Evaluation of a spectrophotometric method for canine serum lipase determination

The British antilewisite butyrate-dithionitrobenzoate (BALB-DTNB) spectrophotometric serum lipase assay was evaluated for precision, accuracy, and diagnostic usefulness in analyzing canine sera. Sera samples from clinically healthy dogs, dogs with experimentally induced pancreatitis, and dogs with spontaneous pancreatitis were analyzed. A titrimetric method of serum lipase determination was used for comparison. Although the BALB-DTNB method was not found to be precise or accurate for determining the lipase activity of canine serum samples, it seemed to be at least as diagnostically useful as the titrimetric procedure. The small sample size requirement and the speed of analysis of the BALB-DTNB procedure are advantages of this method over the titrimetric method, and thus, its use in place of the titrimetric method is justified. A laboratory reference range of 3 to 37 IU/L was determined for canine serum.     

Validation and diagnostic efficacy of a lipase assay using the substrate 1,2-o-dilauryl-rac-glycero glutaric acid-(6' methyl resorufin)-ester for the diagnosis of acute pancreatitis in dogs

BACKGROUND: Increased serum lipase activity has been used historically to support the diagnosis of acute pancreatitis, a common disease in dogs. Most of the lipase assays that are currently in use lack optimum sensitivity and specificity. OBJECTIVE: The objectives of this study were to 1) validate the 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) assay for determination of lipase activity in canine serum and 2) compare results, reference intervals, sensitivity, and specificity of the DGGR assay with a standard 1,2-diglyceride (1,2 DiG) assay for diagnosing acute pancreatitis in dogs. METHODS: Precision, linearity, and interference studies were performed for method validation on a Hitachi 911 analyzer. Lipase results from the DGGR and 1,2 DiG assays were compared by linear regression analysis. Sensitivity, specificity, and diagnostic efficacy were determined for both assays on a population of 30 dogs, 15 of which had acute pancreatitis based on history, clinical signs, and ultrasound findings. RESULTS: Within-run and within-day coefficients of variation (CVs) were low (<3%), with higher day-to-day CVs (< or =14 %). The assay was linear between 8 and 2792 U/L. No significant interference by hemolysis and lipemia was found. Poor correlation was found between the assays (r(s)=0.84). The lipase reference interval was 8-120 U/L for the DGGR assay and 30-699 U/L for the 1,2 DiG assay. Sensitivity and specificity for the diagnosis of pancreatitis were 93% and 53%, respectively, for the DGGR assay and 60% and 73% for the 1,2 DiG assay. Receiver operating characteristic curve analysis showed similar areas under the curve. CONCLUSIONS: On the basis of this study, the DGGR method is considered adequate for assaying serum lipase activity in dogs. The high sensitivity of the DGGR assay suggests it may be a useful screening test for canine pancreatitis.     

Increased canine pancreatic lipase immunoreactivity (cPLI) and 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin) ester (DGGR) lipase in dogs with evidence of portal hypertension and normal pancreatic histology: a pilot study

The clinical presentations of both liver disease and pancreatitis are nonspecific and overlapping, which may cause difficulty in diagnosis. In our retrospective pilot study, we assessed whether dogs with evidence of portal hypertension and absence of pancreatitis on pancreatic histology have increases in canine pancreatic lipase immunoreactivity (cPLI) and 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin) ester (DGGR) lipase. We included dogs that had been presented between 2008 and 2019 if they had normal pancreatic histology, histologically confirmed hepatopathy, and if canine pancreas-specific lipase (Spec cPL; Idexx) or DGGR lipase had been measured. Only dogs with portal hypertension were included. Six dogs fulfilled the inclusion criteria. Four of 6 and 2 of 6 dogs had Spec cPL and DGGR lipase exceeding the upper reference limit, respectively. From the 4 dogs with increased Spec cPL, 2 had concentrations of 200–400 µg/L and 2 had concentrations ≥ 400 µg/L. Our results suggest that canine portal hypertension might lead to increased Spec cPL and DGGR lipase values in the absence of pancreatitis on histology. Until more evidence in a larger number of dogs with portal hypertension is available, both tests should be interpreted cautiously in the presence of portal hypertension.     

Serum amylase and lipase activities in the diagnosis of pancreatitis in dogs

To determine the usefulness of information provided by measurement of serum amylase activity in the evaluation of dogs for pancreatitis, the relationship of amylase activity to lipase activity in 713 paired serum samples was investigated by statistical analysis. Little change in mean amylase concentration was found until lipase values exceeded 800 U/L. The ranges of amylase activity (mean +/- 2 SD) were essentially the same for dogs with no pancreatitis (0 to 100 U of lipase activity/L) as for dogs with a high probability for the disease (700 to 799 U of lipase activity/L), 0 to 4,029 U/L and 857 to 4,869 U/L, respectively. Pathologic findings from biopsy and necropsy reports from 92 dogs for which serum lipase determinations were done indicated that serum lipase increased not only with pancreatitis, but also with other medical problems, such as renal and hepatic disease. It was concluded that determination of serum amylase activity without knowledge of serum lipase activity was of little value to diagnose pancreatitis. High amylase activity was not specific for pancreatitis and low amylase activity could not rule out the disease. The results of this study also showed that low serum lipase values almost always eliminated the possibility of pancreatitis and that high values were often, but not always, diagnostic for pancreatitis.     

Evaluation of an automated colorimetric assay for the measurement of lipase activity in canine sera.

An automated colorimetric method for determining lipase activity in canine sera was evaluated for precision, linearity and correlation to existing assay methods. The colorimetric method was a commercial reagent that used a series of enzymatic reactions based on the hydrolysis of 1,2 diglyceride by pancreatic lipase. Within-run and between-run coefficients of variation were < 6.8% and < 8.3%, respectively. Linearity was determined to be at least 1366 U/L. Canine serum lipase concentrations attained using the colorimetric method were compared to both titrimetric and dry-film methods for measuring serum lipase activity, resulting in significant (P < or = 0.05) correlation coefficients of 0.92 and 0.77, respectively. Canine serum lipase concentrations measured using the colorimetric assay on 2 different automated analyzers had a significant (P < or = 0.05) correlation coefficient of 0.92. A laboratory reference range using serum samples from 56 healthy dogs (0-561 U/L) was established. There were no significant (P < or = 0.05) differences in mean serum lipase concentrations comparing male and female dogs or comparing young dogs (< or = 3 y) to mature (4-7 y) and older (> 7 y) dogs using this assay. It was concluded that the automated colorimetric assay was a reliable indicator of canine serum lipase activity and offered several advantages, including small sample volume and short analysis time.     

Effects of dexamethasone on pancreatic tissue and on serum amylase and lipase activities in dogs

The effects of dexamethasone on the pancreas and on pancreatic amylase and lipase activities were determined in clinically normal dogs and in dogs with neurologic disease. Dexamethasone increased serum lipase activity without any histologic damage to the pancreas in either group of dogs. It decreased serum amylase activity in the normal dogs and had a variable effect in dogs with neurologic disease, with or without confirmed pancreatitis. It was suggested that high serum lipase activity in dexamethasone-treated dogs may not be attributable to pancreatitis and that the reasons are still unknown. It was concluded that high serum lipase activity is an unreliable basis for diagnosis of pancreatitis in dogs treated with dexamethasone. The data allowed no conclusion about an additive effect of dexamethasone and neurologic disease causing pancreatitis.     

Analytical validation of an ELISA for measurement of canine pancreas‐specific lipase

Background: The diagnosis of canine pancreatitis is challenging. Clinical presentation often includes nonspecific clinical signs, such as vomiting, anorexia, and abdominal discomfort. Increased serum lipase activity can be indicative of pancreatitis; however, it can also be increased with other conditions. An immunoassay for measurement of canine pancreas‐specific lipase in canine serum that would be suitable for commercial application and provide rapid results would be beneficial. Objective: The goal of this study was to validate the Spec cPL assay, a commercially available ELISA for the quantitative measurement of canine pancreas‐specific lipase. Methods: Dynamic range, dilutional linearity, precision, interfering substances, assay stability, and reproducibility were investigated for analytical validation. The method was compared with the reference assay, canine pancreatic lipase immunoreactivity (cPLI), and included evaluation of a sample population of dogs and bias. Results: Analytical validation showed a dynamic range of 36–954 μg/L; good precision (intra‐ and interassay coefficient of variation <12%); absence of interference from lipid, hemoglobin, or bilirubin; 12‐month kit stability; and good reproducibility. Method comparison showed a positive bias relative to the cPLI reference method; however, the bias can be accommodated by adjustment of decision limits. The upper limit of the reference interval for Spec cPL was determined to be 216 μg/L based on the upper 97.5th percentile of results from 93 clinically healthy, kennel‐housed dogs. Conclusions: Validation data demonstrated that the Spec cPL assay provides reproducible results for canine pancreas‐specific lipase. A readily available assay for measurement of this enzyme allows broader clinical utilization of this analytical tool, generating timely results to aid in the diagnosis of canine pancreatitis.     

Assessing the severity of canine pancreatitis

The objective of this study was to determine whether laboratory testing currently available is able to provide prognostic information in canine pancreatitis. A prospective study of dogs with naturally occurring pancreatitis was undertaken. Twenty-two cases with histologically confirmed pancreatic inflammation were included in the study. Each dog had routine haematology parameters, serum biochemistry (including lipase and amylase), serum trypsin-like immunoreactivity and trypsinogen activation peptides (TAP) in urine and plasma measured. Twelve of the dogs were classified as having severe disease. These dogs had statistically significant increases in urinary TAP-creatinine ratio (UTCR) measurement, serum lipase, serum phosphate and serum creatinine concentrations. Additionally dogs with severe pancreatitis had significantly decreased urine specific gravity levels. The most sensitive and specific test to assess the severity of pancreatitis was the measurement of UTCR.     

Serum Phospholipase A2 in Canine Acute Pancreatitis

During 3 years 28 cases of acute pancreatitis were diagnosed in dogs. In 26 of these dogs, the disease was fatal. The most frequent symptoms were vomiting, anorexia and lethargy. Two thirds showed tenderness upon abdominal palpation. Ascites was found in 3 cases. Of the blood, parameters, serum amylase level was elevated in 86 % and lipase in 89 % of the cases. Sixteen dogs were uremic and half of the dogs were hyperglycemic. Two thirds of the dogs had leukocytosis. Using stepwise multiple regression the best blood parameters explaining acute pancreatitis were leukocytes together with lipase and glucose.In an attempt to find a more specific serum test for dogs to diagnose acute pancreatitis serum phospholipase A2 (PLA₂) activity was measured. In sixteen out of the 28 dogs with acute pancreatitis, serum PLA₂ activity was increased. The ascites fluids were rich in PLA2. Serum PLA₂ is more often increased in the severe necrotizing pancreatitis (80 %) than in the milder forms of acute pancreatitis (44 %). All dogs with increased serum PLA₂ had also increased serum amylase and lipase activities. The dogs with an increased serum PLA₂ and dogs with ascites had fat necrosis in the vicinity of the pancreas. Experimental pancreatitis was induced in 4 dogs by injecting Na-taurocholate and trypsin into the pancreas. In these cases, very high PLA₂ activities in the serum and ascites fluids were detected, but none seemed to be present in the urine samples.Key words: dog, acute pancreatitis, phospholipase A2     

Diagnosis of pancreatitis.

In summary, pancreatitis is common in dogs and cats, but it seems that most cases remain undiagnosed. Serum amylase and lipase activities are useful as a quick screening test for pancreatitis in the dog only. Serum amylase or lipase activity must be at least three to five times the upper limit of the reference range to suggest a diagnosis of pancreatitis. Furthermore, the diagnosis must be confirmed by other diagnostic modalities, and normal test results do not eliminate the possibility of pancreatitis. Abdominal ultrasound is highly specific for pancreatitis in dogs and cats but is not particularly sensitive, especially in cats. Serum cPLI concentration is highly specific for exocrine pancreatic function and is also highly sensitive for pancreatitis. Similarly, initial data would suggest that serum fPLI is the most sensitive and specific diagnostic test for feline pancreatitis. Until further data are available, however, serum fPLI should be used in conjunction with other diagnostic tests to arrive at a diagnosis of feline pancreatitis. Histopathologic evidence of pancreatitis is conclusive for a diagnosis of pancreatitis. In most cases, however, lesions are localized, and the lack of histopathologic evidence of pancreatitis does not eliminate a diagnosis of pancreatitis.     

Tissue gamma-glutamyl transpeptidase activity and hepatic ultrastructural alterations in dogs with experimentally induced glucocorticoid hepatopathy

Glucocorticoid hepatopathy was induced in 2 Beagle dogs with IM injections of prednisone, 4.4 mg/kg, given once daily for 14 consecutive days. Serum enzymatic activities were monitored and percutaneous hepatic biopsy materials were collected. A placebo-treated group of 2 Beagle dogs was given saline solution injections. Treated dogs showed clinical signs of Cushing's disease and alterations in mean serum enzymatic activities consistent with glucocorticoid hepatopathy. The gamma-glutamyl transpeptidase activities in placebo-treated dogs were highest for kidney, less (but marked) for pancreas, and least for liver, gallbladder, jejunum, duodenum, spleen, heart, lung, skeletal muscle, and erythrocytes. In treated dogs, the gamma-glutamyl transpeptidase activities were highest for pancreas and were decreased for kidney and slightly increased for the other tissues. Results of hepatic biopsy and necropsy examinations of treated dogs showed swollen, pale, friable hepatic parenchyma with hepatocytic cytoplasmic vacuolation and glycogen accumulation. Extrahepatic lesions included gastric ulceration, catarrhal enteritis, splenic and lymph node atrophy and lymphocytolysis, and vacuolation of adrenal cortical cells in the zona fasciculata. Ultrastructural study of liver from dogs with glucocorticoid hepatopathy indicated that the principle alterations were abundant glycogen accumulation and few mitochondria in hepatocytes.     

Rapid turbidimetric determination of serum pancreatic lipase in the dog

A rapid and highly reproducible turbidimetric method for the determination of serum pancreatic lipase activity in the dog is described. Values of 0 to 50 IU/L of serum were obtained in 35 healthy mature dogs, and the maximum values of 325 to 800 IU/L were observed in 8 dogs with induced pancreatitis.     

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.     

Serum values of amylase and pancreatic lipase in healthy mature dogs and dogs with experimental pancreatitis

Serum values of amylase and pancreatic lipase were determined by the iodometric and the turbidimetric methods, respectively, in 44 mature healthy dogs and in 8 dogs with experimentally induced pancreatitis (plus 1 sham-operated control). Serum value of amylase in mature healthy dogs varied from 250 to 1,500 Caraway units/dl and that of pancreatic lipase varied from 0 to 50 IU/L. Maximal serum values of amylase and pancreatic lipase in the dogs with experimentally induced pancreatitis varied from 4,540 to 14,000 Caraway units/dl and 325 to 810 IU/L, respectively. Following pancreatic damage, serum values of amylase and pancreatic lipase increased rapidly in the 8 dogs and ran parallel to each other in 6 of the 8 dogs studied. However, the serum value of amylase returned to within normal range earlier than the serum value of pancreatic lipase in 2 dogs; the reverse was true in 2 other dogs.     

Serum lipase activity in young dogs with acute enteritis or gastroenteritis

Blood serum lipase activity was determined in 48 young dogs with acute enteritis or gastroenteritis due to canine parvovirus (16 Cases) and presumably to other infectious agents (32 cases). Elevated serum lipase activity (> 500 U/L) was found in 13 dogs (27.1 %) with values ranging from 800 to 2,780 U/L. The hyperlipasemia of these cases may be attributed to acute pancreatitis secondary to acute gastroenteritis or to gastrointestinal upset.     

COMBINED USE OF ULTRASONOGRAPHY AND CONTRAST ENHANCED COMPUTED TOMOGRAPHY TO EVALUATE ACUTE NECROTIZING PANCREATITIS IN TWO DOGS

The imaging findings in two miniature schnauzers with acute necrotizing pancreatitis are described. Both dogs were treated previously for diabetes mellitus and hyperlipidemia. Vomiting, anorexia, and lethargy were observed in both dogs at presentation. Laboratory evaluations supportive of pancreatitis included left shift, abnormally high serum amylase and lipase activities, hypocalcemia, and abnormally high serum activities of liver enzymes. Sonographically, both dogs had diffusely enlarged hypoechoic pancreatic tissue with anechoic foci compatible with necrosis, abscessation, phlegmon, and pseudocysts formation. Contrast-enhanced computed tomography (CT) findings in both dogs were compatible with pancreatic necrosis. Dog 1 was managed medically for 11 days. Follow-up CT scan in this dog disclosed decreased pancreatic size and increased contrast enhancement compatible with partial resolution of pancreatitis.     

Evaluation of 1,2-O-dilauryl-rac-glycero glutaric acid-(6′-methylresorufin) ester (DGGR) and 1,2-diglyceride lipase assays in dogs with naturally occurring hypercortisolism

1,2-O-dilauryl-rac-glycero glutaric acid-(6′-methylresorufin) ester (DGGR) lipase activity has been proposed as a faster and less expensive test used in the diagnosis of acute pancreatitis (AP) compared to canine pancreatic lipase immunoreactivity (cPLI), which is considered the most sensitive and specific serum test available for dogs. Elevations in lipase activity have been observed in dogs with naturally occurring hypercortisolism (HC) and in those treated with exogenous steroids, which complicates the diagnosis of AP in dogs with HC. We compared lipase activity measured by DGGR and 1,2-diglyceride (1,2-DiG) assays in 22 dogs with HC, 22 with AP, and 22 healthy dogs. The dogs with HC had no clinical signs or ultrasonographic findings consistent with AP. DGGR lipase activity was elevated in 64% and 73% of the dogs with HC and AP, respectively, and in 18% of healthy dogs. 1,2-DiG lipase activity was high in 23% and 36% of the dogs with HC and AP, respectively, and in 5% of the healthy dogs. Both DGGR and 1,2-DiG lipase activities were significantly different between the healthy dogs and the other 2 groups, whereas no differences were detected between the dogs with HC and those with AP. Our results support a lack of specificity for both DGGR and 1,2-DiG lipase activity assays in aiding the diagnosis of AP in dogs with HC.     

Patient-side assay of lipase activity correlating with pancreatic lipase immunoreactivity in the dog

Pancreatitis is a common exocrine pancreatic disease in dogs, and the pancreatic lipase immunoreactivity (PLI) test is used for diagnosis. Enzyme catalytic assay is thought to have low specificity, but a lipase activity assay with increased specificity has been developed in human clinical chemistry. We measured serum lipase activity of 65 client-owned dogs using the newly developed FUJI DRI-CHEM slide and compared the results with their PLI concentrations. The results showed a good correlation (r = 0.91), and the normal and pancreatitis dogs identified based on the PLI values were correctly separated based on lipase activity. The present study suggests that FUJI DRI-CHEM lipase activity would be helpful for diagnosis of pacreatitis in dogs and, in particular, that it can be used as a patient-side assay and contributes to immediate treatment.     

Model of chronic pancreatitis in the dog

A model of chronic pancreatitis was developed and characterized in the dog. Pancreatitis was produced by infusion of oleic acid through a cannula in the accessory pancreatic duct. Biochemical changes included early and marked increases in serum amylase and lipase activities which returned to base line within 3 weeks, at which time the dogs were clinically normal. In dogs euthanatized within 2 weeks, pathologic changes included massive necrosis and hemorrhage, cystic spaces filled with fluid, and abscesses. Histologic features revealed acute exudative pancreatitis with pancreatic atrophy and fibrosis. In dogs killed between weeks 3 to 12, changes included: marked atrophy with remaining acini surrounded by remnants consisting of collapsed stroma, blood vessels, and pancreatic ducts; marked coarse fibrosis with scattered inflammatory cells and occasional acinar tissue; and large irregular pseudocysts.     

Serum Pancreatic Lipase Immunoreactivity Concentrations in Dogs Treated with Potassium Bromide and/or Phenobarbital

Potassium bromide, phenobarbital, or a combination of both is commonly used in the treatment of canine epilepsy. Several cases of clinical pancreatitis have been reported in dogs after treatment with potassium bromide, but the risk of elevated serum canine pancreatic lipase immunoreactivity concentrations in dogs treated with potassium bromide and/or phenobarbital has not previously been evaluated in a large group of dogs. This study suggests an increased risk for elevated serum canine pancreatic lipase immunoreactivity concentrations and possibly pancreatitis in dogs treated with potassium bromide or phenobarbital alone or in combination.