter size were not significant risk factors for adult obesity. Male sex, body weight around 15 weeks of age and being born during the increasing photoperiod were significantly associated with being overweight at 9 years. The growth equation created can be used to predict peak body weight, when sex and season of birth are included. Thus, the trajectory to obesity is determined early in life in cats, and early intervention appears essential to prevent obesity in pet cats.

Felis catus papillomavirus type 2 DNA loads on kittens are transient and do not reflect their susceptibility to infection.

Objectives Felis catus papillomavirus type 2 (FcaPV-2) commonly infects the skin of domestic cats, and mounting evidence suggests that the virus could be involved in a subset of feline skin cancers. The reason why some cats develop FcaPV-2-induced disease and others do not is currently unknown. However, it has been shown that kittens in different litters have markedly different FcaPV-2 DNA loads and the aim of this study was to determine whether these differences could be due to inherent differences in susceptibility to infection. Such differences could potentially explain why only a small proportion of cats develop FcaPV-2-associated skin disease. Methods Repeated skin swabs were taken to measure FcaPV-2 DNA loads in queens and kittens in a research colony. The kittens either stayed in their original litters or were moved between litters; eventually, all of the kittens were housed together. A subset of samples was also analysed for FcaPV-2 mRNA. Results While there were initially large differences in FcaPV-2 DNA loads between litters of kittens, these differences disappeared when the kittens were moved between litters or housed together. Importantly, the viral DNA loads changed too rapidly to be due to the acquisition or clearance of infection. In contrast, the differences in viral DNA loads between the different queens were sustained throughout the experiment. FcaPV-2 mRNA was also detected in samples from 1- to 8-day-old kittens. Conclusions and relevance The results suggest that the FcaPV-2 DNA load in a swab sample from an individual kitten largely reflects the overall level of FcaPV-2 shedding in the group of in-contact cats, rather than the infection status of the individual kitten. Therefore, there was no evidence for inherent differences in susceptibility to infection. However, the finding of FcaPV-2 mRNA suggests that at least some kittens do become infected with FcaPV-2 early in life.

Amino Acid Oxidation Increases with Dietary Protein Content in Adult Neutered Male Cats as Measured Using [1-13C]Leucine and [15N2]Urea.

BACKGROUND: Cats are unique among domestic animals in that they are obligate carnivores and have a high protein requirement. However, there are few data on protein turnover and amino acid (AA) metabolism in cats. OBJECTIVE: The aim of this study was to examine the effects of dietary protein content on urea production and Leu metabolism in cats. METHODS: Eighteen neutered male cats (4.4 ± 0.11 kg body weight, aged 4.6 ± 0.41 y) fed to maintain body weight for 3 wk with 15%, 40%, or 65% metabolizable energy intake as crude protein (CP) had [1-(13)C]Leu administered in the fed state. Urea production was measured by the infusion of [(15)N2]urea. Leu flux, nonoxidative Leu disposal (NOLD; protein synthesis), Leu rate of appearance (Ra; protein degradation), and Leu oxidation were determined. RESULTS: Urea production and Leu oxidation were both ∼ 3 times greater in cats fed 65% CP compared with those fed 15% CP, whereas those fed 40% CP were ∼ 1.6 times greater (P < 0.05). Leu flux was 1.9 and 1.3 times greater in cats fed 65% CP compared with those fed 15% and 40% CP (P < 0.001). Almost 39% of total Leu flux was oxidized by cats fed 15% CP, whereas this increased to 58% in cats fed 65% CP (P < 0.002). There were no differences for Ra, but cats fed 65% CP tended to have 30% greater NOLD (P = 0.09) and to be in positive protein balance (P = 0.08) compared with those fed 15% CP. CONCLUSION: The high protein requirement of cats combined with a low rate of whole-body protein synthesis ensures that an obligate demand of AAs for energy or glucose (or both) can be met in an animal that evolved with a diet high in protein with very little or no carbohydrate.

Assessment of a carbon dioxide laser for the measurement of thermal nociceptive thresholds following intramuscular administration of analgesic drugs in pain-free female cats.

OBJECTIVE: To assess the potential of a thermal carbon dioxide (CO2) laser to explore antinociception in pain-free cats. STUDY DESIGN: Experimental, prospective, blinded, randomized study. ANIMALS: Sixty healthy adult female cats with a (mean±standard deviation) weight of 3.3±0.6 kg. METHODS: Cats were systematically allocated to one of six treatments: saline 0.2 mL per cat; morphine 0.5 mg kg(-1); buprenorphine 20 µg kg(-1); medetomidine 2 µg kg(-1); tramadol 2 mg kg(-1), and ketoprofen 2 mg kg(-1). Latency to respond to thermal stimulation was assessed at baseline and at intervals of 15-30, 30-45, 45-60, 60-75, 90-105 and 120-135 minutes. Thermal thresholds were assessed using time to respond behaviourally to stimulation with a 500 mW CO2 laser. Within-treatment differences in response latency were assessed using Friedman's test. Differences amongst treatments were assessed using independent Kruskal-Wallis tests. Where significant effects were identified, pairwise comparisons were conducted to elucidate the direction of the effect. RESULTS: Cats treated with morphine (χ2=12.90, df=6, p=0.045) and tramadol (χ2=20.28, df=6, p=0.002) showed significant increases in latency to respond. However, subsequent pairwise comparisons indicated that differences in latencies at specific time-points were significant (p<0.05) only for tramadol at 60-75 and 90-105 minutes after administration (21.9 and 43.6 seconds, respectively) in comparison with baseline (11.0 seconds). No significant pairwise comparisons were found within the morphine treatment. Injections of saline, ketoprofen, medetomidine or buprenorphine showed no significant effect on latency to respond. CONCLUSIONS AND CLINICAL RELEVANCE: The CO2 laser technique may have utility in the assessment of thermal nociceptive thresholds in pain-free cats after analgesic administration and may provide a simpler alternative to existing systems. Further exploration is required to examine its sensitivity and comparative utility.

Urinary felinine excretion in intact male cats is increased by dietary cystine.

Felinine is a branched-chain sulfur amino acid present in the urine of certain Felidae, including domestic cats. The objective of the present study was to determine if additional cystine and/or dietary N would increase felinine and N-acetylfelinine excretion by intact male cats fed a low-protein(LP) diet. Feeding five adult intact male cats an LP diet (18.8% of metabolisable energy (ME) as protein) v. a high-protein diet (38.6% of ME as protein) resulted in a trend (P=0.08) for decreased urinary felinine and no change in N-acetylfelinine excretion. In a 23 d study, when the LP diet was supplemented with L-cystine at 9.3 g/kg DM, urinary felinine:creatinine ratio showed a linear two-fold (121 %) increase (P<0.01) from 0.24 (SEM 0.05) to 0.53 (SEM 0.13) after 10 d. Subsequent feeding of the LP diet resulted in a decrease in felinine excretion to base levels. Plasma gamma-glutamyl felinylglycine concentrations were consistent with the excretion of felinine. Supplementation of the LP diet with L-cystine (9.3 g/kg DM),dispensable amino acids and arginine to a second group (n 5) also resulted in a significant (P<0.01) but smaller (+72 %) increase in the daily felinine:creatinine ratio (0.25 (SEM 0.04) to 0.43 (SEM 0.05)). The degree of felinine N-acetylation within groups was unaffected by dietary addition and withdrawal of amino acids. The results indicate that felinine synthesis is regulated by cystine availability, and that arginine may be physiologically important in decreasing felinine biosynthesis in intact male cats.