• View in gallery
    Figure 1—

    Box-and-whisker plots of TX metabolite concentrations in voided urine samples (n = 10; A) and urine samples collected via cystocentesis (11; B) from apparently healthy cats. Each box represents the 25th to 75th percentiles, the horizontal line in each box represents the median, whiskers represent the 10th to 90th percentiles, and circles represent outliers.

  • View in gallery
    Figure 2—

    Correlation between concentrations of TXB2 (squares), 2,3 dinorTXB2 (circles), and 11-dehydroTXB2 (triangles) in voided urine samples and urine samples collected via cystocentesis from apparently healthy cats. Correlations were evaluated via Pearson product moment correlation. Each symbol represents results for 1 cat. The dotted line represents equivalence.

  • View in gallery
    Figure 3—

    Box-and-whisker plots of the interday variation in concentrations of 11-dehydroTXB2 in voided urine samples collected from 8 apparently healthy cats over a 5-day period. Each box represents the 25th to 75th percentiles, the horizontal line in each box represents the median, and whiskers represent the 10th to 90th percentiles.

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  • 40. Westlund P, Granstrom E, Kumlin M, et al. Identification of 11-dehydro-TXB2 as a suitable parameter for monitoring thromboxane production in the human. Prostaglandins 1986; 31: 929960.

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Concentrations of thromboxane metabolites in feline urine

Brittany Heggem-PerryDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Stephanie A. SmithDepartment of Biochemistry, School of Molecular and Cellular Biology, University of Illinois, Urbana, IL 61802.

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Maureen A. McMichaelDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Mauria O'BrienDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Anne SaundersDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Jason TarriconeDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Abstract

OBJECTIVE To determine the predominant thromboxane (TX) metabolite in urine of healthy cats, evaluate whether the method of sample collection would impact concentration of that metabolite, and propose a reference interval for that metabolite in urine of healthy cats.

ANIMALS 17 cats (11 purpose-bred domestic shorthair cats, 5 client-owned domestic shorthair cats, and 1 client-owned Persian cat).

PROCEDURES All cats were deemed healthy on the basis of results for physical examination, a CBC, serum biochemical analysis, urinalysis, and measurement of prothrombin time and activated partial thromboplastin time. Voided and cystocentesis urine samples (or both) were collected. Aliquots of urine were stored at −80°C until analysis. Concentrations of TXB2, 11-dehydroTXB2, and 2,3 dinorTXB2 were measured with commercially available ELISA kits. Urinary creatinine concentration was also measured.

RESULTS 11-dehydroTXB2 was the most abundant compound, representing (mean ± SD) 59 ± 18% of the total amount of TX detected. In all samples, the concentration of 11-dehydroTXB2 was greater than that of 2,3 dinorTXB2 (mean, 4.2 ± 2.7-fold as high). Mean concentration of 11-dehydroTXB2 for the 17 cats was 0.57 ± 0.47 ng/mg of creatinine. A reference interval (based on the 5% to 95% confidence interval) of 0.10 to 2.1 ng of 11-dehydroTXB2/mg of creatinine was proposed for healthy cats.

CONCLUSIONS AND CLINICAL RELEVANCE In this study, 11-dehydroTXB2 was the major TX metabolite in feline urine. Measurement of this metabolite may represent a noninvasive, convenient method for monitoring in vivo platelet activation in cats at risk for thromboembolism.

Abstract

OBJECTIVE To determine the predominant thromboxane (TX) metabolite in urine of healthy cats, evaluate whether the method of sample collection would impact concentration of that metabolite, and propose a reference interval for that metabolite in urine of healthy cats.

ANIMALS 17 cats (11 purpose-bred domestic shorthair cats, 5 client-owned domestic shorthair cats, and 1 client-owned Persian cat).

PROCEDURES All cats were deemed healthy on the basis of results for physical examination, a CBC, serum biochemical analysis, urinalysis, and measurement of prothrombin time and activated partial thromboplastin time. Voided and cystocentesis urine samples (or both) were collected. Aliquots of urine were stored at −80°C until analysis. Concentrations of TXB2, 11-dehydroTXB2, and 2,3 dinorTXB2 were measured with commercially available ELISA kits. Urinary creatinine concentration was also measured.

RESULTS 11-dehydroTXB2 was the most abundant compound, representing (mean ± SD) 59 ± 18% of the total amount of TX detected. In all samples, the concentration of 11-dehydroTXB2 was greater than that of 2,3 dinorTXB2 (mean, 4.2 ± 2.7-fold as high). Mean concentration of 11-dehydroTXB2 for the 17 cats was 0.57 ± 0.47 ng/mg of creatinine. A reference interval (based on the 5% to 95% confidence interval) of 0.10 to 2.1 ng of 11-dehydroTXB2/mg of creatinine was proposed for healthy cats.

CONCLUSIONS AND CLINICAL RELEVANCE In this study, 11-dehydroTXB2 was the major TX metabolite in feline urine. Measurement of this metabolite may represent a noninvasive, convenient method for monitoring in vivo platelet activation in cats at risk for thromboembolism.

Thromboxane A2 is a cyclooxygenase product of arachidonic acid produced from the intermediary prostaglandin H2. Thromboxane A2 can be produced by multiple cell types, including smooth muscle cells, neutrophils, macrophages, hepatocytes, and endothelial cells, but TXA2 is most widely associated with release from activated platelets.1 Investigators of 1 study2 correlated an increase in the TXA2 concentration as a result of release from feline platelets with an increase in platelet activity. Once released, TXA2 can trigger various physiologic responses, including platelet aggregation, bronchoconstriction, and vasoconstriction.3

Biochemical assessment of TXA2 in blood is difficult because of the transitory nature of TXA2, which is rapidly hydrolyzed to TXB2.4,5 Once formed, TXB2 is then metabolized at the tissue level to 11-dehydroTXB2 and 2,3 dinorTXB2. Both of these compounds are then further metabolized and excreted in the urine.6 Phlebotomy-induced platelet activation does not cause additional production of the metabolites because they are produced enzymatically in the tissues. Therefore, concentrations of the metabolites are considered to be more reliable (compared with concentrations of TXB2) as indicators of platelet thromboxane release in vivo.6,7 Measurement of metabolite concentrations in urine has the further advantage of being a time-averaged indicator of platelet activity for the period during which the urine was produced. In humans, 11-dehydroTXB2 is the major metabolite of TXB2,8,9 whereas 2,3 dinorTXB2 is the major metabolite in dogs.5,10 To our knowledge, TX metabolism in cats has not been evaluated.

Cats with cardiac disease have an increased risk of developing arterial thromboembolism, which accounts for substantial morbidity among this population.11,12 Results for an early study13 of experimentally induced thrombosis in cats suggest that platelets may be a major mediator in the pathogenesis of thromboembolic events that are sequelae to heart disease of cats, and some studies14,15 have suggested platelet hyperreactivity in cats with heart disease. Evaluation of urinary TX metabolite concentrations may consequently be a useful, noninvasive, and convenient method for monitoring in vivo platelet activation in cats with cardiac disease.

The objectives of the study reported here were to determine the predominant TX metabolite in feline urine, evaluate whether the method of sample collection would impact the concentration of that metabolite, and propose an initial reference interval for that metabolite in urine from apparently healthy cats.

Materials and Methods

Animals

Seventeen healthy cats provided urine samples for the study. There were 11 purpose-bred facility-owned domestic shorthair cats (10 neutered males and 1 sexually intact female), 5 client-owned domestic shorthair cats (3 neutered males and 2 spayed females), and 1 client-owned sexually intact male Persian. Cats were deemed to be healthy on the basis of results of physical examination and routine laboratory testing (results of a CBC, platelet count, serum biochemical analysis, and urinalysis and measurement of prothrombin time and activated partial thromboplastin time). Cats had not received any antiplatelet or other medications prior to collection of urine samples. Facility-owned cats were housed in accordance with guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International and cared for in accordance with established principles.16 Informed consent was obtained for client-owned cats; the study was approved by an institutional animal care and use committee.

Collection and storage of urine samples

Samples of voided urine were obtained from facility-owned cats by use of nonabsorbent litter prior to other interventions. Subsequently (within a few days to weeks), a blood sample was obtained via venipuncture and an additional urine sample was obtained via cystocentesis from each of these cats. Venipuncture and cystocentesis were performed with only manual restraint in 6 cats, but 5 cats required sedation with a combination of butorphanol tartrate (0.4 mg/kg, IM) and dexmedetomidine (3 μg/kg, IM) to enable investigators to perform venipuncture and cystocentesis. For client-owned cats, urine samples were obtained by use of nonabsorbent litter (voided samples; n = 2) and by use of cystocentesis (4). Aliquots of urine were stored at −80°C until batch analysis.

Subsequently (several weeks later), urine samples were collected by means of cystocentesis from 4 facility-owned cats that were not sedated and that were not subjected to concurrent venipuncture; these samples were collected within 2 days of collection of voided samples from the same 4 cats. Concentrations of TX compounds were measured in those samples. Finally, 3 voided samples were collected from each of 8 facility-owned cats over a 5-day period (days 1, 3, and 5) and used to evaluate day-to-day variation in 11-dehydroTXB2 concentrations.

Measurement of urinary TX metabolite concentrations

Urine samples were thawed at ambient temperature (21°C) immediately before evaluation. Urinary TX and TX metabolite concentrations were measured with commercially available ELISA kits used in accordance with the manufacturer's instructions. Kits for measurement of 11-deydroTXB2,a TXB2,b and 2,3 dinorTXB2c have not been validated for use on feline urine but have been used on dog,5,17–20 mouse,21 rat,22 and human23,24 urine. Because of the lack of interspecies variation in the structure of TXs, it was expected that the ELISA antibodies would bind to these compounds in feline urine. Purification of urine samples was not necessary for use with the ELISA as determined on the basis of previous studies and recommendations of the kit manufacturer.5,17,18,25 Samples were assayed in duplicate at 2 dilutions. Results for urinary TXB2, 11-dehydroTXB2, and 2,3 dinorTXB2 were corrected for cross-reactivity among the 3 molecules as described elsewhere.5

To account for individual variations in urine concentrating capacity, TXB2, 11-dehydroTXB2, and 2,3 dinorTXB2 concentrations were normalized on the basis of the urinary creatinine concentration by calculation of the urinary TX-to-creatinine ratio as described elsewhere.5,26 Urinary creatinine concentrations of all samples were determined at the University of Illinois Veterinary Diagnostic Laboratory by use of an automated dry chemistry system.

Statistical analysis

Normality was determined by use of the Shapiro-Wilk test. Comparisons between compounds were performed with a repeated-measure 1-way ANOVA. Correlations were evaluated via Pearson product moment correlation. Tests were performed by use of a statistical software package.d Values of P < 0.05 were considered significant.

Results

Comparison of urinary TX and TX metabolite concentrations

Initial experiments focused on determining the evaluated compound that was the most abundant in feline urine. Concentrations of TXB2, 11-dehydroTXB2, and 2,3 dinorTXB2 were measured in voided urine samples of facility-owned cats (Figure 1). In all samples, except for 1, the most abundant of the 3 compounds was 11-dehydroTXB2 (P = 0.016; repeated-measure 1-way ANOVA); it represented (mean ± SD) 59 ± 18% of the amount of TX detected. For all urine samples, the concentration of 11-dehydroTXB2 was greater than that of 2,3 dinorTXB2 (mean, 4.2 ± 2.7-fold as high).

Figure 1—
Figure 1—

Box-and-whisker plots of TX metabolite concentrations in voided urine samples (n = 10; A) and urine samples collected via cystocentesis (11; B) from apparently healthy cats. Each box represents the 25th to 75th percentiles, the horizontal line in each box represents the median, whiskers represent the 10th to 90th percentiles, and circles represent outliers.

Citation: American Journal of Veterinary Research 77, 12; 10.2460/ajvr.77.12.1340

Concentrations of the 3 TX compounds were also measured in urine samples collected via cystocentesis from facility-owned cats, and results were compared with those for voided urine samples (Figure 1). In all samples, TXB2 was the most abundant (P < 0.001; repeated-measure 1-way ANOVA) of the 3 compounds; it represented (mean ± SD) 80 ± 5% of the amount of TX detected. For all urine samples, the concentration of 11-dehydroTXB2 was greater than that of 2,3 dinorTXB2 (mean, 5.7 ± 5.4-fold as high).

Concentrations for each compound were compared between the voided samples and those collected via cystocentesis (Figure 2). There was no significant difference in concentrations of 11-dehydroTXB2 or 2,3 dinorTXB2 between the 2 collection methods as determined by use of a repeated-measures 1-way ANOVA. Cystocentesis samples contained significantly (P = 0.002) more TXB2 than did voided samples. However, all of the cystocentesis samples were collected within 10 minutes after venipuncture, and some (5/11) were collected from sedated cats. None of the cystocentesis samples obtained from each of the 4 facility-owned cats that were not sedated and were not subjected to concurrent venipuncture, and which were obtained within 2 days of the collection of voided samples, contained more TXB2 than did the corresponding voided sample.

Figure 2—
Figure 2—

Correlation between concentrations of TXB2 (squares), 2,3 dinorTXB2 (circles), and 11-dehydroTXB2 (triangles) in voided urine samples and urine samples collected via cystocentesis from apparently healthy cats. Correlations were evaluated via Pearson product moment correlation. Each symbol represents results for 1 cat. The dotted line represents equivalence.

Citation: American Journal of Veterinary Research 77, 12; 10.2460/ajvr.77.12.1340

Concentration of 11-dehydroTXB2

Results of comparisons suggested that 11-dehydroTXB2 was the most abundant TX metabolite in feline urine; thus, further experiments focused on this compound. The mean ± SD concentration of 11-dehydroTXB2 in urine samples obtained from the 17 cats was 0.57 ± 0.47 ng/mg of creatinine. Thus, on the basis of the 5% to 95% confidence interval, a proposed initial reference interval of 0.10 to 2.1 ng of 11-dehydroTXB2/mg of creatinine was determined; however, a larger sample size will be needed to determine reference intervals for clinical use.

Day-to-day variation in 11-dehydroTXB2 concentrations was determined by use of 3 voided samples collected over a 5-day period from each of 8 cats (Figure 3). Mean ± SD coefficient of variation among the 3 samples was 0.40 ± 0.26. There was intra-individual variation over the 5-day period. There was no significant correlation between urinary 11-dehydroTXB2 concentration and the platelet concentration.

Figure 3—
Figure 3—

Box-and-whisker plots of the interday variation in concentrations of 11-dehydroTXB2 in voided urine samples collected from 8 apparently healthy cats over a 5-day period. Each box represents the 25th to 75th percentiles, the horizontal line in each box represents the median, and whiskers represent the 10th to 90th percentiles.

Citation: American Journal of Veterinary Research 77, 12; 10.2460/ajvr.77.12.1340

Discussion

Platelet activation potentially contributes to the pathophysiologic mechanisms of a variety of disease processes in cats, including thromboembolic complications of neoplasia and cardiac disease. Unfortunately, collection of feline platelets may be problematic for a number of reasons, including the fact restraint for venipuncture is challenging in some cats, stress associated with venipuncture can lead to complications in clinically unstable patients, and feline platelets can be activated during the blood collection process.27 Consequently, it would be useful to identify a diagnostic marker for in vivo activation of platelets that is noninvasive and not subject to artifacts from sample collection or ex vivo changes. Because feline urine samples can be easily collected by clients in at-home settings, use of this type of sample eliminates the need for venipuncture. Furthermore, sampling-associated platelet changes are not an issue. Assays for metabolites of TXB2 in human urine have been extensively described as markers of in vivo platelet activation,28–30 particularly with respect to evaluation of the response to antiplatelet agents.24 Elevated 11-dehydroTXB2 concentrations have been detected in urine from humans with a variety of conditions associated with platelet activation, including diabetes mellitus,31 atherosclerosis,32 unstable angina,33 occluded retinal veins,34 stroke,35 and myocardial infarction.36 Consequently, we wanted to evaluate feline urine for concentrations of TX metabolites.

Thromboxane A2 released from platelets during the activation process is rapidly converted to TXB2 in the blood. Plasma TXB2 is converted via tissue enzymes to 2,3 dinorTXB2 and 11-dehydroTXB2 and then to other downstream metabolites, which are then excreted in the urine. Concentrations of TXB2 and its metabolites have been measured in plasma or serum of dogs and cats as evidence of platelet activity.10,37,38 It must be mentioned that TXB2 can be generated as a result of ex vivo platelet activation during venipuncture, so plasma TXB2 concentrations may not be reflective of in vivo platelet activity. Furthermore, serum TXB2 concentrations are indicative of platelet activity that occurs ex vivo during coagulation of blood in a collection tube; consequently, they are an indicator of the potential for platelet response, rather than an indicator of the ongoing in vivo response.9 Because TXB2 is further metabolized to 2,3 dinorTXB2 and 11-dehydroTXB2 by tissue enzymes, 2,3 dinorTXB2 and 11-dehydroTXB2 are not subject to platelet activation during blood sample collection.6 Therefore, urinary concentrations of 2,3 dinorTXB2 and 11-dehydroTXB2 are most reflective of ongoing systemic in vivo release of TX from platelets.9,39,40

Dogs have markedly higher concentrations of 2,3 dinorTXB2 than 11-dehydroTXB2,5,10 but 11-dehydroTXB2 appears to be the major metabolite in humans9 and other animal species (rabbits, guinea pigs, monkeys, and rats).10 Evaluation of urine from apparently healthy cats of the present study indicated that similar to most other evaluated species, 11-dehydroTXB2 is the predominant TX metabolite in feline urine. Because 11-dehydroTXB2 concentrations were not different between voided and cystocentesis urine samples collected from the same cat, either method of collection should be acceptable for obtaining samples for measurement of this compound. It should be mentioned that most of the healthy cats in the study reported here were males. In humans, gender can impact TX concentrations. However, it was expected that sex would not be a relevant factor in cats because of the lack of influence of sex hormones in feline patients at our facility.

Urine samples collected via cystocentesis in the initial experiments of the present study contained markedly more TXB2 than did the voided urine samples. We suspect that the increased TXB2 concentration in these samples was a function of platelet activation during concurrent venipuncture or renal generation associated with sedation. This interpretation was supported by the lack of difference in TXB2 concentrations between voided and cystocentesis urine samples in the latter experiments in which venipuncture was not performed immediately prior to cystocentesis. Only TXB2 concentrations were measured; these values were required for correction of 2,3 dinorTXB2 concentrations because the antibodies in the ELISA reacted equally with both TXB2 and 2,3 dinorTXB2. Cross-reactivity would be a major disadvantage associated with use of the 2,3 dinorTXB2 ELISA as a clinical assay because TXB2 must also be measured to yield useful 2,3 dinorTXB2 results. Corrections for cross-reactivity are much less important for canine urine because the amount of 2,3 dinorTXB2 far exceeds the amount of TXB2.5 The kidneys also produce TXA2 and TXB2; thus, some of the TXB2 present in the urine is not of platelet derivation.20,41 Consequently, urinary TXB2 concentration is not a useful indicator of in vivo platelet activation. The fact that prior venipuncture or sedation might impact urinary TXB2 concentration is not of consequence with regard to evaluating in vivo platelet activity in cats.

Evaluation of in vivo platelet activation in cats is most likely to be of use for a population at risk for thromboembolic events, such as cats with cardiac disease. The precise underlying mechanism causing thrombogenic events in cats with cardiac disease has not been determined, but platelet function may be a contributing factor. Investigators of several studies14,15,42 have reported evidence of platelet activation in cats with cardiac disease. Those investigators reported increased platelet expression of P-selectin15 and increased responsiveness in vitro to ADP14,15 and collagen42 for platelets obtained from cats with cardiac disease (as compared with results for platelets obtained from apparently healthy cats). However, to obtain samples of feline platelets for in vitro evaluation, venipuncture by necessity must be performed. Therefore, it is possible that platelets from cats with cardiac disease are not actually hyperresponsive in vivo, but rather are primed (during venipuncture or in response to exposure to agonists in vitro) in some manner to be more reactive. Alternatively, it is possible that cats with cardiac disease do not have increased platelet activation in vivo. Investigators of 1 report43 found no difference in closure time between blood samples obtained from clinically normal cats and those with hypertrophic cardiomyopathy. It might be expected that urine samples collected from cats with cardiac disease would contain higher amounts of 11-dehydroTXB2 than would urine samples collected from apparently healthy cats, but future studies will be needed to evaluate platelet responses in cats with cardiac disease.

Because aspirin is sometimes administered to cats at risk for thrombosis,12 measurement of urinary 11-dehydroTXB2 concentrations may be of use as a tool for evaluating aspirin responsiveness. Urinary 11-dehydroTXB2 concentrations have been used to monitor response to aspirin treatment in human patients.23 Aspirin administered at adequate doses to dogs reportedly decreases the amount of 11-dehydroTXB2 in urine.5,17,18 Although it is not currently clear that use of aspirin is of benefit in cats at risk for thrombosis, measurement of urinary 11-dehydroTXB2 concentrations may provide additional information regarding the response to aspirin in this population of cats.

Acknowledgments

Supported by the Morris Animal Foundation (grant No. D13-FE-020).

The authors thank Alyssa Galligan and Jessica Garrett for technical assistance.

ABBREVIATIONS

TX

Thromboxane

Footnotes

a.

11-deydro TXB2 ELISA, Cayman Chemical Co, Ann Arbor, Mich.

b.

TXB2 ELISA, Cayman Chemical Co, Ann Arbor, Mich.

c.

2,3 dinorTXB2 ELISA, Cayman Chemical Co, Ann Arbor, Mich.

d.

SigmaPlot 13, Systat Software, San Jose, Calif.

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Contributor Notes

Address correspondence to Dr. McMichael (mmcm@illinois.edu).