Dog Red Blood Cells Research Articles

Measurement of Redox Biomarkers in the Whole Blood and Red Blood Cell Lysates of Dogs.

The evaluation of the biomarkers of oxidative status is usually performed in serum, however, other samples, such as red blood cells (RBCs) lysates or whole blood (WB), can be used. The objective of this study was to evaluate if a comprehensive panel of redox biomarkers can be measured in the WB and RBCs of dogs, and their possible changes “in vitro” after the addition of different concentrations of ascorbic acid. The panel was integrated by biomarkers of the antioxidant status, such as cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP), Trolox equivalent antioxidant capacity (TEAC), thiol and paraoxonase type 1 (PON-1), and of the oxidant status, such as total oxidant status (TOS), peroxide-activity (POX-Act), reactive oxygen-derived compounds (d-ROMs), advanced oxidation protein products (AOPP) and thiobarbituric acid reactive substances (TBARS). All the assays were precise and accurate in WB and RBCs lysates. In addition, they showed changes after ascorbic acid addition that are in line with previously published results, being WB more sensitive to detect these changes in our experimental conditions. In conclusion, the panel of assays used in this study can be measured in the WB and RBCs of the dog. In particular, the higher sensitivity to detect changes in our experimental conditions and its easier sample preparation makes WB a promising sample for the evaluation of redox status in dogs, with also potential applications to other animal species and humans.

Improving the method of dog red blood cells under the protection of dimethyl sulfoxide

Improving the method of dog red blood cells under the protection of dimethyl sulfoxide

Convolutional neural networks based focal loss for class imbalance problem: a case study of canine red blood cells morphology classification

Morphologies of red blood cells are normally interpreted by a pathologist. It is time-consuming and laborious. Furthermore, a misclassified red blood cell morphology will lead to false disease diagnosis and improper treatment. Thus, a decent pathologist must truly be an expert in classifying red blood cell morphology. In the past decade, many approaches have been proposed for classifying human red blood cell morphology. However, those approaches have not addressed the class imbalance problem in classification. A class imbalance problem---a problem where the numbers of samples in classes are very different---is one of the problems that can lead to a biased model towards the majority class. Due to the rarity of every type of abnormal blood cell morphology, the data from the collection process are usually imbalanced. In this study, we aimed to solve this problem specifically for classification of dog red blood cell morphology by using a Convolutional Neural Network (CNN)---a well-known deep learning technique---in conjunction with a focal loss function, adept at handling class imbalance problem. The proposed technique was conducted on a well-designed framework: two different CNNs were used to verify the effectiveness of the focal loss function and the optimal hyper-parameters were determined by 5-fold cross-validation. The experimental results show that both CNNs models augmented with the focal loss function achieved higher $F_{1}$-scores, compared to the models augmented with a conventional cross-entropy loss function that does not address class imbalance problem. In other words, the focal loss function truly enabled the CNNs models to be less biased towards the majority class than the cross-entropy did in the classification task of imbalanced dog red blood cell data.

STUDI KASUS : PROFIL SEL DARAH MERAH ANJING YANG TERINFEKSI BABESIA SP.

Babesia sp. is a protozoa that infects dogs through tick bite and can cause babesiosis. The purpose of blood tests in case dogs is to find out the profile of the dog's red blood cells with babesiosis. On clinical examination found many ticks on the dog's body. Dogs show clinical symptoms of dehydration and paleness in the mucosa. Blood collection is done through the anterior antibrachii cephalica vein. Blood is taken and collected in EDTA tubes. Then the examination is done by examining blood parasites, examining erythrocyte values, examining hemoglobin concentration and hematocrit values. Examination of blood parasites was done by making reticulocyte preparations, while examining erythrocyte values, hemoglobin concentration and hematocrit values were performed using a Vet Scan® HM 5TM machine. The results of examination of reticulocyte preparations showed the existence of Babesia sp infestations with a presentation of 0.68%. Erythrocyte values, hemoglobin concentration and hematocrit values were 3.52x106 // µL, 6.90 g / dL and 23.81%. Based on history, physical examination, clinical and laboratory examination, it can be concluded that the Beagle dog named Loh has microcytic hypochromatic anemia.

Exploring the Possibility of Cryopreservation of Feline and Canine Erythrocytes by Rapid Freezing with Penetrating and Non-Penetrating Cryoprotectants.

Efficient application of veterinary blood transfusion approaches for small companion animals requires readily available supply of the donor material. This can be achieved by developing of effective biobanking technologies allowing long-term storage of donor blood components via cryopreservation. Transfusion of an erythrocyte concentrate allows the successful correction of various hematological pathologies, severe bleeding, and etc. While in the past there were several approaches to cryopreserve red blood cells of dogs, to our knowledge there is virtually no data on cryopreservation of feline erythrocytes. In this paper, we performed a comprehensive parameter optimization for low temperature storage of RBCs of both species. Here, the efficiency of single-component and multicomponent cryoprotective media as well as necessary time of pre-incubation with penetrating and non-penetrating cryoprotectants prior to rapid freezing is analyzed. This study showed that glycerol was not sufficient for cryopreservation of red blood cells of the studied species under the investigated conditions. Application of 10% (v/v) ME2SO allowed for a significant reduction of canine and feline erythrocytes hemolysis after thawing. 17.5% hydroxyethyl starch demonstrated the highest cryoprotective activity for both species. It was found that dog RBCs should be incubated in cryoprotective media for 30 min at 22°C prior to freezing, while for cat RBCs 20 min is sufficient. Combination of CPAs was less effective. Presented data may be considered in further studies in veterinary transfusion and blood banking optimization.

Comparative evaluation of factors affecting hemagglutinatingactivity of avian influenza (H9) virus

Influenza virus H9 was standardized for hemagglutination assay using different factors such as red blood cell (RBC) types, concentrations, diluent types, and storage times. Avian influenza virus H9 was grown in embryonated chicken eggs and confirmed by spot agglutination. A significant (P 0.05) were found between human blood type O, chicken, and dog RBCs, as well as among rabbit, pigeon, sheep, and parrot. The highest titer (9.00 ± 0.00) with a nonsignificant difference was found using virus stored at 4 °C and -20 °C while 37 °C showed the lowest significant mean hemagglutinin (HA) titer (11.08 ± 188.21). Nonsignificant differences were observed in HA titers against H9 virus stored for 3, 4, 5, and 6 months. Nonsignificant differences were found between the use of normal saline and 0.5% peptone water with the lowest HA titers of 7.83 ± 0.40 and 8.00 ± 0.00, respectively, while the highest HA titer (9.00 ± 0.00) with nonsignificant difference was observed by using HA-HI buffer and phosphate buffer saline as diluents. RBCs with 0.5% and 1% concentrations showed nonsignificant difference in HA titer but significant difference with 0.1% RBCs.

Synergistic hemolysins of coagulase-negative staphylococci (CoNS).

A total of 104 coagulase negative staphylococci, belonging to S. capitis, S. hominis, S. haemolyticus and S. warneri, originating from the collection of the Department of Pharmaceutical Microbiology (ZMF), Medical University of Lodz, Poland, were tested for their synergistic hemolytic activity. 83% of strains produced δ-hemolysin, however, the percentage of positive strains of S. haemolyticus, S. warneri, S. capitis and S. hominis was different - 98%, 78%, 75% and 68%, respectively. Highly pure hemolysins were obtained from culture supernatants by protein precipitation with ammonium sulphate (0-70% of saturation) and extraction by using a mixture of organic solvents. The purity and molecular mass of hemolysins was determined by TRIS/Tricine PAGE. All CoNS hemolysins were small peptides with a molar mass of about 3.5 kDa; they possessed cytotoxic activity against the line of human foreskin fibroblasts ATCC Hs27 and lysed red cells from different mammalian species, however, the highest activity was observed when guinea pig, dog and human red blood cells were used. The cytotoxic effect on fibroblasts occurred within 30 minutes. The S. cohnii ssp. urealyticus strain was used as a control. The antimicrobial activity was examined using hemolysins of S. capitis, S. hominis, S. cohnii ssp. cohnii and S. cohnii ssp. urealyticus. Hemolysins of the two S. cohnii subspecies did not demonstrate antimicrobial activity. Cytolysins of S. capitis and S. hominis had a very narrow spectrum of action; out of 37 examined strains, the growth of only Micrococcus luteus, Corynebacterium diphtheriae and Pasteurella multocida was inhibited.

Red blood cell size and the Fåhraeus–Lindqvist effect

The reason for the large interspecies variation in vertebrate red blood cell (RBC) size is poorly understood. To test the effect of RBC size on blood viscosity, blood samples from two vertebrates, the dog (Canis familiaris L., 1758) and the greater siren (Siren lacertina L., 1766), with extremely different RBC sizes were examined. In this study, we examined whether RBC size altered the relationship of viscosity and viscometer tube diameter, the well-known Fåhraeus–Lindqvist effect (FLE). We used a glass capillary viscometer that incorporates tubes with a diameter narrow enough to evoke the FLE. At similar RBC concentrations, viscosity of suspensions with the larger siren RBCs were greater than the viscosity of suspensions with the smaller dog RBCs. However, the relationship between viscosity and tube diameter is independent of RBC size. The results of this study allow us to conclude that the FLE is not related to RBC size. While packaging hemoglobin in RBCs appears adaptive by reducing blood viscosity through the FLE, RBC size does not contribute to the reductions in tube-relative viscosity.

Na+-inhibitory sites of the Na+/H+ exchanger are Li+ substrate sites

Amiloride-inhibitable Li+ influx in dog red blood cells is mediated by the Na+/H+ exchanger, NHE. However, there are substantial differences between the properties of Li+ transport and Na+ transport through the NHE. Li+ influx is activated by cell shrinkage, and Na+ influx is not, as we reported previously (Dunham PB, Kelley SJ, and Logue PJ. Am J Physiol Cell Physiol 287: C336-C344, 2004). Li+ influx is a sigmoidal function of its concentration, and Na+ activation is linear at low Na+ concentrations. Li+ does not inhibit its own influx; in contrast, Na+ inhibits Na+ influx. Li+ prevents this inhibition by Na+. Na+ is a mixed or noncompetitive inhibitor of Li+ influx, implying that both a Na+ and a Li+ can be bound at the same time. In contrast, Li+ is a competitive inhibitor of Na+ influx, suggesting Li+ binding at one class of sites on the transporter. Because the properties of Li+ transport and Na+ transport are different, a simple explanation is that Na+ and Li+ are transported by separate sites. The similarities of the properties of Li+ transport and the inhibition of Na+ transport by Na+ suggest that Li+ is transported by the Na+-inhibitory sites.

Evidence for stanniocalcin binding activity in mammalian blood and glomerular filtrate

The 50 kD form of the hormone stanniocalcin-1 (STC50) is widely distributed in organs such as kidney, lung, and liver. Kidney collecting duct cells produce STC50 for local targeting to proximal tubule cells to increase phosphate reabsorption. As such the current dogma is that in most organs STC50 is a purely local mediator that is not released into the circulation. However, liver hepatocytes contain high levels of both STC50 and its receptor but little evidence of STC production, suggesting that the hormone may in fact be delivered to hepatocytes systemically. Moreover, previous data suggest that red blood cells may in fact bind STC. In this report, we have sought to identify STC binding activity in mammalian blood. Human, pig, and dog red blood cells were analyzed in STC receptor binding assays. Mouse red blood cells and adult mouse kidney were also analyzed histologically for the presence of STC ligand and receptor. Saturable, high affinity STC receptors were identified on red blood cells from all species. More intriguingly, STC binding activity was also identified in glomerular filtrate, indicative of a soluble, filterable STC binding protein. This binding protein was subsequently observed being reabsorbed in proximal straight tubules. These findings suggest that our inability to detect STC in mammalian serum is due to its being attached to soluble and tethered forms of a high-affinity binding protein. This could be a means of delivering STC to distant targets as well as a mechanism for removing unwanted hormone from the circulation.

Extracellular Na+ inhibits Na+/H+ exchange: cell shrinkage reduces the inhibition.

Na+/H+ exchangers (NHE) are ubiquitous transporters participating in regulation of cell volume and pH. Cell shrinkage, acidification, and growth factors activate NHE by increasing its sensitivity to intracellular H+ concentration. In this study, the kinetics were studied in dog red blood cells of Na+ influx through NHE as a function of external Na+ concentration ([Na+](o)). In cells in isotonic media, [Na+](o) inhibited Na+ influx >40 mM. Osmotic shrinkage activated NHE by reducing this inhibition. In cells in isotonic media + 120 mM sucrose, there was no inhibition, and influx was a hyperbolic function of [Na+](o). The kinetics of Na+-inhibited Na+ influx were analyzed at various extents of osmotic shrinkage. The curves for inhibited Na+ fluxes were sigmoid, indicating more than one Na+ inhibitory site associated with each transporter. Shrinkage significantly increased the Na+ concentration at half-maximal velocity of Na+-inhibited Na+ influx, the mechanism by which shrinkage activates NHE.

Thiopurine Methyltransferase Activity in Red Blood Cells of Dogs

Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of thiopurine medications such as azathioprine. In humans, activity varies widely among individuals, primarily because of genetic polymorphisms. Low TPMT activity increases the risk of myelosuppression from azathioprine and 6‐mercaptopurine, whereas high TPMT activity is associated with poor drug efficacy. The purpose of this study was to determine whether dogs also show a wide range of TPMT activity. Heparinized blood samples were obtained from 177 dogs associated with a veterinary teaching hospital. Red blood cell (RBC) TPMT activity was measured by means of a modification of a radiochemical method as established for use in people. TPMT activity varied across a 9‐fold range (7.9–71.8 U of RBC per milliliter; median, 21.7). Variation in TPMT activity was not associated with age, sex, or neutering status. Giant Schnauzers had much lower TPMT activity (7.9–20 U of RBC per milliliter; median, 13.1;P< .001) than did other breeds, and Alaskan Malamutes had much higher TPMT activity (22.7–71.8 U of RBC per milliliter; median, 36.0;P< .001) than did other breeds. Such variations in TPMT activity in the canine population and within groups of related dogs could affect thiopurine drug toxicity and efficacy in canine patients.

Thiopurine methyltransferase activity in red blood cells of dogs.

Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of thiopurine medications such as azathioprine. In humans, activity varies widely among individuals, primarily because of genetic polymorphisms. Low TPMT activity increases the risk of myelosuppression from azathioprine and 6-mercaptopurine, whereas high TPMT activity is associated with poor drug efficacy. The purpose of this study was to determine whether dogs also show a wide range of TPMT activity. Heparinized blood samples were obtained from 177 dogs associated with a veterinary teaching hospital. Red blood cell (RBC) TPMT activity was measured by means of a modification of a radiochemical method as established for use in people. TPMT activity varied across a 9-fold range (7.9-71.8 U of RBC per milliliter; median, 21.7). Variation in TPMT activity was not associated with age, sex, or neutering status. Giant Schnauzers had much lower TPMT activity (7.9-20 U of RBC per milliliter; median, 13.1; P < .001) than did other breeds, and Alaskan Malamutes had much higher TPMT activity (22.7-71.8 U of RBC per milliliter; median, 36.0; P < .001) than did other breeds. Such variations in TPMT activity in the canine population and within groups of related dogs could affect thiopurine drug toxicity and efficacy in canine patients.

Standardisation of the haemagglutination test for peste des petits ruminants (PPR)

The different conditions which affect haemagglutination (HA) by peste des petits ruminants (PPR) virus were investigated. The conditions investigated include how sensitive red blood cells (RBCs) of different animal species are to the PPR haemagglutinin, the agglutination time for the chicken RBC, the temperature at which PPR virus gives the highest HA titre and the effect of temperature of the specimen containing the PPR virus on the HA titre. Human group “O” RBCs were found to give the highest HA titre when compared with chicken RBC, goat RBC, pig RBC and dog RBC using the same specimen (antigen) and under the same conditions. Following human group “O” in sensitivity were chicken RBC, goat RBC and pig RBC in descending order. Dog RBC was completely insensitive (i.e. gave no HA reaction). For chicken RBC, mean agglutination time was 25min, when HA test was done at 25°C (room temperature). When phosphate buffered saline (PBS) was used at different pH levels, highest HA titre was obtained at pH 6.8. Higher HA titre was obtained when the test was done at 4°C than when it was performed at 25°C. Temperature of the specimen just before the test was done, had no effect on the HA titre. So, it has been recommended that to make HA test for diagnosis of PPR standard, it should be performed with either human group “O’ or chicken RBCs. The diluent for the test should be PBS of pH 6.8 and the test should be performed at 4°C. It should be read within 1h of setting-up the test.

Comparison of cation transport and regulatory volume decrease between red blood cells from the japanese black bear and the dog

Cation transport and regulatory volume decrease (RVD) were investigated in the red blood cells (RBC) of the Japanese black bear and compared with that of the normal dog (LK dog), which possesses RBC with high sodium (Na) and low potassium (K). Unlike the RBC of the American black bear, RBC from the Japanese black bear failed to exhibit K congener rubidium (Rb) influxes through Na–K pump and K-chloride (Cl) cotransport. In the RBC of the Japanese black bear, treatment with N-ethyl maleimide (NEM), calcium (Ca) ionophore A23187 and EGTA, and hyposmotic medium failed to elicit K–Cl cotransport, whereas these perturbations elicited K–Cl cotransport in the LK dog RBC. The Ca uptake was measured in RBC of both the Japanese black bear and the LK dog, and the fluxes were enhanced in hyposmotic medium and inhibited with quinidine, an inhibitor of Na/Ca exchange transporter. Extracellular Ca-dependent Na efflux was detected in RBC of both the Japanese black bear and the LK dog, and it was also inhibited with quinidine. The Ca uptake and Na efflux were probably mediated by Na/Ca exchange transporter. RBC of both the Japanese black bear and the LK dog were restored to their original volume after being swollen in hyposmotic medium with, but not without, Ca. Thus, Na/Ca exchange transporter causes regulatory volume decrease in the RBC of both the Japanese black bear and the LK dog.

Comparative Nuclear Magnetic Resonance Studies of Diffusional Water Permeability of Red Blood Cells from Different Species. XII. Dog (Canis familiaris) and Cat (Felis domestica)

The diffusional water permeability (P d ) of dog and cat red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulphonate (PCMBS). The values of P d were in the case of cat RBC ∼3.0 × 10−3 cm/s at 15 °C, 3.5 × 10−3 cm/s at 20 °C, 4.2 × 10−3 cm/s at 25 °C, 4.4 × 10−3 cm/s at 30 °C and 5.9 × 10−3 cm/s at 38 °C. In case of dog RBC the values of P d were higher ∼3.8 × 10−3 cm/s at 15 °C, 4.6 × 10−3 cm/s at 20 °C, 5.0 × 10−3 cm/s at 25 °C, 5.9 × 10−3 cm/s at 30 °C and 7.9 × 10−3 cm/s at 37 °C. Systematic studies of the effect of PCMBS on water diffusion indicated that in the case of dog RBCs the maximal inhibition was reached in 15–30 min with 1 mm PCMBS, whereas in the case of cat RBCs in 60 min with 1 mm PCMBS or in 30 min with 2 mm PCMBS. The values of maximal inhibition in the case of cat RBC were in the range of 55–60% at 15 °C, 60–68% at 20 °C and 25 °C, 50–60% at 30 °C and 50–55% at 37 °C. In the case of dog RBC the corresponding values were higher, 75–80% at 15 °C, 70–80% at 20 °C and 25 °C, 65–70% at 30 °C and 55–60% at 37 °C. The basal permeability to water was estimated to be ∼1 × 10−3 cm/s −2 × 10−3 cm/s in the range of temperatures of 25–37 °C. The activation energy of water diffusion E a,d was ∼19 kJ/mol for the dog RBC and ∼23 kJ/mol for the cat RBC. After incubation with PCMBS the values of E a,d increased, reaching 40 kJ/mol in conditions of maximal inhibition of water exchange. The membrane polypeptide electrophoretic pattern of dog and cat RBCs has been compared with its human counterpart. Dog and cat RBCs contained higher amounts of spectrin (band 1 and 2) and lower amounts of bands 4.4, 4.2, band 5 and band 7 compared to human RBCs. Band 4.9 was decreased only in the cat RBCs, whereas band 6 was decreased only in the dog RBCs.

Determination of ATP release from erythrocytes using microbore tubing as a model of resistance vessels in vivo.

A biomimetic model is described for the detection of adenosine triphosphate (ATP) release from red blood cells (RBCs) as they traverse fused-silica tubing ranging in i.d. from 25 to 75 microm. A continuous flow system is employed to create stress on the RBCs once they have entered the microbore tubing. This stress induces release of RBC-derived ATP, which is known to stimulate nitric oxide production in endothelial cells, resulting in eventual dilation of arterial smooth muscle. In this study, the RBCs were subjected to variations in tubing length and inside diameter. In a 25-microm-i.d. tube, the amount of ATP released from the RBCs increased from 3.74 +/- 0.56 to 9.55 +/- 0.73 microM as the tubing length was increased from 35 to 100 cm. In addition, for a 100-cm-length tube, the amount of ATP released from the RBCs increased from 3.03 +/- 0.49 to 9.55 +/- 0.73 microM as the inside diameter of the tubing decreased from 75 to 25 microm. To demonstrate that the erythrocytes were not being lysed inside the fused-silica tubing, dog RBCs, which are known to contain amounts of ATP similar to those of rabbit but do not release that ATP under physiological conditions, were investigated. It was determined that the dog RBCs released < 1 microM of ATP when passed through tubing with a 25-microm i.d. and a length of 100 cm.

GSH depletion, K-Cl cotransport, and regulatory volume decrease in high-K/high-GSH dog red blood cells.

Thiol reagents activate K-Cl cotransport (K-Cl COT), the Cl-dependent and Na-independent ouabain-resistant K flux, in red blood cells (RBCs) of several species, upon depletion of cellular glutathione (GSH). K-Cl COT is physiologically active in high potassium (HK), high GSH (HG) dog RBCs. In this unique model, we studied whether the same inverse relationship exists between GSH levels and K-Cl COT activity found in other species. The effects of GSH depletion by three different chemical reactions [nitrite (NO(2))-mediated oxidation, diazene dicarboxylic acid bis-N,N-dimethylamide (diamide)-induced dithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB)] were tested on K-Cl COT and regulatory volume decrease (RVD). After 85% GSH depletion, all three interventions stimulated K-Cl COT half-maximally with the following order of potency: diamide > NO(2) > CDNB. Repletion of GSH reversed K-Cl COT stimulation by 50%. Cl-dependent RVD accompanied K-Cl COT activation by NO(2) and diamide. K-Cl COT activation at concentration ratios of oxidant/GSH greater than unity was irreversible, suggesting either nitrosothiolation, heterodithiol formation, or GST-mediated dinitrophenylation of protein thiols. The data support the hypothesis that an intact redox system, rather than the absolute GSH levels, protects K-Cl COT activity and cell volume regulation from thiol modification.

Participation of cAMP in a signal-transduction pathway relating erythrocyte deformation to ATP release.

Previously, we reported that red blood cells (RBCs) of rabbits and humans release ATP in response to mechanical deformation and that this release of ATP requires the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). It was reported that cAMP, acting through a cAMP-dependent protein kinase, PKA, is an activator of CFTR. Here we investigate the hypothesis that cAMP stimulates ATP release from RBCs. Incubation of human and rabbit RBCs with the direct activator of adenylyl cyclase, forskolin (10 or 100 microM), with IBMX (100 microM), resulted in ATP release and increases in intracellular cAMP. In addition, epinephrine (1 microM), a receptor-mediated activator of adenylyl cyclase, stimulated ATP release from rabbit RBCs. Moreover, incubation of human and rabbit RBCs with an active cAMP analog [adenosine 3'5'-cyclic monophosphorothioate Sp-isomer (Sp-cAMP, 100 microM)] resulted in ATP release. In contrast, forskolin and Sp-cAMP were without effect on dog RBCs, cells known not to release ATP in response to deformation. When rabbit RBCs were incubated with the inactive cAMP analog and inhibitor of PKA activity, adenosine 3',5'-cyclic monophosphorothioate Rp-isomer (100 microM), deformation-induced ATP release was attenuated. These results are consistent with the hypothesis that adenylyl cyclase and cAMP are components of a signal-transduction pathway relating RBC deformation to ATP release from human and rabbit RBCs.

Thiopurine Methyltransferase in Red Blood Cells of Dogs, Cats, and Horses

Our objective was to determine if thiopurine methyltransferase (TPMT), the enzyme important in the metabolism of azathioprine in human beings, is detectable in red blood cell lysates (RBCL) of healthy dogs, cats, and horses. Values for TPMT activity were determined from blood collected from 20 healthy dogs, cats, and horses. The TPMT activity in each animal's RBCL was determined using a radioenzymatic end point involving TPMT‐facilitated metabolism of 6‐mercaptopurine to 6‐methylmercaptopurine (6‐MMP). One unit of TPMT activity represents the formation of 1 nmol of 6‐MMP per milliliter of packed red blood cells per hour of incubation at 37°C. TPMT activity in RBCL was detectable in all species, with mean RBC values ± standard deviation of 17.9 ± 3.79 U/mL in dogs; 2.76 ± 0.70 U/mL in cats; and 2.185 ± 0.36 U/mL in horses. Values for TPMT in the 3 species were significantly (P&lt; .05) different from one another. TPMT values for dogs were significantly higher than the other species, and TPMT values for cats were significantly higher than those for horses. We conclude that RBCL TPMT values are measurable in dogs, cats, and horses and that dogs have higher values than cats or horses. These findings are consistent with the lower tolerance for azathioprine in cats as compared with dogs. It remains to be determined whether RBCL TPMT values in these species correlate with TPMT activity in the liver, where most of the metabolization of azathioprine is believed to occur.