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  • Author or Editor: Jan S. Suchodolski x
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Abstract

Objective—To purify and partially characterize feline pepsinogen (fPG) from the gastric mucosa and compare fPG with PGs of other species.

Sample Population—Stomachs of 6 cats.

Procedure—A crude protein extract was prepared from the gastric mucosa of feline stomachs. Feline PG A was purified by ammonium sulfate precipitation, weak-anion-exchange chromatography, size-exclusion chromatography, and strong-anion exchange chromatography. Partial characterization consisted of estimation of molecular weights (MWs) and isoelectric points, N-terminal amino acid sequencing, and investigation of susceptibility to pepstatin inhibition.

Results—Several fPG A-group isoforms were identified. The MWs of the isoforms ranged from 37,000 to 44,820. Isoelectric points were all < pH 3.0. The proteolytic activity of the activated PGs was inhibited completely by pepstatin in a range of equimolar to 10- fold molar excess. The specific absorbance of fPG A was 1.29. The N-terminal amino acid sequence of the first 25 residues of the predominant fPG A7 had 75%, 72%, 64%, and 56% homology with PG A of dogs, rabbits, cattle, and humans, respectively. Sequences of 4 other fPG A-group isoforms were similar to fPG A7. All isoforms were immunologically cross-reactive with sheep anti-fPG A7 antiserum.

Conclusions and Clinical Relevance—PG A is the only identified type of PG in cats and, similar to pg in other species, comprises multiple isoforms. The availability of fPG A may be used to facilitate the development of an immunoassay to quantify serum fPG A as a potential marker for gastric disorders in cats. (Am J Vet Res 2004;65:1195–1199)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the prevalence of hypocobalaminemia or methylmalonic acidemia (or both) in dogs with chronic gastrointestinal disease.

Sample—Serum samples from 56 dogs with chronic gastrointestinal disease and 43 control dogs.

Procedures—Serum cobalamin and methylmalonic acid (MMA) concentrations were measured in all samples and compared between groups. A correlation between serum cobalamin and MMA concentrations and the canine chronic enteropathy clinical activity index was evaluated via the Spearman rank correlation.

Results—20 of 56 (36%) dogs with gastrointestinal disease had hypocobalaminemia. Serum cobalamin concentrations were significantly lower in dogs with gastrointestinal disease than in control dogs. Five of 56 (9%) dogs with chronic gastrointestinal disease and 5 of 20 (25%) hypocobalaminemic dogs had increased MMA concentrations. There was a significant negative correlation (Spearman r = −0.450) between serum cobalamin and MMA concentrations in dogs with gastrointestinal disease. No correlation was found between the canine chronic enteropathy clinical activity index and serum cobalamin or MMA concentrations.

Conclusions and Clinical Relevance—These data indicated the prevalence of hypocobalaminemia in dogs with chronic gastrointestinal disease was 20 of 56 (36%). Five of 20 (25%) hypocobalaminemic dogs had increased serum MMA concentrations, which indicated that although hypocobalaminemia was common in these dogs, it did not always appear to be associated with a deficiency of cobalamin on a cellular level. Hypocobalaminemia is a risk factor for negative outcome in dogs with chronic gastrointestinal disease and should be considered in every patient with corresponding clinical signs.

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 validate an ELISA for measurement of serum canine pepsinogen A (cPG A) as a diagnostic marker of gastric disorders in dogs and to measure serum cPG A in healthy dogs after food deprivation and after feeding.

Sample Population—Sera from 72 healthy dogs.

Procedure—A sandwich ELISA was developed and validated. The reference range for serum concentrations of cPG A was determined in 64 healthy dogs. Postprandial changes in serum concentrations of cPG A were evaluated in 8 healthy dogs.

Results—Assay sensitivity was 18 µg/L, and the maximum detectable concentration was 1,080 µg/L. The observed-to-expected ratio (O:E) for 3 serial dilutions of 3 serum samples ranged from 69.3 to 104.1%. The O:E for 3 serum samples spiked with 8 concentrations of cPG A ranged from 58.8 to 120.4%. Coefficients of variation for intra- and interassay variability of 3 serum samples ranged from 7.6 to 11.9% and from 10.1 to 13.1%, respectively. Mean ± SD serum concentration of cPG A in healthy dogs was 63.8 ± 31.0 µg/L and the reference range was 18 to 129 µg/L. Significant increases in serum concentrations of cPG A were observed between 1 and 7 hours after feeding.

Conclusions and Clinical Relevance—The ELISA for measuring cPG A was sufficiently sensitive, linear, accurate, precise, and reproducible for clinical use. Serum concentrations of cPG A increase substantially after feeding, which should be taken into account when conducting clinical studies. (Am J Vet Res 2003;64:1146–1150)

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate the serine protease inhibitor, Kazal type 1 (SPINK1) gene for variants and to determine their possible association with pancreatitis in Miniature Schnauzers.

Animals—39 Miniature Schnauzers with pancreatitis, 25 healthy Miniature Schnauzers, and 23 healthy dogs of other breeds.

Procedures—The entire canine SPINK1 gene with its intron-exon boundaries was initially sequenced in 22 Miniature Schnauzers. Then, 2 regions of the gene were sequenced in 65 additional canine DNA samples at the locations of variants identified in the initial sequencing of the entire SPINK1 gene.

Results—Analysis of the SPINK1 gene in Miniature Schnauzers revealed 3 closely associated variants in healthy Miniature Schnauzers and Miniature Schnauzers with pancreatitis. These variants consisted of 2 missense mutations in the second exon (N20K and N25T) and a poly T insertion in the third intron that was near the boundary of exon 3 (IVS3+26–27ins(T)33–39,15_61dup11). Pancreatitis was significantly associated with homozygous alleles for these 3 variants in Miniature Schnauzers. In healthy dogs of other breeds, only the 2 exon variants were identified.

Conclusions and Clinical Relevance—Variants of the SPINK1 gene may be associated with the development of pancreatitis in Miniature Schnauzers.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To analytically validate a gas concentration of chromatography–mass spectrometry (GC-MS) method for measurement of 6 amino acids in canine serum samples and to assess the stability of each amino acid after sample storage.

SAMPLES Surplus serum from 80 canine samples submitted to the Gastrointestinal Laboratory at Texas A&M University and serum samples from 12 healthy dogs.

PROCEDURES GC-MS was validated to determine precision, reproducibility, limit of detection, and percentage recovery of known added concentrations of 6 amino acids in surplus serum samples. Amino acid concentrations in serum samples from healthy dogs were measured before (baseline) and after storage in various conditions.

RESULTS Intra- and interassay coefficients of variation (10 replicates involving 12 pooled serum samples) were 13.4% and 16.6% for glycine, 9.3% and 12.4% for glutamic acid, 5.1% and 6.3% for methionine, 14.0% and 15.1% for tryptophan, 6.2% and 11.0% for tyrosine, and 7.4% and 12.4% for lysine, respectively. Observed-to-expected concentration ratios in dilutional parallelism tests (6 replicates involving 6 pooled serum samples) were 79.5% to 111.5% for glycine, 80.9% to 123.0% for glutamic acid, 77.8% to 111.0% for methionine, 85.2% to 98.0% for tryptophan, 79.4% to 115.0% for tyrosine, and 79.4% to 110.0% for lysine. No amino acid concentration changed significantly from baseline after serum sample storage at −80°C for ≤ 7 days.

CONCLUSIONS AND CLINICAL RELEVANCE GC-MS measurement of concentration of 6 amino acids in canine serum samples yielded precise, accurate, and reproducible results. Sample storage at −80°C for 1 week had no effect on GC-MS results.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate the qualitative variation in bacterial microflora among compartments of the intestinal tract of dogs by use of a molecular fingerprinting technique.

Animals—14 dogs (similarly housed and fed identical diets).

Procedure—Samples of intestinal contents were collected from the duodenum, jejunum, ileum, colon, and rectum of each dog. Bacterial DNA was extracted from the samples, and the variable V6 to V8 region of 16S ribosomal DNA (gene coding for 16S ribosomal RNA) was amplified by use of universal bacterial primers; polymerase chain reaction amplicons were separated via denaturing gradient gel electrophoresis (DGGE). Similarity indices of DGGE banding patterns were used to assess variation in the bacterial microflora among different compartments of the intestine within and among dogs. Bacterial diversity was assessed by calculating the Simpson diversity index, the Shannon-Weaver diversity index, and evenness.

Results—DGGE profiles indicated marked differences in bacterial composition of intestinal compartments among dogs (range of similarity, 25.6% to 36.6%) and considerable variation among compartments within individual dogs (range of similarity, 36.7% to 57.9%). Similarities between neighboring intestinal compartments were significantly greater than those between non-neighboring compartments. Diversity indices for the colon and rectum were significantly higher than those of the duodenum, jejunum, and ileum.

Conclusions and Clinical Relevance—Results indicated that the different intestinal compartments of individual dogs appear to host different bacterial populations, and these compartmental populations vary among dogs. In dogs, fecal sample analysis may not yield accurate information regarding the composition of bacterial populations in compartments of the gastrointestinal tract. (Am J Vet Res 2005;66:1556–1562)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To measure serum calprotectin concentration in dogs with inflammatory bowel disease (IBD) before and after initiation of treatment and evaluate its correlation with a clinical scoring system (canine IBD activity index), serum canine C-reactive protein concentration, and severity of histopathologic changes.

Animals—34 dogs with idiopathic IBD and 139 healthy control dogs.

Procedures—From dogs with IBD, blood samples were collected immediately before (baseline) and 3 weeks after initiation of 1 of 2 treatments: prednisone (1 mg/kg, PO, q 12 h; n = 21) or a combination of prednisone and metronidazole (10 mg/kg, PO, q 12 h; 13). Blood samples were collected once from each of the control dogs. For all samples, serum calprotectin concentration was determined via radioimmunoassay.

Results—Mean serum calprotectin concentrations for dogs with IBD at baseline (431.1 μg/L) and 3 weeks after initiation of treatment (676.9 μg/L) were significantly higher, compared with that (219.4 μg/L) for control dogs, and were not significantly correlated with the canine IBD activity index, serum C-reactive protein concentration, or severity of histopathologic changes. The use of a serum calprotectin concentration of ≥ 296.0 μg/L as a cutoff had a sensitivity of 82.4% (95% confidence interval, 65.5% to 93.2%) and specificity of 68.4% (95% confidence interval, 59.9% to 76.0%) for distinguishing dogs with idiopathic IBD from healthy dogs.

Conclusions and Clinical Relevance—Serum calprotectin concentration may be a useful biomarker for the detection of inflammation in dogs, but the use of certain drugs (eg, glucocorticoids) appears to limit its clinical usefulness.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE

To determine breed-specific reference intervals for whole blood (WB) and plasma taurine concentrations in adult, overtly healthy Cavalier King Charles Spaniels (CKCSs) and determine whether taurine concentrations differ across preclinical myxomatous mitral valve disease (MMVD) stages or between CKCSs eating diets that meet World Small Animal Veterinary Association (WSAVA) nutritional guidelines versus other diets.

ANIMALS

200 privately owned CKCSs.

PROCEDURES

Clinically healthy adult CKCSs were recruited prospectively. Diet and supplement history was collected. Dogs were staged by echocardiography using MMVD consensus guidelines. Taurine concentrations were measured in deproteinized lithium heparin WB and plasma samples with the postcolumn ninhydrin derivatization method on a dedicated amino acid analyzer.

RESULTS

There were 12 stage A (6%), 150 stage B1 (75%), and 38 stage B2 (19%) CKCSs. Seventy-eight dogs (39%) were reported by their owners to be eating diets meeting WSAVA nutritional guidelines; 116 (58%) were not. Taurine concentrations in plasma (P = .444) and WB (P = .073) were not significantly different across MMVD stages or between CKCSs eating diets meeting WSAVA nutritional guidelines versus other diets (P = .345 and P = .527, respectively). Reference intervals for WB taurine (152 to 373 µM) and plasma taurine (51 to 217 µM) concentrations in CKCSs were generated.

CLINICAL RELEVANCE

In CKCSs, taurine concentrations do not differ significantly based on preclinical MMVD stage, nor do they differ significantly based on consumption of a diet that does or does not meet WSAVA nutritional guidelines.

Full access
in Journal of the American Veterinary Medical Association

Abstract

Objective—To determine whether hypertriglyceridemia in Miniature Schnauzers is associated with insulin resistance.

Design—Case-control study.

Animals—28 Miniature Schnauzers with hypertriglyceridemia and 31 Miniature Schnauzers for which serum triglyceride concentrations were within the reference range (control dogs).

Procedures—All dogs had no history of chronic disease, were free of clinical signs for at least 3 months prior to blood collection, and were not receiving any medications known to affect lipid metabolism or serum insulin concentration. Food was withheld from each dog for ≥ 12 hours; a 5- to 10-mL blood sample was collected and allowed to clot to obtain serum. Serum insulin and glucose concentrations were measured, and the homeostasis model assessment (HOMA) score was calculated (ie, [basal serum insulin concentration {mU/L} × basal serum glucose concentration {mmol/L}]/22.5).

Results—Median serum insulin concentration was significantly higher in hypertriglyceridemic Miniature Schnauzers (21.3 mU/L) than it was in control dogs (12.5 mU/L). The percentage of dogs with high serum insulin concentrations was significantly greater in the hypertriglyceridemic group (28.6%) than it was in the control group (6.5%; odds ratio, 5.8; 95% confidence interval, 1.1 to 30.2). Median HOMA score for hypertriglyceridemic Miniature Schnauzers (4.9) was significantly higher than that for control dogs (2.8).

Conclusions and Clinical Relevance—Results indicated that hypertriglyceridemia in Miniature Schnauzers is often associated with insulin resistance. Further studies are needed to determine the prevalence and clinical importance of insulin resistance in hypertriglyceridemic Miniature Schnauzers.

Full access
in Journal of the American Veterinary Medical Association