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  • Author or Editor: Jan S. Suchodolski x
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Abstract

Objective—To develop and analytically validate a radioimmunoassay (RIA) for the quantification of canine calprotectin (cCP) in serum and fecal extracts of dogs.

Sample Population—Serum samples (n = 50) and fecal samples (30) were obtained from healthy dogs of various breeds and ages.

Procedures—A competitive, liquid-phase, double-antibody RIA was developed and analytically validated by assessing analytic sensitivity, working range, linearity, accuracy, precision, and reproducibility. Reference intervals for serum and fecal cCP concentrations were determined.

Results—Sensitivity and upper limit of the working range were 29 and 12,774 μg/L for serum and 2.9 and 1,277.4 μg/g for fecal extracts, respectively. Observed-to-expected ratios for serial dilutions of 6 serum samples and 6 fecal extracts ranged from 95.3% to 138.2% and from 80.9% to 118.1%, respectively. Observed-to-expected ratios for spiking recovery for 6 serum samples and 6 fecal extracts ranged from 84.6% to 121.5% and from 80.3% to 132.1%, respectively. Coefficients of variation for intra-assay and interassay variability were < 3.9% and < 8.7% for 6 serum samples and < 8.5% and < 12.6% for 6 fecal extracts, respectively. Reference intervals were 92 to 1,121 μg of cCP/L for serum and < 2.9 to 137.5 μg of cCP/g for fecal extracts.

Conclusions and Clinical Relevance—The RIA described here was analytically sensitive, linear, accurate, precise, and reproducible for the quantification of cCP in serum and fecal extracts. This assay should facilitate research into the clinical use of serum and fecal cCP measurements in dogs with inflammatory bowel disease.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To develop and analytically validate a gas chromatography–mass spectrometry (GC-MS) method for the quantification of lactulose, rhamnose, xylose, 3-O-methylglucose, and sucrose in canine serum.

Sample Population—Pooled serum samples from 200 dogs.

Procedures—Serum samples spiked with various sugars were analyzed by use of GC-MS. The method was analytically validated by determination of dilutional parallelism, spiking recovery, intra-assay variability, and interassay variability.

Results—Standard curves ranging from 0.5 to 500 mg/L for each sugar revealed a mean r 2 of 0.997. The lower detection limit was 0.03 mg/L for lactulose, rhamnose, xylose, and methylglucose and 0.12 mg/L for sucrose. The observed-to-expected ratios for dilutional parallelism had a mean ± SD of 105.6 ± 25.4% at dilutions of 1:2, 1:4, and 1:8. Analytic recoveries for the GC-MS assays of sugars ranged from 92.1% to 124.7% (mean ± SD, 106.2 ± 13.0%). Intra-assay coefficients of variation ranged from 6.8% to 12.9% for lactulose, 7.1% to 12.8% for rhamnose, 7.2% to 11.2% for xylose, 8.9% to 11.5% for methylglucose, and 8.9% to 12.0% for sucrose. Interassay coefficients of variation ranged from 7.0% to 11.5% for lactulose, 6.4% to 9.4% for rhamnose, 6.8% to 13.2% for xylose, 7.0% to 15.9% for methylglucose, and 5.5% to 9.4% for sucrose.

Conclusions and Clinical Relevance—The GC-MS method described here was accurate, precise, and reproducible for the simultaneous measurement of sugar probes in canine serum.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To purify neutrophil elastase (NE) from dog blood and develop and validate an ELISA for the measurement of canine NE (cNE) in canine serum as a marker for gastrointestinal tract inflammation.

Sample Population—Neutrophils from 6 dogs immediately after they were euthanatized and serum from 54 healthy dogs.

Procedures—cNE was purified from blood by use of dextran sedimentation, repeated cycles of freezing-thawing and sonication, cation-exchange chromatography, and continuous elution electrophoresis. Antibodies against cNE were generated in rabbits, and an ELISA was developed and validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and interassay variability. A reference range was established by assaying serum samples from the 54 healthy dogs and by use of the lower 97.5th percentile.

Results—cNE was successfully purified from blood, and antibodies were successfully generated in rabbits. An ELISA was developed with a sensitivity of 1,100 μg/L. The reference range was established as < 2,239 μg/L. Ratios of observed-to-expected results for dilutional parallelism for 4 serum samples ranged from 85.4% to 123.1%. Accuracy, as determined by spiking recovery, ranged from 27.1% to 114.0%. Coefficient of variation for 4 serum samples was 14.2%, 16.0%, 16.8%, and 13.4%, respectively, for intra-assay variability and 15.4%, 15.0%, 10.5%, and 14.6%, respectively, for interassay variability.

Conclusions and Clinical Relevance—The purification protocol used here resulted in rapid and reproducible purification of cNE with a high yield. The novel ELISA yielded linear results and was accurate and precise. Additional studies are needed to evaluate the clinical usefulness of this assay.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the optimal sample handling and processing conditions for the carbon 13 (13C)-labeled aminopyrine demethylation blood test (ADBT; phase 1) and determine the reference range for test results (phase 2) in apparently healthy dogs.

Animals—44 apparently healthy dogs (phase 1, 19 dogs; phase 2, 44 dogs).

Procedures—In phase 1, a blood sample from each dog was collected before and 45 minutes after (day 0) IV administration of 13C-labeled aminopyrine (2 mg/kg); aliquots were immediately transferred into tubes containing sodium heparin and hydrochloric acid (samples A and B), sodium heparin alone (samples C, D, and E), or sodium fluoride (sample F). Hydrochloric acid was added to samples C through F at days 7, 14, 21, and 21, respectively. The baseline and 45-minute samples' absolute 13C:12C ratios were determined via fractional mass spectrometry on day 0 (control sample A) or 21 (samples B through F) and used to calculate the percentage dose of 13C recovered in CO2 extracted from samples (PCD). In phase 2, blood samples from each dog were collected into tubes containing sodium fluoride and processed within 3 weeks.

Results—Compared with the control sample value, PCDs for samples C through E differed significantly, whereas PCD in sample F did not. The 13C-ADBT–derived PCD reference range (central 95th percentile) for apparently healthy dogs was 0.08% to 0.2%.

Conclusions and Clinical Relevance—Glycolytic CO2 production in canine blood samples collected during 13C-ADBTs was sufficiently inhibited by sodium fluoride to allow delayed sample analysis and avoid transportation of hydrochloric acid–treated samples.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE

To develop and analytically validate a liquid chromatography–tandem mass spectrometry method for measurement of endogenous trans-4-hydroxy-l-proline concentrations in canine serum and to assess serum trans-4-hydroxy-l-proline concentrations in dogs with chronic hepatitis.

SAMPLE

Serum samples obtained from 20 dogs with histopathologically confirmed chronic hepatitis and 20 healthy control dogs.

PROCEDURES

A liquid chromatography–tandem mass spectrometry method for quantification of trans-4-hydroxy-l-proline concentration was developed and assessed for analytic sensitivity, linearity, accuracy, precision, and reproducibility. Serum concentration of trans-4-hydroxy-l-proline in dogs with chronic hepatitis and healthy control dogs was measured.

RESULTS

Observed-to-expected ratios for dilutional parallelism ranged from 72.7% to 111.5% (mean ± SD, 91.3 ± 19.6%). Intra-assay and interassay coefficients of variation ranged from 2.1% to 3.0% and 3.2% to 5.3%, respectively. Relative error ranged from −2.3% to 7.8%. Trans-4-hydroxy-l-proline concentrations were significantly lower in serum obtained from dogs with chronic hepatitis (median, 0.24 ng/mL; range, 0.06 to 1.84 ng/mL) than in serum obtained from healthy control dogs (median, 0.78 ng/mL; range, 0.14 to 4.90 ng/mL).

CONCLUSIONS AND CLINICAL RELEVANCE

The method described here for the quantification of trans-4-hydroxy-l-proline concentration in canine serum was found to be sensitive, specific, precise, accurate, and reproducible. Dogs with chronic hepatitis had significantly lower serum trans-4-hydroxy-l-proline concentrations than did healthy control dogs, possibly as a result of altered hepatic metabolism of amino acids.

Full access
in American Journal of Veterinary Research