Diagnosis of canine exocrine pancreatic insufficiency by the assay of serum trypsin-like immunoreactivity

A radioimmunoassay for canine serum trypsin-like immunoreactivity (TLI) has been developed and evaluated for use in the diagnosis of exocrine pancreatic insufficiency (EPI). The maximum fasting TLI concentration in 14 dogs with EPI was 1.8 μg/l, compared with a minimum value of 7.0 μg/l in a control group of 75 dogs. The measurement of serum TLI therefore provides a sensitive test for the diagnosis of EPI in the dog.     

Serum feline trypsin-like immunoreactivity in cats with exocrine pancreatic insufficiency

Exocrine pancreatic insufficiency is thought to occur rarely in cats. This assumption has been made based on the lack of a specific test for this disease in the cat. Clinical data from the 1st 20 cats with serum feline trypsin-like immunoreactivity (fTLI) concentrations < or = 8 microg/L are presented. In 17 of these 20 cats compelling evidence for a diagnosis of exocrine pancreatic insufficiency (EPI) was present and in the remaining 3 supportive evidence for a diagnosis of EPI was available. The conclusion was made that serum fTLI concentration is a specific test for EPI in the cat.     

High serum trypsin-like immunoreactivity secondary to pancreatitis in a dog with exocrine pancreatic insufficiency

A 13-year-old spayed Doberman Pinscher with acute vomiting of 24 hours' duration and concurrent 2-week history of polyphagia with weight loss had diabetic ketoacidosis complicated by acute pancreatitis and exocrine pancreatic insufficiency. Diagnostic testing for exocrine pancreatic insufficiency, by determining serum trypsin-like immunoreactivity, revealed an unexpectedly high result when a low result was anticipated. High trypsin-like immunoreactivity was attributed to acute pancreatic inflammation.     

Breed associations for canine exocrine pancreatic insufficiency

BACKGROUND: Knowledge of breed associations is valuable to clinicians and researchers investigating diseases with a genetic basis. HYPOTHESIS: Among symptomatic dogs tested for exocrine pancreatic insufficiency (EPI) by canine trypsin-like immunoreactivity (cTLI) assay, EPI is common in certain breeds and rare in others. Some breeds may be overrepresented or underrepresented in the population of dogs with EPI. Pathogenesis of EPI may be different among breeds. ANIMALS: Client-owned dogs with clinical signs, tested for EPI by radioimmunoassay of serum cTLI, were used. METHODS: In this retrospective study, results of 13,069 cTLI assays were reviewed. RESULTS: An association with EPI was found in Chows, Cavalier King Charles Spaniels (CKCS), Rough-Coated Collies (RCC), and German Shepherd Dogs (GSD) (all P < .001). Chows (median, 16 months) were younger at diagnosis than CKCS (median, 72 months, P < .001), but not significantly different from GSD (median, 36 months, P = .10) or RCC (median, 36 months, P = .16). GSD (P < .001) and RCC (P = .015) were younger at diagnosis than CKCS. Boxers (P < .001), Golden Retrievers (P < .001), Labrador Retrievers (P < .001), Rottweilers (P = .022), and Weimaraners (P = .002) were underrepresented in the population with EPI. CONCLUSIONS AND CLINICAL IMPLICATIONS: An association with EPI in Chows has not previously been reported. In breeds with early-onset EPI, immune-mediated mechanisms are possible or the disease may be congenital. When EPI manifests later, as in CKCS, pathogenesis is likely different (eg, secondary to chronic pancreatitis). Underrepresentation of certain breeds among dogs with EPI has not previously been recognized and may imply the existence of breed-specific mechanisms that protect pancreatic tissue from injury.     

A survey of the diagnosis and treatment of canine exocrine pancreatic insufficiency

A questionnaire was sent to veterinary surgeons who had diagnosed exocrine pancreatic insufficiency (EPI) by assay of low serum concentration of trypsin-like immunoreactivity in 302 dogs. Two-thirds of cases were German shepherd dogs, in which EPI was found equally in males and females. The disease was also diagnosed in 33 other breeds, most commonly in crossbreeds, spaniels, small terriers and collies, and was more common in females. Detailed replies on 160 cases were returned; 79 dogs were alive and 41 dead, with the remainder lost to long term follow-up. The major reasons for death were euthanasia because of the cost, the prognosis, and failure to respond to treatment. An adequate response to treatment was obtained in 92 per cent of dogs. Uncoated preparations of pancreatic enzyme replacement were found to be more successful, and the use of dietary manipulation and antibiotics were frequently reported to be useful adjunctive therapies.     

Further evaluation of bentiromide in the diagnosis of canine exocrine pancreatic insufficiency

The primary objective of this study was to evaluate the accuracy of the bentiromide test in differentiating between dogs with exocrine pancreatic insufficiency (EPI) and those with primary intestinal disease (PID). A secondary objective was to correlate the results of the commonly used diagnostic techniques with the results of the bentiromide test. This test consists of the oral administration of a synthetic peptide that is cleaved only by chymotrypsin. A subsequent rise in the plasma concentration of p-aminobenzoic acid (PABA) indicates the degree of cleavage, providing an in vivo assessment of chymotrypsin activity. Fourteen dogs with EPI and five dogs with PID were categorized on the basis of clinical signs, laboratory evaluations, and histologic examination of intestinal biopsies. Six normal dogs served as controls. The bentiromide test clearly identified the dogs with EPI and distinguished them from the dogs with PID and the control dogs. The results of the bentiromide test correlated well with the results of the clinical and laboratory evaluations. On the basis of these observations and conclusions, recommendations for the pragmatic application of the bentiromide test are offered.     

One-step immunochromatography assay for detection of high-level canine serum trypsin-like immunoreactivity

We developed a one-step immunochromatography assay kit to measure high levels of canine trypsin-like immunoreactivity (cTLI) for bedside estimation of canine pancreatitis. The serum cTLI level can be determined within 10 min by visual comparison of color strengths in the test and reference zones. The serum cTLI levels determined by this method correlate well with canine TLI-ELISA and can be classified into 3 categories: cTLI levels higher than 60 ng/ml were considered positive; 20-60 ng/ml, weakly positive; and less than 20 ng/ml, negative. Twelve dogs suspected of pancreatitis were examined using this method; 4 dogs were positive, 2 were weakly positive, and 6 were negative. This test can detect a high level of serum cTLI and a positive result in the TLIH test will provide critical information for evaluation of pancreatitis in dogs.     

Two unusual cases of canine exocrine pancreatic insufficiency

Exocrine pancreatic insufficiency (EPI) was diagnosed in two German shepherd dogs and treatment given for three months and two years. The owners later discontinued therapy because the dogs' condition improved and both had a normal life for 2.5 and 5.5 years without medication or modified diet. This suggests that dogs with EPI do not always exhibit clinical signs and that the disease can occur in a ‘subclinical’ form. The atrophic process of the pancreas was followed by serial histopathological examination of biopsies in one of the dogs.     

Long term management of canine exocrine pancreatic insufficiency

Twenty dogs with exocrine pancreatic insufficiency (EPI) were managed for an initial four-month period on a low fat diet in conjunction with a non-enteric coated enzyme replacer. At the end of this initial period the clinical signs had been reversed in all the dogs and their body-weights had increased by an average of 24 per cent. Owners were subsequently encouraged to determine the most cost effective form of management; progress was followed up for between five and 39 months. Contact was maintained with 17 of the dogs of which 11 were progressing well (three had been euthanased because of unsatisfactory progress and three for unrelated reasons]. A range of dietary regimens were being implemented and in all cases the requirement for a daily enzyme replacer was less; in terms of absolute enzyme activity, between six and 58 per cent of the original requirement was being used at the follow-up. The low fat diet proved effective in the initial stabilisation of the dogs and was invaluable in their long term management.     

Development and validation of a radioimmunoassay for the measurement of canine pancreatic lipase immunoreactivity in serum of dogs

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 microg/L, respectively. Observed-to-expected ratios for serial dilutions ranged from 84.9 to 116.5% for 4 samples. Observed-to-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 microg/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.     

Serum lipase activities and pancreatic lipase immunoreactivity concentrations in dogs with exocrine pancreatic insufficiency

OBJECTIVE: To determine serum lipase activities and pancreatic lipase immunoreactivity (PLI) concentrations in dogs with exocrine pancreatic insufficiency (EPI). ANIMALS: 74 healthy dogs and 25 dogs with EPI. PROCEDURES: A diagnosis of EPI was made on the basis of clinical signs, low serum trypsin like immunoreactivity (TLI) concentration, and response to treatment with enzyme replacement. Median values for fasting serum lipase activity and serum PLI concentrations were compared between the 2 groups with a Mann-Whitney U test. RESULTS: Median fasting serum lipase activity was not significantly different between dogs with EPI (366.0 U/L) and healthy dogs (294.5 U/L), and only 1 dog with EPI had a serum lipase activity less than the lower limit of the reference range. Median serum PLI concentration was significantly lower in dogs with EPI (0.1 microg/L) than in healthy dogs (16.3 microg/L). All dogs with EPI had serum PLI concentrations less than the lower limit of the reference range. CONCLUSION AND CLINICAL RELEVANCE: Serum lipase activity is not limited to the exocrine pancreas in origin, whereas serum PLI is derived only from the exocrine pancreas. Unlike in serum TLI concentrations, there was a small degree of overlap in serum PLI concentrations between healthy dogs and dogs with EPI. Serum TLI concentration remains the test of choice for diagnosis of EPI.     

Evaluation of serum feline trypsin-like immunoreactivity for the diagnosis of pancreatitis in cats

OBJECTIVE: To evaluate serum feline trypsin-like immunoreactivity (fTLI) concentration and results of abdominal ultrasonography, CBC, and serum biochemical analyses for diagnosis of pancreatitis in cats. DESIGN: Prospective study. ANIMALS: 28 cats with clinical signs compatible with pancreatitis. PROCEDURE: Serum fTLI concentrations were determined, and abdominal ultrasonography, CBC, and serum biochemical analyses were performed prior to histologic evaluation of pancreatic, hepatic, and intestinal specimens. On the basis of histologic results, cats were categorized as having a normal pancreas (n = 10), pancreatic fibrosis with ongoing inflammation (9), pancreatic fibrosis without inflammation (4), and acute necrotizing pancreatitis (5). Serum fTLI concentrations and results of CBC, serum biochemical analyses, and histologic evaluation of hepatic and intestinal specimens were compared among groups. RESULTS: Significant differences in serum fTLI concentrations or any hematologic or biochemical variable were not detected among the 4 groups of cats. Median serum fTLI concentrations were 51 micrograms/L (range, 18 to 200 micrograms/L) in cats with a normal pancreas, 32 micrograms/L (range, 12 to > 200 micrograms/L) in cats with pancreatic fibrosis and ongoing inflammation, 124 micrograms/L (range, 36 to > 200 micrograms/L) in cats with pancreatic fibrosis without ongoing inflammation, and 30 micrograms/L (range, 24 to 84 micrograms/L) in cats with acute necrotizing pancreatitis. We detected a high prevalence of concurrent hepatic and intestinal tract disease in cats with pancreatitis. CONCLUSIONS AND CLINICAL RELEVANCE: In cats with clinical signs of pancreatitis, serum fTLI concentration is poorly associated with histopathologic diagnosis.     

A new test for the diagnosis of exocrine pancreatic insufficiency in the dog

A new approach to the diagnosis of exocrine pancreatic insufficiency in the dog has been evaluated. The synthetic peptide, N-benzoyl-L-tyrosyl-p-aminobenzoic acid, administered orally, is specifically cleaved by pancreatic chymotrypsin to release p-aminobenzoic acid (PABA) which is subsequently absorbed and excreted in the urine. The assay of plasma PABA or the estimation of urinary excretion over a 6-hour period was found to give a clear differentiation between two small groups of healthy dogs and animals with pancreatic insufficiency. The absorption of orally administered sodium PABA was similar in the two groups confirming that the rate limiting process following oral peptide is hydrolysis not absorption.     

An immunoassay for canine pancreatic elastase 1 as an indicator for exocrine pancreatic insufficiency in dogs.

The detection of pancreatic elastase 1 in stool samples has become the noninvasive gold standard for the diagnosis of pancreatic insufficiency in humans. Accordingly, the development of a sandwich-ELISA specific for canine pancreatic elastase 1, based on monoclonal antibodies, is presented here. The test has a detection range of 4-240 microg canine pancreatic elastase l/g feces. The intraassay coefficient of variation is 7.4%, and the interassay coefficient of variation is 7.7%. Spiking experiments show that canine elastase 1 is quantitatively detectable in fecal samples. Interestingly, the range of the elastase 1 concentration in canine feces within several days is higher as compared with humans. As the proposed cutoff of 10 microg/g is below this variation range in 96.1% of the tested samples, the effect on the test specificity is negligible. Because the test detects neither human nor bovine and porcine elastase 1, pancreatic function can be monitored without interrupting an enzyme replacement therapy.     

Development and analytical validation of a radioimmunoassay for the measurement of feline pancreatic lipase immunoreactivity in serum

Pancreatitis is recognized as an important cause for morbidity and mortality in cats, but diagnosis remains difficult in many cases. As a first step in trying to identify a better diagnostic tool for feline pancreatitis the objective of this project was to develop and analytically validate a radioimmunoassay for the measurement of feline pancreatic lipase immunoreactivity (fPLI). Feline pancreatic lipase (fPL) was purified from pancreatic tissue and antiserum against fPL was raised in rabbits. Tracer was produced by iodination of fPL using the chloramine T method. A radioimmunoassay was established and analytically validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and interassay variability. A control range for fPLI in cat serum was established from 30 healthy cats using the central 95th percentile. The sensitivity of the assay was 1.2 microg/L. Observed to expected ratios for serial dilutions ranged from 98.8% to 164.3% for 3 different serum samples. Observed to expected ratios for spiking recovery ranged from 76.9% to 147.6% for 3 different serum samples. Coefficients of variation for intra- and interassay variability for 4 different serum samples were 10.1%, 4.5%, 2.2%, and 3.9% and 24.4%, 15.8%, 16.6%, and 21.3%, respectively. A reference range for fPLI was established as 1.2 to 3.8 microg/L. We conclude that the assay described is sensitive, accurate, and precise with limited linearity in the lower and limited reproducibility in the lower and higher end of the working range. Further studies to evaluate the clinical usefulness of this assay are needed and in progress.     

Influence of feeding on serum feline trypsin-like immunoreactivity.

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 micrograms/L when cats were fed diet 1 and 21.1 +/- 5.0 micrograms/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 micrograms/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.     

Development of a canine trypsin-like immunoreactivity assay system using monoclonal antibodies

The radioimmunoassay (RIA) for trypsin-like immunoreactivity (TLI) is one of the most sensitive and specific tests for detecting exocrine pancreatic insufficiency (EPI). An abnormally low serum TLI concentration (<2.5 ng/ml) indicates end-stage EPI. Although RIA methods can be used to detect canine serum TLI, these procedures are beyond the capabilities of most veterinary clinics and general laboratories. Using monoclonal antibodies (mAbs), we developed an enzyme-linked immunosorbent assay (ELISA) for canine TLI and incorporated it into an immunochromatographic test (ICT) for the diagnosis of EPI. The ELISA was linear over TLI concentrations of 1-100 ng/ml. Levels of intra-assay coefficients of variance (CVs) were 1.8-6.1%, inter-assay CVs were 5.1-9.8%, and the recovery of TLI added to two samples of canine serum ranged from 89 to 111 and 93 to 108%, respectively. Good correlation (correlation coefficient, 0.974) occurred between the TLI values obtained by the ELISA method and those by RIA from 56 clinical samples. Serum TLI values in clinically healthy dogs ranged from 7.8 to 29.2 ng/ml by ELISA, and those from dogs with EPI were 0.0-0.6 ng/ml. The values were 0.0-287.4 ng/ml for dogs with pancreatitis, and those from dogs with gastrointestinal disease were 5.5-58.9 ng/ml. The only statistically significant difference (P<0.01) occurred between the TLI level of healthy dogs and those with EPI. The ICT kit showed high reproducibility, and the TLI values yielding negative results differed significantly (P<0.01) from those returning positive results. The ICT kit yielded negative results (indicating EPI) from clinical serum samples with TLI concentrations of 0.0-4.1 ng/ml by ELISA. Both the ELISA and ICT kit are useful tools in the diagnosis of canine EPI.