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Abstract

Objective—To develop and validate a radioimmunoassay (RIA) for measuring canine pancreatic lipase immunoreactivity (cPLI) in serum obtained from dogs.

Sample Population—Serum samples from 47 healthy dogs.

Procedures—Canine pancreatic lipase (cPL) was purified from pancreatic specimens of dogs. Antibodies against cPL were raised in rabbits and purified by use of affinity chromatography. A tracer was produced by iodination of cPL with 125I. An RIA was established and validated by determination of sensitivity, working range, dilutional parallelism, spiking recovery, and intra- and interassay variability. A reference range for cPLI in serum was established by use of the central 95th percentile for samples obtained from 47 healthy dogs.

Results—Sensitivity and upper limit of the working range were 0.88 and 863 µg/L, respectively. Observed-to-expected ratios for serial dilutions ranged from 84.9 to 116.5% for 4 samples. Observedto- expected ratios for spiking recovery ranged from 82.8 to 128.6% for 4 samples. Coefficients of variation for intra-assay variability for 4 serum samples were 18.3, 4.2, 3.5, and 8.9%, whereas interassay coefficients of variation were 29.2, 6.2, 3.9, and 4.4%, respectively. The reference range was 4.4 to 276.1 µg/L.

Conclusions and Clinical Relevance—We conclude that the RIA described is sensitive, linear, accurate, precise, and reproducible, with limited accuracy in the high end of the working range and limited precision and reproducibility in the low end of the working range. Additional studies are needed to evaluate whether this degree of accuracy, precision, and reproducibility will negatively impact clinical use of this assay. (Am J Vet Res 2003;64:1237–1241)

Full access
in American Journal of Veterinary Research

Abstract

Objectives

To establish values for gastrointestinal tract permeation by chromium 51-labeled ethylenediaminetetraacetate (51Cr-labeled EDTA) in healthy adult dogs, and to evaluate the time course for 51Cr-labeled EDTA absorption over a 24-hour period after its administration, in an effort to define a shorter, more practical collection method.

Animals

6 healthy adult mixed-breed dogs.

Procedure

After an 18-hour nonfeeding period, each dog was given a solution containing 50 μCi of 51Cr-labeled EDTA in deionized water (10 ml/kg of body weight) by stomach tube. Complete urine collection was done at 2, 4, 6, and 24 hours after 51Cr-labeled EDTA administration. Five-milliliter samples of urine were counted for 15 minutes in a gamma counter, and radioactivity in urine was expressed as a percentage of the orally administered dose.

Results

Median (range) 24-hour urinary recovery of 51Cr-labeled EDTA after 24 hours was 15.1 (12.7 to 20.3)%. Urine collected at 2, 4, and 6 hours contained 1.0 (0.2 to 3.5)%, 6.5 (2.2 to 8.7)%, and 10.0 (8.1 to 11.7)% of the administered 51Cr-labeled EDTA, respectively. Urine passed during the first 6 hours contained, on average, 67 (54 to 77)% of the total 24-hour urine recovery.

Conclusions

6-hour urinary recovery of 51Cr-EDTA provides a potential alternative to 24-hour recovery. This shorter collection period may more specifically reflect small intestinal permeability. (Am J Vet Res 1998; 59:1113-1115)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether feeding causes a change in feline trypsin-like immunoreactivity (fTLI) in serum from healthy cats.

Animals

6 healthy domestic shorthair cats.

Procedures

For the first 12 days of the study, 3 cats were fed a high-protein, high-fat (diet 1), and the other 3 were fed a maintenance (diet 2). On day 12, diets were switched, and cats were fed the other diet for the remaining 12 days of the study. On days 11 and 23, food was withheld for 24 hours, and baseline serum fTLI was measured. Cats were offered food equivalent to half their daily caloric maintenance requirements, and serum fTLI was measured 1, 2, 4, 6, 12, and 24 hours later. Uneaten food was removed after 1 hour.

Results

Overall mean ± SD serum fTLI was 22.7 ± 5.8 µg/L when cats were fed diet 1 and 21.1 ± 5.0 µg/L when cats were fed diet 2. There was no significant difference in serum fTLI over time or between diets. However, there was a statistically significant, but clinically unimportant (mean increase, 1.7 µg/L), increase in serum fTLI, compared with baseline values, 1 hour after cats were fed diet 2 but not when cats were fed diet 1.

Conclusions and Clinical Relevance

A maintenance diet may cause a clinically unimportant increase in serum fTLI 1 hour after feeding in healthy cats. Results suggest that for healthy cats, it is not necessary to withhold food before collecting samples for determination of fTLI in serum. Whether feeding changes fTLI in serum from cats with disorders of the exocrine portion of the pancreas remains to be determined. (Am J Vet Res 1999;60:895–897)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To improve a previously described purification process by producing a higher yield and purity of α1-protease inhibitor (α1-PI) from canine plasma.

Animals

Plasma pool from 10 clinically normal male dogs.

Procedure

Canine α1-PI was purified by use of ammonium sulfate precipitation, ion-exchange chromatography, and 3 affinity chromatographic procedures: concanavalin A-Sepharose, thiol, and hemoglobin-Sepharose. Characterization was performed by gel electrophoresis, isoelectric focusing, and immunoblot analysis. The N-terminal amino acid sequence was obtained by use of the Edman degradation method and a gas amino acid sequencer.

Results

Canine α1-PI was purified with a yield of approximately 7% and a 54-fold increase in specific inhibitory activity. The inhibitor had a molecular weight of 59,000 and had 2 major patterns after isoelectric focusing: fast and intermediate in homozygous and/or heterozygous forms. Edman degradation revealed glutamic acid as the starting amino acid from the N-terminal sequence. Homologies of the N-terminal sequence of canine α1-PI with those of sheep, horse, and human α1-protease inhibitors were 54, 46, and 41 %, respectively.

Conclusions

Canine protease inhibitor is analogous to the α1-protease inhibitors of sheep, human beings, and mice in terms of molecular weight, amino acid composition, and inhibitory activity against trypsin. Although the method described had a yield of 7%, the final product retained inhibitory activity and was pure.

Clinical Relevance

The availability of pure canine α1-PI, as well as the specific antibodies, will facilitate studies on the fecal excretion and structural heterogeneity of this protein in dogs with naturally acquired protein-losing enteropathy.(Am J Vet Res 1996; 57:258-263)

Free access
in American Journal of Veterinary Research

Summary

Fecal proteolytic activity determined in single samples collected on each of 3 consecutive days from each of 20 clinically normal cats ranged from 19 to 363 azocasein units (acu)/g of feces when determined colorimetrically, using azocasein substrate, and ranged from undetectable (in 1 sample from 1 cat) to 21 mm of gel-clearing when determined using radial enzyme diffusion in agar gels containing a casein substrate. Corresponding mean 3-day values for each cat ranged from 29 to 207 acu/g and from 5 to 16 mm, respectively. Studies of proteolytic activity remaining after treatment of fecal extracts with a specific trypsin inhibitor indicated that trypsin accounted for 0 to 77% of proteolytic activity. In a cat with exocrine pancreatic insufficiency, fecal proteolytic activity was 0, 0, and 3 acu/g in a sample of feces collected from each of 3 consecutive days and was undetectable by use of radial enzyme diffusion. Assay of fecal proteolytic activity by use of either azocasein hydrolysis or radial enzyme diffusion allows evaluation of pancreatic function in cats, provided that several samples of feces are tested.

Free access
in Journal of the American Veterinary Medical Association

Abstract

Objective—To validate an automated chemiluminescent immunoassay for measuring serum cobalamin concentration in cats, to establish and validate gas chromatography-mass spectrometry techniques for use in quantification of methylmalonic acid, homocysteine, cysteine, cystathionine, and methionine in sera from cats, and to investigate serum concentrations of methylmalonic acid, methionine, homocysteine, cystathionine, and cysteine as indicators of biochemical abnormalities accompanying severe cobalamin (vitamin B12) deficiency in cats.

Sample Population—Serum samples of 40 cats with severe cobalamin deficiency (serum cobalamin concentration < 100 ng/L) and 24 control cats with serum cobalamin concentration within the reference range.

Procedure—Serum concentrations of cobalamin were measured, using a commercial automated chemiluminescent immunoassay. Serum concentrations of methylmalonic acid, methionine, homocysteine, cystathionine, and cysteine were measured, using gas chromatography-mass spectrometry, selected ion monitoring, stable-isotope dilution assays.

Results—Cats with cobalamin deficiency had significant increases in mean serum concentrations of methylmalonic acid (9,607 nmol/L), compared with healthy cats (448 nmol/L). Affected cats also had substantial disturbances in amino acid metabolism, compared with healthy cats, with significantly increased serum concentrations of methionine (133.8 vs 101.1 µmol/L) and significantly decreased serum concentrations of cystathionine (449.6 vs 573.2 nmol/L) and cysteine (142.3 vs 163.9 µmol/L). There was not a significant difference in serum concentrations of homocysteine between the 2 groups.

Conclusions and Clinical Relevance—Cats with gastrointestinal tract disease may have abnormalities in amino acid metabolism consistent with cobalamin deficiency. Parenteral administration of cobalamin may be necessary to correct these biochemical abnormalities. (Am J Vet Res 2001;62:1852–1858)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine cellular immunolocalization of canine gastric lipase (cGL) and canine pancreatic lipase (cPL) in various tissues obtained from clinically healthy dogs.

Sample Population—Samples of 38 tissues collected from 2 climically healthy dogs.

Procedures—The cGL and cPL were purified from gastric and pancreatic tissue, respectively, obtained from dogs. Antisera against both proteins were developed, using rabbits, and polyclonal antibodies were purified by use of affinity chromatography. Various tissues were collected from 2 healthy dogs. Primary antibodies were used to evaluate histologic specificity. Replicate sections from the collected tissues were immunolabeled for cGL and cPL and examined by use of light microscopy.

Results—Mucous neck cells and mucous pit cells of gastric glands had positive labeling for cGL, whereas other tissues did not immunoreact with cGL. Pancreatic acinar cells had positive labeling for cPL, whereas other tissues did not immunoreact with cPL.

Conclusions and Clinical Relevance—We concluded that cGL and cPL are exclusively expressed in gastric glands and pancreatic acinar cells, respectively. Also, evidence for cross-immunoreactivity with other lipases or related proteins expressed by other tissues was not found for either protein. Analysis of these data suggests that gastric lipase is a specific marker for gastric glands and that pancreatic lipase is a specific marker for pancreatic acinar cells. These markers may have clinical use in the diagnosis of gastric and exocrine pancreatic disorders, respectively. (Am J Vet Res 2002;63:722–727).

Full access
in American Journal of Veterinary Research

Abstract

Objective—To investigate postprandial changes in serum concentrations of unconjugated bile acids in healthy Beagles.

Animals—7 healthy Beagles.

Procedure—Blood samples were obtained from dogs at regular intervals up to 8 hours after consumption of a meal. Serum concentrations of 5 unconjugated bile acids were determined at each time point, using gas chromatography-mass spectrometry with selected ion monitoring.

Results—Total serum unconjugated bile acid concentration was significantly increased, relative to baseline values, at 360, 420, and 480 minutes after feeding. Unconjugated cholic acid was significantly increased at 360, 420, and 480 minutes. The proportion of total unconjugated bile acids represented by cholic acid was significantly increased at 240 to 480 minutes. Deoxycholic acid was significantly increased at 360 and 420 minutes. Chenodeoxycholic acid was significantly increased at 360 to 480 minutes. Lithocholic acid was significantly increased at 180 minutes, whereas no significant changes in ursodeoxycholic acid were detected at any time point.

Conclusion and Clinical Relevance—Healthy Beagles had significant increases in serum concentrations and changes in the profile of unconjugated bile acids after a meal. These increases persisted > 8 hours, indicating that prolonged withholding of food is necessary when to avoid the risk of a false-positive diagnosis when assessing serum unconjugated bile acid concentrations in dogs. (Am J Vet Res 2002;63:789–793

Full access
in American Journal of Veterinary Research

Abstract

Objective—To describe the kinetics of urinary recovery (UR) of 5 sugars used for gastrointestinal permeability and mucosal function testing following orogastric administration of lactose, rhamnose, xylose, methylglucose, and sucrose.

Animals—7 healthy male Beagles.

Procedure—A sugar solution containing lactulose, rhamnose, xylose, methylglucose, and sucrose was administered by orogastric intubation to healthy dogs. Urine samples were collected immediately before sugar solution administration (baseline) and at 2-hour intervals thereafter. The UR of the 5 sugars was determined from urine concentrations measured by high pressure liquid chromatography and pulsed amperometric detection. Percent urinary recovery (%UR) of the total UR up to 12 hours after sugar solution administration was calculated for each sugar at 2-hour intervals.

Results—Mean %UR exceeded 85% for all 5 sugars at 6 hours after orogastric administration of the sugar solution and exceeded 90% after 8 hours.

Conclusion and Clinical Relevance—In healthy dogs, a urine collection period of 6 hours is sufficient for gastrointestinal permeability and mucosal function testing following orogastric administration of lactulose, rhamnose, xylose, methylglucose, and sucrose. (Am J Vet Res 2002;63:845–848)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To develop and validate a gas chromatography–mass spectrometry (GC-MS) method for determination of Nτ-methylhistamine (NMH) concentration in canine urine and fecal extracts and to assess urinary NMH concentrations in dogs with mast cell neoplasia and fecal NMH concentrations in dogs with protein-losing enteropathy.

Sample Population—Urine specimens were collected from 6 healthy dogs and 7 dogs with mast cell neoplasia. Fecal extracts were obtained from fecal specimens of 28 dogs with various severities of protein-losing enteropathy, as indicated by fecal concentration of α1-proteinase inhibitor.

Procedures—NMH was extracted directly from urine, and fecal specimens were first extracted into 5 volumes of PBSS containing 1% newborn calf serum. Nτ-methylhistamine in specimens was quantified via stable isotope dilution GC-MS. The assay was validated via determination of percentage recovery of known amounts of NMH and interassay coefficients of variation. Urinary excretion of NMH was evaluated by means of NMH-to-creatinine concentration ratios.

Results—Recovery of NMH in urine and fecal extracts averaged 104.6% and 104.5%, respectively. Interassay coefficients of variation ranged from 5.4% to 11.7% in urine and 12.6% to 18.1% in fecal extracts. Urinary NMH excretion was significantly increased in dogs with mast cell neoplasia, compared with that in healthy dogs. No correlation was detected between severity of protein-losing enteropathy and fecal NMH concentration.

Conclusions and Clinical Relevance—This method provided a sensitive, reproducible means of measuring NMH in canine urine and fecal extracts. High urinary NMH-to-creatinine concentration ratios in dogs with mast cell neoplasia are consistent with increased histamine release in this disease.

Full access
in American Journal of Veterinary Research