• View in gallery
    Figure 1—

    Regression lines showing the linearity of PON1 activity in each of 2 canine serum samples at various dilutions by use of 3 assays: lactonase activity via measurement of TBBL hydrolysis (A and B), arylesterase activity via measurement of phenyl acetate hydrolysis (C and D), and arylesterase activity via measurement of p-nitrophenyl acetate hydrolysis (E and F). Regression equations and coefficients of determination (R2) are shown.

  • View in gallery
    Figure 2—

    Interferograms corresponding to the effect of hemoglobin concentration on PON1 activity as measured by use of 3 assays: lactonase activity via measurement of TBBL hydrolysis (A), arylesterase activity via measurement of phenyl acetate hydrolysis (B), and arylesterase activity via measurement of p-nitrophenyl acetate hydrolysis (C). Values for each given concentration (Vf) are reported as a percentage of the original value (V0).

  • View in gallery
    Figure 3—

    Interferograms corresponding to the effect of triglycerides concentration on PON1 activity as measured by use of the assays in Figure 2. See Figure 2 for remainder of key.

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Validation of spectrophotometric assays for serum paraoxonase type-1 measurement in dogs

Asta TvarijonaviciuteDepartment of Animal Medicine and Surgery, Veterinary School, University of Murcia, 30100 Murcia, Spain

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Fernando TeclesDepartment of Animal Medicine and Surgery, Veterinary School, University of Murcia, 30100 Murcia, Spain

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Marco CaldinSan Marco Veterinary Hospital, Padova, Italy.

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Silvia TascaSan Marco Veterinary Hospital, Padova, Italy.

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José CerónDepartment of Animal Medicine and Surgery, Veterinary School, University of Murcia, 30100 Murcia, Spain

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Abstract

Objective—To evaluate and validate 3 spectrophotometric assays for measuring serum activity of paraoxonase type-1 (PON1), an enzyme associated with high-density lipoproteins, in dogs.

Animals—22 healthy adult dogs and 10 dogs with eccentrocytosis.

Procedures—2 methods were adapted for use in 96-well microplates with phenyl acetate and 5-thiobutyl butyrolactonase as substrates, and 1 was adapted for use in an automated analyzer with p-nitrophenyl acetate as substrate. Blood samples were collected from all dogs, serum was harvested, and serum PON1 activity was measured with each method.

Results—Imprecision was low for all 3 methods, with the exception of interassay imprecision for 5-thiobutyl butyrolactonase, and results were linear across serial sample dilutions. The 3 methods were able to detect low PON1 activity when EDTA was used for blood sample collection, yielded lower PON1 values in sick dogs with eccentrocytosis than in healthy dogs, and yielded highly correlated results.

Conclusions and Clinical Relevance—The methods described here may allow a wider use of PON1 activity as a biomarker of oxidative stress in dogs in clinical and research settings. Results of each method were robust and precise (with the exception of the interassay values for the lactonase method), and the methods were easy to set up in a laboratory.

Abstract

Objective—To evaluate and validate 3 spectrophotometric assays for measuring serum activity of paraoxonase type-1 (PON1), an enzyme associated with high-density lipoproteins, in dogs.

Animals—22 healthy adult dogs and 10 dogs with eccentrocytosis.

Procedures—2 methods were adapted for use in 96-well microplates with phenyl acetate and 5-thiobutyl butyrolactonase as substrates, and 1 was adapted for use in an automated analyzer with p-nitrophenyl acetate as substrate. Blood samples were collected from all dogs, serum was harvested, and serum PON1 activity was measured with each method.

Results—Imprecision was low for all 3 methods, with the exception of interassay imprecision for 5-thiobutyl butyrolactonase, and results were linear across serial sample dilutions. The 3 methods were able to detect low PON1 activity when EDTA was used for blood sample collection, yielded lower PON1 values in sick dogs with eccentrocytosis than in healthy dogs, and yielded highly correlated results.

Conclusions and Clinical Relevance—The methods described here may allow a wider use of PON1 activity as a biomarker of oxidative stress in dogs in clinical and research settings. Results of each method were robust and precise (with the exception of the interassay values for the lactonase method), and the methods were easy to set up in a laboratory.

Serum paraoxonase type-1 is an enzyme associated with high-density lipoproteins. Synthesized by the liver, PON1 protects low-density lipoprotein and high-density lipoprotein particles from oxidative stress by promoting degradation of lipid peroxides contained in cholesteryl esters and phospholipids.1 This enzyme has received increasing attention in human medicine, largely because alterations in circulating PON1 are associated with various diseases involving oxidative stress, such as renal or liver disease or neoplasia.2–4 In addition, PON1 has anti-inflammatory properties.5 The activity of PON1 is reportedly low in humans with type 2 diabetes mellitus6 and obesity,7,8 and low activity may contribute to the development of cardiovascular disease.9 Because of the important role PON may play in lipid metabolism, there is a need for reliable PON assays for clinical and epidemiological studies.10

Activity of PON1 can be assessed by use of various substrates, such as phenyl acetate and its derivates, paraoxon, and butyrolactones, which can be used to measure the arylesterase, triesterase, and lactonase activities of this enzyme, respectively.7,11,12 Use of the paraoxon method in clinical practice is limited because of the extreme toxic effects of paraoxon and the strong influence of PON1 gene polymorphisms on the enzyme's activity.2 Measurements of PON1 lactonase and arylesterase activities are free of these limitations. Although lactonase measurements would better reflect the main activity of PON1, correlation between arylesterase and lactonase activities has been observed in human studies.7

Serum PON1 activity has been measured in dogs with a manual method that involves paraoxon as substrate.13 To the authors' knowledge, no methods for measuring PON1 activity in canine serum by use of substrates other than the toxic paraoxon or through automated or semiautomated assays have been described. The purpose of the study reported here was to evaluate and validate 3 spectrophotometric assays of PON1 activity in canine serum: 2 adapted to a 96-well microplate format with phenyl acetate and TBBL as substrates, and 1 adapted to an automated analyzer with p-nitrophenyl acetate as substrate. The development of facile enzymatic assays with nontoxic substrates that are suitable for high-throughput screening tests could be useful for evaluating the role of PON1 in dogs.

Materials and Methods

Animals—Sixteen 6- to 8-year-old healthy adult dogs (5 sexually intact females, 3 spayed females, 7 sexually intact males, and 1 castrated male) were evaluated at San Marco Veterinary Hospital and included in the study. Breeds represented included mixed (n = 9), Golden Retriever (2), American Cocker Spaniel (2), Yorkshire Terrier (2), and German Shepherd Dog (1). All dogs had no evident acute or chronic disease. Results of physical and clinical examinations were unremarkable, as were results of routine hematologic and serum biochemical analyses. Also included were 6 healthy 6- to 8-year-old Beagles (3 sexually intact females and 3 sexually intact males) from the Murcia Animal Resources Centres.

In addition to the healthy dogs, ten 4- to 15-year old sick dogs (4 sexually intact females, 1 spayed female, 3 sexually intact males, and 2 castrated males) were included in which eccentrocytosis (an indicator of oxidative cell damage) was diagnosed at the San Marco Veterinary Hospital during 2009. These dogs were of various breeds and had a broad range of hematologic abnormalities. Diagnoses were as follows: diabetes mellitus (n = 3), onion intoxication (2), acute leukemia (2), rodenticide intoxication (1), hemangioma (1), and brain tumor (1).

All procedures involving the Beagles from Murcia Animal Resources Centres were performed in accordance with the animal care guidelines of the University of Murcia. Consent for study participation was obtained from owners of the 26 dogs that were evaluated at San Marco Veterinary Hospital.

Sample collection and processing—After food was withheld from the dogs for at least 12 hours overnight, blood samples were collected from each via jugular or lateral saphenous venipuncture into tubes containing EDTA for hematologic analysis and tubes with clotting acceleratora for serum biochemical analysis. Samples were centrifuged at 2,000 × g for 10 minutes at room temperature (20° to 22°C), and serum was harvested. The CBCs and serum biochemical analyses were performed with automatic analyzers.b,c

Total antioxidant capacity was determined as described elsewhere.14 The method used is based on 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate)d decolorization by antioxidants according to their concentrations and antioxidant capacities. The color change is measured as a change in light absorbance at 660 nm. For the process, an automated analyzere was used, and the assay was calibrated with 6-hydroxy-2,5,7,8-tetra-methylchroman-2-carboxylic acid.f

Biochemical measurement of PON1 activity—Serum lactonase activity was analyzed by measuring the hydrolysis of TBBL (assay A). The method involves use of a chromogenic lactone that structurally resembles the proposed natural lipolactone substrates.2 Serum samples were prepared in sample buffer consisting of 50 mM Trisg and 1mM CaCl2h (pH, 8.0) in a 20-fold dilution. One microliter of 100 mM 5,5′-dithio-bis-2-nitrobenzoic acidi in dimethyl sulfoxide,j 45 μL of 4% acetonitrilek solution in sample buffer, 5 μL of diluted serum, and 50 μL of sample buffer then were added to the wells of a 96-well microplate.l Finally, 100 μL of freshly made substrate buffer containing 0.4mM TBBLm solution in sample buffer was added to the wells to initiate the reaction. Two minutes after TBBL addition, the reaction was monitored at 412 nm in an automated microplate readern at 37°C. The nonenzymatic hydrolysis of TBBL, which was based on the hydrolysis rate in the absence of serum, was subtracted from the total hydrolysis rate. Lactonase activity was expressed as units per milliliter of serum, in which 1 unit equals 1 mmol of TBBL hydrolyzed/min. The molar extinction coefficient used to calculate the rate of hydrolysis was 7,000 M−1 cm−1. A path-length correction was applied for the use of microtiter plates.

Serum arylesterase activity was analyzed by measuring the hydrolysis of phenyl acetateo into phenol (assay B) as described elsewhere.11 Serum samples were prepared in sample buffer consisting of 20mM Trisg and 1mM CaCl2h (pH, 8.0) in a 40-fold dilution. Five micro-liters of diluted serum was added to 200 μL of freshly made substrate buffer containing 20mM Tris, 1mM CaCl2, and 1mM phenyl acetate (pH, 8.0). The reaction was monitored on a microtiter platel at 260 nm in an automated microplate readern at 37°C. The nonenzymatic hydrolysis of phenyl acetate, which was based on the hydrolysis rate in the absence of serum, was subtracted from the total hydrolysis rate. Arylesterase activity is expressed as units per milliliter of serum, in which 1 U equals 1 μmol of phenyl acetate hydrolyzed/min. The molar extinction coefficient used to calculate the rate of hydrolysis was 1,310 M−1 cm−1. A path-length correction was applied for the use of microtiter plates.

Serum arylesterase activity was also analyzed by measuring the hydrolysis of p-nitrophenyl acetate to p-nitrophenol as described elsewhere15 but with a modification to remove substrate from the working reagent buffer and prepare it in water as a separate starting reagent that remained colorless (assay C).16 This modification was made because p-nitrophenylacetate is subject to considerable spontaneous hydrolysis in the reagent buffer system originally described, and this hydrolysis was grossly apparent in the yellow color of the reagent. The starting reagent was added to initiate the kinetic reaction.

Because p-nitrophenyl acetatep is insoluble in water, 63 mg of this compound was dissolved in 10 mL of methanolq and stored at 2° to 8°C. In our experience, this stock solution can be kept for approximately 1 week with only a small increase in free p-nitrophenol. Afterward, 1 mL of this solution was slowly added to 20 mL of distilled water with strong agitation to prevent precipitation. The aqueous solution was freshly prepared each day.

To perform assay C, serum samples were each mixed with 307 μL of buffer containing 50mM Tris and 1mM CaCl2 (pH, 8.0) and then freshly made substrate containing 2.5mM p-nitrophenyl acetate in distilled water was added. After 100 seconds, the reaction was monitored at 405 nm at 37°C for 210 seconds in an automated biochemistry analyzer.r The nonenzymatic hydrolysis of phenyl acetate, which was based on the hydrolysis rate in the absence of serum, was subtracted from the total hydrolysis rate. The activity was expressed as units per milliliter of serum, in which 1 U equals 1 μmol of phenyl acetate hydrolyzed/min. The molar extinction coefficient used to calculate the rate of hydrolysis was 14,000 M−1 cm−1.

Assay validation—For analytic validation of the 3 methods used for PON1 activity measurement, the following characteristics were calculated: precision, intra-assay CV, accuracy, and limit of detection. Two pools of sera with different PON1 activities were prepared from the group of healthy dogs and the group of dogs with eccentrocytosis and used for precision evaluations. To determine interassay precision, these pools were divided in aliquots and stored in plastic vials at −20°C until analysis. On the day of analysis, samples were brought to room temperature prior to PON1 measurement. Intra-assay CV was calculated after analysis of the 2 serum pools 6 times in 1 assay run. Interassay CV was determined by analyzing the same pools in 6 separate runs performed on different days.

Because of the lack of a criterion-referenced standard or commercially available certified reference material for canine PON activity, assay accuracy was evaluated indirectly through assessment of dilutional linearity. For this process, 2 canine serum samples were serially diluted with saline (0.9% NaCl) solution and analyzed by use of the procedures previously described. Limit of detection was calculated on the basis of data from 20 replicate determinations of the zero standard (buffer of assays) as mean value plus 2 SDs.

To determine whether results of the 3 assays were correlated, serum samples from all study dogs were evaluated with each assay. All samples used for repetitive analysis were frozen in aliquots, and to avoid possible changes due to repetitive thawing and freezing, only vials needed for each run were used.

Effects of hemolysis and lipemia—To investigate effect of hemolysis and lipemia on results of the 3 assays for PON1 activity, 3 serum samples from 3 healthy Beagles were mixed with various concentrations of hemoglobin or lipid solution as described,17 and each preparation was run in duplicate. For hemolysis investigation, a fresh hemolysate was prepared by addition of distilled water to packed, saline solution–washed RBCs from 1 Beagle. Hemoglobin concentration in hemolysate was determined by use of a veterinary animal blood cell counterb and adjusted to 200 g/L by adding assay buffer to create a stock solution. The stock solution was serially diluted with sample buffer, and 10 μL of each dilution was added to three 90-μL samples of canine serum. The final hemoglobin concentrations were 8, 4, 2, 1, 0.5, and 0.0 g/L. The 0.0 g/L concentration was achieved by adding 10 μL of sample diluent buffer. These hemoglobin concentrations were intended to correspond to slight hemolysis (0.5 g/L), moderate hemolysis (1 and 2 g/L), and marked hemolysis (4 and 8 g/L). Prepared samples were used to measure PON1 activity with the 3 assays.

For the lipids investigation, commercial fat emulsions with a triglycerides concentration of 200 g/L was serially diluted with sample diluent buffer and 10 μL of each dilution was added to two 90-μL samples of canine serum. Homogeneity was achieved by mixing with a vortex device. The final triglycerides concentrations were 5, 2.50, 1.25, 0.625, and 0.3125 g/L, which were intended to correspond to slight lipemia (0.3125 and 0.625 g/L), moderate lipemia (1.25 and 2.5 g/L), and marked lipemia (5 g/L). Prepared samples were used to measure PON1 activity with the 3 assays.

Effects of EDTA—Blood samples (8 mL each) from 6 Beagles were obtained via jugular venipuncture by use of a disposable syringe and 21-gauge needle. Aliquots of the samples were placed in 2 types of tubes: one containing 0.072 mL of 7.5% tripotassium meth-ylenediaminetetraacetic acid (EDTA K3)t for EDTA-treated plasma and the other containing clot activator (5-mL tubea) for serum. All samples were centrifuged at 2,000 × g for 10 minutes, and serum and EDTA-treated plasma were immediately separated prior to analysis on the same day. The interval between collection and centrifugation was approximately 30 minutes. All plasma and serum samples were analyzed by use of the 3 study methods to measure PON1 activity.

Statistical analysis—Arithmetic means, medians, and intra-assay and interassay CVs were calculated by use of routine descriptive statistical procedures and computer software.u Dilutional linearity was evaluated through ordinary linear regression analysis in which the measured activities of PON1 were compared with the expected activities. Correlation among results obtained with the 3 methods was assessed by use of linear regression. Interferograms were prepared to show the differences in PON1 activities when hemoglobin or triglycerides were added. The influence of hemoglobin or triglycerides on PON1 activity was investigated by use of 1-way ANOVA and Dunnett posttests. Comparisons of the results for serum and EDTA-treated plasma were made by use of a Student t test for repeated measurements. A Student t test was used to evaluate the difference of TAC and PON1 activities between healthy dogs and dogs with eccentrocytosis. The correlation between PON1 activity and TAC was evaluated by calculation of the Spearman correlation coefficient (ρ). Values of P < 0.05 were considered significant for all analyses.

Results

Assays—For assay A (serum lactonase activity by use of microplates), intra-assay and interassay CV ranges were 5.5% to 6.7% and 11.3% to 17.7%, respectively (Table 1). Serial dilution of 2 serum samples resulted in linear regression equations with correlation coefficients close to 1 (r = 0.997 and 0.953; Figure 1). The assay detection limit was 1.0 U/mL (mean ± SD, 0.4 ± 0.2 U/mL). For assay B, intra-assay and interassay CV ranges were 4.0% to 4.8% and 5.4% to 9.5%, respectively. Serial dilution of 2 serum samples resulted in linear regression equations with correlation coefficients close to 1 (r = 0.999 and 0.996). The assay detection limit was 9.1 U/mL (mean ± SD, 1.8 ± 2.5 U/mL). For assay C, intra- and interassay CV ranges were 4.5% to 5.6% and 7.3% to 8.2%. Serial dilution of 2 serum samples resulted in linear regression equations with correlation coefficients close to 1 (r = 0.997 and 0.995). The assay detection limit was 0.6 U/mL (mean ± SD, 0.49 ± 0.05 U/mL).

Figure 1—
Figure 1—

Regression lines showing the linearity of PON1 activity in each of 2 canine serum samples at various dilutions by use of 3 assays: lactonase activity via measurement of TBBL hydrolysis (A and B), arylesterase activity via measurement of phenyl acetate hydrolysis (C and D), and arylesterase activity via measurement of p-nitrophenyl acetate hydrolysis (E and F). Regression equations and coefficients of determination (R2) are shown.

Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.34

Table 1—

Intra-assay and interassay variation in PON1 activity in 2 pools of canine serum samples (healthy dogs [pool 1] and dogs with eccentrocytosis [pool 2]) as measured by use of lactonase activity via measurement of TBBL hydrolysis (assay A), arylesterase activity via measurement of phenyl acetate hydrolysis (assay B), and arylesterase activity via measurement of p-nitrophenyl acetate hydrolysis (assay C).

AssayComparisonPoolMean ± SD activity (U/mL)Mean CV (%)
AIntra-assay14.3 ± 0.25.5
  21.0 ± 0.26.7
 Interassay12.0 ± 0.917.7
  24.4 ± 0.511.3
BIntra-assay120.1 ± 0.84.0
  215.7 ± 0.84.8
 Interassay121.7 ± 1.25.4
  217.5 ± 1.79.5
CIntra-assay15.1 ± 0.24.5
  22.6 ± 0.25.6
 Interassay15.5 ± 0.47.3
  23.1 ± 0.38.2

When results for 38 serum samples analyzed with the 3 assays were used, linear regression analysis revealed significant correlations between the results of assays A and B (P < 0.001; r = 0.852), A and C (P < 0.001; r = 0.850), and B and C (P < 0.001; r = 0.870).

Effects of hemolysis and lipemia on assay results—For assay A, a significant decrease in PON1 activities was evident when hemoglobin was added to the serum samples at a concentration of 8 g/L or when triglycerides were added at a concentration of 5 g/L (Figures 2 and 3). For assay B, a significant decrease in PON1 activities appeared when triglycerides at a concentration of 5 g/L were added to the samples. No substantial interference in PON1 activities by hemoglobin was evident at concentrations < 8 g/L. For assay C, a significant decrease in PON1 activities was evident when triglycerides at a concentration of ≥ 1.25 g/L were added to the samples. No significant interference by hemoglobin was evident at concentrations < 8 g/L.

Figure 2—
Figure 2—

Interferograms corresponding to the effect of hemoglobin concentration on PON1 activity as measured by use of 3 assays: lactonase activity via measurement of TBBL hydrolysis (A), arylesterase activity via measurement of phenyl acetate hydrolysis (B), and arylesterase activity via measurement of p-nitrophenyl acetate hydrolysis (C). Values for each given concentration (Vf) are reported as a percentage of the original value (V0).

Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.34

Figure 3—
Figure 3—

Interferograms corresponding to the effect of triglycerides concentration on PON1 activity as measured by use of the assays in Figure 2. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.34

Effects of EDTA on assay results—Results obtained when serum and EDTA-treated plasma samples were evaluated by use of the 3 methods were summarized (Table 2). Significant (P < 0.001) decreases in PON1 activity were detected in EDTA-treated plasma samples versus serum samples for each assay.

Table 2—

Activities of PON1 in serum and EDTA-treated plasma sample aliquots as measured by use of 3 assays.

AssaySampleActivity in serum (U/mL)Activity in plasma with EDTA (U/mL)
A14.591.03
 24.360.46
 34.251.26
 43.670.34
 54.711.95
 64.360.69
 Mean ± SD4.32 ± 0.360.96 ± 0.60*
B121.289.10
 221.289.10
 318.789.10
 418.789.10
 520.0311.27
 615.029.10
 Mean ± SD19.20 ± 2.339.46 ± 0.89*
C16.511.87
 24.831.09
 35.334.22
 44.921.05
 55.632.38
 64.691.17
 15.32 ± 0.681.96 ± 1.22*

Value differs significantly (P < 0.001) from value for serum.

Assay discrimination—Activities of PON1 as well as TAC in dogs with eccentrocytosis were significantly lower than those in healthy dogs when analyzed with the 3 methods (Table 3). The Spearman correlation test revealed a positive, significant correlation between TACs and results of assays A (ρ = 0.64; P = 0.002), B (ρ = 0.49; P = 0.027), and C (ρ = 0.48; P = 0.033).

Table 3—

Mean ± SD PON1 activity determined by use of 3 assays and TAC in 10 dogs with eccentrocytosis and 22 healthy dogs.

AnalyteDogs with eccentrocytosisHealthy dogsP value
PON1 activity (U/mL)   
   Assay A4.10 ± 1.224.82 ± 0.500.048
   Assay B14.97 ± 6.1320.00 ± 2.370.023
   Assay C4.82 ± 1.446.31 ± 0.680.010
TAC (mmol)0.35 ± 0.290.66 ± 0.180.011

A value of P < 0.05 was considered significant.

Discussion

In the study reported here, 3 spectrophotometric methods for measurement of PON1 activity were evaluated and validated. A different substrate was used in each assay: TBBL for lactonase activity and p-nitrophenyl acetate and phenyl acetate for arylesterase activity. Paraoxon is a highly toxic and unstable compound that is unsuitable for routine high-throughput use,2 so measurements of paraoxonase activity of PON1 were not included in the present study. In humans, arylesterase or lactonase activities are less variable between subjects than is paraoxonase activity, which is influenced by genetic variation.2,6,10

Although PON1 was long considered to be an arylesterase and paraoxonase and its activity was measured accordingly, it recently became apparent that PON1 can catalyze the hydrolysis and formation of various lactones. Lactonase activity is the only activity shared by all other members of the PON family, some of which have no paraoxonase or arylesterase activity.18 Some investigators have consequently postulated that use of phenyl acetate or paraoxon would not have physiologic relevance and have suggested that assays of PON1 activity should mainly address the lactonase activity.12 However, because the substrate for lactonase activity measurements is not widely available and a correlation between arylesterase and lactonase activities has been reported for humans,7 we aimed to validate assays not only for lactonase activity but also for arylesterase activity.

Initially, we intended to fully automate all 3 methods to improve the assay precision and decrease the time needed to analyze PON1 activity in each sample. However, the assays that involved phenylacetate and lactonase were validated in a 96-well ELISA plate format because the absorbance of phenylacetate at < 340 nm and instability of lactonase in aqueous solutions precluded its adaptation to automated analyzers. Although these assays were not fully automated, analysis involving a 96-well ELISA plate is fast and enables simultaneous processing of large batches of serum samples. In addition, spectrophotometric readers for 96-well ELISA plates are widely available in biochemistry laboratories, so this technology could be suitable for use in veterinary diagnostic laboratories.19

The validation results showed that the 3 methods were practical, rapid, and allowed evaluation of multiple samples at the same time; they also required a low volume of sample. These characteristics are also important if the measurement is to be included in a battery of several tests or in situations in which sample volume might be limited. Both arylesterase assays had CVs < 10% in all assessed situations, which is similar to assay validation findings in humans.20 The low CVs suggest a good assay reproducibility and support their suitability for use in other studies. However, the lactonase method had an interassay CV of 17.7%, which is similar to values reported when the method is used in humans.2 Ideally, when lactonase activity is the analyte of interest, serum samples should be analyzed in the same batch to avoid these interassay variations. Perhaps because its suitability for automation diminishes analytic errors, p-nitrophenyl acetate is reportedly more sensitive than other substrates for detecting variations in PON1 activity.21 Moreover, p-nitrophenyl acetate is a nontoxic ester that is less volatile than lactone and thus would be more suitable for routine measurement of PON1 activity.21

Hemolysis did not result in substantial interferences in the arylesterase assays, and only a significant decrease in serum PON1 activity was evident with the lactonase method at a high hemoglobin concentration (8 g/L). This finding is similar to findings in humans regarding the lactonase assay; in humans, results are not affected by hemolysis when hemoglobin values of < 6 g/L are present,2 indicating hemolysis has no influence on PON1 assay results unless it is severe. In lipemic serum samples, significant decreases in PON1 activities were observed with increasing triglycerides concentration with all 3 methods. A negative correlation between serum PON1 activity and triglycerides has been reported for humans.22 In addition, lipemic samples can interfere with many clinical chemistry tests through various mechanisms, the most common of which is the scattering of light rays by the lipids (mainly chylomicrons and very low-density lipoproteins).23

Results of all the methods used in the present study were highly correlated. In humans, similarly significant correlations exist between results of arylesterase and lactonase assays.7 However, results of PON1 activity testing in humans with certain diseases can vary depending of the substrate used, so the simultaneous use of at least 2 substrates to measure PON1 activity is recommended to improve the reliability of the results.24

To test the ability of the methods to detect and differentiate between PON1 activities, we performed in vitro assays to assess the influence of EDTA and oxidative dog illness (eccentrocytosis) on PON1 activity. Reportedly, PON1 has an absolute requirement for Ca2+ for activity and stability, so use of EDTA can inactivate PON1.25 Our results are in agreement with previous findings26 that a significant decrease of PON1 activity occurs when EDTA-treated plasma is used instead of serum.

Eccentrocytes are erythrocytes in which hemoglobin is concentrated on 1 side of the cell, leaving a pale or clear eccentric space. These cells are associated with oxidative injury of erythrocytes in humans and animals.27–29 Increased amounts of endogenous oxidants are generated in various disorders, such as inflammation, neoplasia, and diabetes, or when reducing pathways in erythrocytes are defective, as occurs with glucose-6-phosphate dehydrogenase and flavin adenine dinucleotide deficiencies.30,31 In the present study, dogs with eccentrocytosis had lower TAC values than healthy dogs, indicating oxidative damage.32,33 The decrease of PON1 in dogs with eccentrocytosis in the present study indicates that the validated methods were able to detect lower PON1 values associated with oxidative stress. As has been reported for humans,34 a significant correlation was detected between PON1 activities and TACs. These data indicate that the PON1 methods described here can be used to evaluate oxidative status in dogs.

The 3 assays described here will allow a wider use of PON1 activity as a biomarker of oxidative stress in dogs because the assays were robust, precise (with the exception of the interassay values for lactonase method), and easy to set up in laboratories. As in human medicine, these assays could allow additional research into PON1 behavior in situations that can alter oxidative status, such as sepsis and renal or chronic liver disease in dogs; eventually, such studies could lead to applications already described for humans, such as the use of PON1 as a prognostic marker of disease progress and recovery in patients with chronic liver disease2,35 or sepsis.18,36

ABBREVIATIONS

CV

Coefficient of variation

PON

Serum paraoxonase

TAC

Total antioxidant capacity

TBBL

5-thiobutyl butyrolactonase

a.

TapVal, Aquisel, Barcelona, Spain.

b.

Advia 120 hematology system, Siemens, Italy.

c.

AU 400 automated biochemical analyzer, Olympus, Minneapolis, Minn.

d.

2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), diammonium salt, Sigma-Aldrich Co, St Louis, Mo.

e.

Cobas Mira Plus multiparametric autoanalyser, ABX Diagnostics Inc, Montpellier, France.

f.

(R)-(+)-6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Sigma-Aldrich Co, St Louis, Mo.

g.

Tris(hydroxymethyl)aminomethane, Sigma-Aldrich Co, St Louis, Mo.

h.

Calcium chloride dihydrate, Sigma-Aldrich Co, St Louis, Mo.

i.

5,5′-Dithiobis(2-nitrobenzoic acid), Sigma-Aldrich Co, St Louis, Mo.

j.

Dimethyl sulfoxide, Sigma-Aldrich Co, St Louis, Mo.

k.

Acetonitrile, Mulrisolvent HPLC grade ACS, Sharlau Chemie SA, Sentmenat, Spain.

l.

Microtiter plates, polystyrene 96-wells, Sigma-Aldrich Co, St Louis, Mo.

m.

Provided by Dr. Khersonsky, Weizmann Institute of Science, Israel.

n.

PowerWave XS, Bio-Tek Instruments Inc, Winooski, Vt.

o.

Phenyl acetate, Sigma-Aldrich Co, St Louis, Mo.

p.

4-nitrophenyl acetate, Sigma-Aldrich Co, St Louis, Mo.

q.

Methanol, Sigma-Aldrich Co, St Louis, Mo.

r.

Coulter CPA, Kemia Cientifica SA, Madrid, Spain.

s.

Lipofundina 20%, Braun Medical SA, Barcelona, Spain.

t.

BD Vacutainer, BD, Madrid, Spain.

u.

GraphPad Prism, version 5 for Windows, Graph Pad Software Inc, San Diego, Calif.

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Contributor Notes

Address correspondence to Dr. Cerón (jjceron@um.es).