New Developments in Gastrointestinal Function Testing in Small Animals
World Small Animal Veterinary Association World Congress Proceedings, 2003
Jörg M. Steiner,, PhD, DACVIM, DECVIM-CA
GI Laboratory, Texas A&M University
College Station, TX, USA


The clinical usefulness of diagnostic tests is determined by sensitivity, specificity, positive predictive value, and negative predictive value. For diagnostic tests with a quantitative result these parameters are dependent on the cut-off value that is set for the diagnosis of a specific disease. For diagnostic tests used as screening tests the cut-off value should be chosen to maximize sensitivity, while for true diagnostic tests the cut-off value should be chosen to maximize specificity. It should be noted that most tests utilized in veterinary gastroenterology are true diagnostic tests.

Serum trypsin-like immunoreactivity (TLI)1,2

A severely decreased serum TLI is specific for exocrine pancreatic insufficiency (EPI) in both dogs and cats (dogs: cTLI < 2.5 µg/L; cats: fTLI < 8 µg/L). In addition serum TLI is elevated in some patients with pancreatitis (dogs: cTLI > 50 µg/L; cats: fTLI > 100 µg/L). While an elevated serum TLI concentration is specific for pancreatitis it is not very sensitive (sensitivity 30-60%). With the recent development of serum tests for both canine and feline pancreatic lipase immunoreactivity (PLI) more sensitive diagnostic tests for pancreatitis are now available. However, at least in the cat serum fTLI is still being used in the diagnosis of pancreatitis until more data about the fPLI are available. Under physiologic conditions a small quantity of trypsinogen leaks into the vascular space and can be quantified by immunoassays. When more than 90% of the secretory reserve of the exocrine pancreas is lost clinical signs of EPI ensue. At the same time the amount of trypsinogen leaking into the vascular space is decreased leading to a decreased serum TLI concentration. In contrast, when pancreatic tissue is inflamed an increased amount of trypsinogen and trypsin leak into the vascular space increasing serum TLI concentrations.

Serum cobalamin and folate concentrations3,4

Serum folate concentration can be decreased in proximal small intestinal disorders, while serum cobalamin concentration can be decreased in distal small intestinal disorders and EPI in both dogs and cats. In dogs and cats with diffuse small intestinal disorders both serum folate and cobalamin concentrations can be decreased. Finally, a decreased serum cobalamin concentration and an increased serum folate concentration can be seen in dogs with small intestinal bacterial overgrowth (SIBO). Serum cobalamin is not only of diagnostic but also of therapeutic interest. Cobalamin deficiency in human patients has been shown to cause systemic as well as gastrointestinal changes. Villous atrophy, inflammatory infiltrates of the intestinal mucosa, cobalamin malabsorption, and malabsorption of other nutrients have all been described. Systemic changes include polyneuropathies and suppression of the immune system. Cats and dogs with cobalamin deficiency often do not respond to therapy of the underlying gastrointestinal disorder unless cobalamin is supplemented.

Folate and cobalamin are both water-soluble vitamins that are plentiful in both canine and feline diets. Therefore, nutritional deficiencies are uncommon. However, dietary folate is poorly absorbed, because it occurs as folate polyglutamate and needs to be deconjugated by folate deconjugase, a jejunal brush border enzyme. Folate monoglutamate is absorbed by specific carriers in the proximal small intestine. Therefore, longstanding and severe disorders of the proximal small intestine can lead to a depletion of folate body stores and a decreased serum folate concentration. In contrast, in SIBO microorganisms in the small intestine synthesize folic acid, which can lead to an increase in folate absorption and in turn to an increased serum folate concentration. Dietary cobalamin is bound to dietary protein. In the stomach dietary protein is partially digested by pepsin and HCl and cobalamin is released. However, cobalamin immediately binds to R-protein. The R-protein in turn is digested by pancreatic proteases in the small intestine. Free cobalamin binds to intrinsic factor released mostly by the exocrine pancreas. The cobalamin-intrinsic factor complexes are absorbed through specific receptors in the ileum. Therefore, severe and longstanding disorders of the distal small intestine as well as exocrine pancreatic insufficiency will lead to depletion of cobalamin body stores and to a decreased serum cobalamin concentration. Finally, microorganisms, present in excessive numbers in the small intestine in patients with SIBO, will utilize cobalamin and compete with the host for dietary cobalamin.

Serum pancreatic lipase immunoreactivity (PLI)5,6

Cells of many different tissues synthesize lipases and traditional assays for serum lipase activity cannot discern between lipases of different tissue origins. In contrast, new assays for the measurement of canine and feline pancreatic lipase immunoreactivity concentration (PLI) exclusively measure lipase that originates from the exocrine pancreas. Serum cPLI has been shown to be specific for exocrine pancreatic function in the dog and is also highly sensitive for canine pancreatitis (82%). Thus, serum cPLI is the most sensitive diagnostic test available for canine pancreatitis. Similarly, serum fPLI has been shown to be the most sensitive and specific diagnostic test available for feline pancreatitis.

Fecal a1-proteinase inhibitor (a1-PI)7

Fecal α1-PI clearance is used for the diagnosis of protein-losing enteropathy (PLE) in human beings after hepatic disease, renal protein loss, and occult GI bleeding have been excluded. Fecal α1-PI concentrations were increased in 3 dogs with increased fractional 51Cralbumin excretion. During PLE macromolecules leak across the intestinal mucosa. Most plasma proteins are quickly digested by pancreatic proteases in the intestinal lumen. Alpha1-PI, which has a similar molecular weight as albumin is a proteinase inhibitor and is therefore not digested and can be measured in feces. This test is not only useful to confirm intestinal protein loss in dogs with clinical signs of gastrointestinal disease but can also be used in animals suspected of having subclinical gastrointestinal disease. For example, an increased fecal α1-PI concentration is one of the earliest findings in soft-coated Wheaten Terriers with PLE.

Gastrointestinal permeability and mucosal function testing8,9

A wide range of mono- and disaccharides have been used as permeability markers for the small intestine while sucrose has been used as a specific permeability marker for the stomach. Other monosaccharides have been used as markers for small intestinal mucosal absorptive capacity. After the bladder of the patient is emptied a sugar solution is given orally or by gastric gavage. Urine produced over the following 6 hours is collected and assayed. Under physiologic conditions there are large numbers of small pores in the intestinal mucosa that are probably transcellular in location and permeable for monosaccharides, such as rhamnose. There is also a small number of larger pores that are probably located in the area of the tight junctions and permeable for disaccharides, such as lactulose. During intestinal disease the mucosal surface area decreases significantly leading to a decrease in number of small pores and also a decrease in transport-proteins for carrier-mediated transport processes. In turn the permeability of monosaccharides and the carrier-mediated transport of other monosaccharides is decreased. At the same the tight junctions get weakened and the permeability for disaccharides increases. Gastrointestinal permeability and mucosal function testing is highly sensitive for altered gastrointestinal function and may be abnormal in patients with subclinical GI disease.

Breath hydrogen testing10

Hydrogen can only be produced by bacteria, but not by mammalian cells. After oral application of a sugar solution (e.g., glucose, lactulose, or xylose) bacteria in the intestinal lumen metabolize these sugars and release hydrogen. Some of the hydrogen diffuses through the mucosa and into the blood stream. Hydrogen gets carried into the lungs and is expired. Expired gases can be collected using a cone, a nonrebreathing valve, and a special collection bag. The sample can be analyzed with a breath hydrogen analyzer. Under normal conditions most of the carbohydrates get absorbed in the small intestine and do not reach the intestinal bacterial flora, mostly located in the colon. However, in dogs with SIBO there is a large bacterial load in the proximal intestine, which metabolizes the carbohydrates and produces hydrogen, leading to an early peak of hydrogen concentration in the expiratory air. In contrast, in animals with malabsorption the carbohydrates reach the bacteria in the large intestine and a late peak of hydrogen concentration in the expiratory air is observed. Hydrogen breath testing can be performed in conjunction with gastrointestinal permeability and mucosal function testing as the same sugars can be utilized for both tests. Finally, by using a non-metabolizable carbohydrate, oro-colonic transit time can be calculated. Breath samples are extremely stable in glass tubes and can be safely shipped.

Serum unconjugated cholic acid (SUCA)11

Serum total unconjugated bile acids are increased in human patients with small intestinal bacterial overgrowth (SIBO). Serum unconjugated cholic acid was also mildly to severely increased in 9 of 10 dogs with culture-proven SIBO. Conjugated BAs are secreted into the duodenum and deconjugated by bile salt hydrolase-producing bacteria. Unconjugated BAs are then reabsorbed and circulate in the vascular space and can be quantified. Most bacterial species involved in small intestinal bacterial overgrowth produce bile salt hydrolase. Thus the concentration of unconjugated bile acids is increased in dogs with SIBO.

13C-aminopyrine demethylation blood test for hepatic function testing12

Currently there is no hepatic function test available for use in small animals that is both sensitive and specific for an altered hepatic function. 13C-aminopyrine breath tests have been used to evaluate hepatic function in both human beings and laboratory animals. These techniques have been adjusted for use in dogs and cats and for the use of blood instead of breath samples. 13C-aminopyrine is administered intravenously and reaches the liver where it is demethylated by microsomal enzymes. 13C is oxidized, diffuses into the blood, and is transported in the blood, mostly as bicarbonate. By addition of a strong acid to the blood sample CO2 can be extracted and the fraction of 13CO2 in the total CO2 can be determined by fractional mass spectrometry. In initial clinical studies dogs and cats treated with phenobarbital had an increased 13C-aminopyrine demethylation, while dogs with severe hepatic disease had a decreased aminopyrine demethylation. Further clinical studies are under way to evaluate the overall clinical usefulness of this new diagnostic tool.


1.  Williams DA, Batt RM. Sensitivity and specificity of radioimmunoassay of serum trypsin-like immunoreactivity for the diagnosis of canine exocrine pancreatic insufficiency. J Am Vet Med Assoc 1988; 192:195-201.

2.  Steiner JM, Williams DA. Serum feline trypsin-like immunoreactivity in cats with exocrine pancreatic insufficiency. J Vet Intern Med 2000; 14:627-629.

3.  Batt RM, Morgan JO. Role of serum folate and vitamin B12 concentrations in the differentiation of small intestinal abnormalities in the dog. Res Vet Sci 1982; 32:17-22.

4.  Simpson KW, Fyfe J, Cornetta A, et al. Subnormal concentrations of serum cobalamin (Vitamin B12) in cats with gastrointestinal disease. J Vet Int Med 2001; 15:26-32.

5.  Steiner JM, Broussard J, Mansfield CS, Gumminger SR, Williams DA. Serum canine pancreatic lipase immunoreactivity (cPLI) concentrations in dogs with spontaneous pancreatitis. J.Vet.Int.Med. 2001;15:274 (abstract).

6.  Steiner JM, Gumminger SR, Rutz GM, Williams DA. Serum canine pancreatic lipase immunoreactivity (cPLI) concentrations in dogs with exocrine pancreatic insufficiency. J.Vet.Int.Med. 2001;15:274 (abstract).

7.  Melgarejo T, Tamayo A, Williams DA. Fecal alpha1-protease inhibitor (α1-PI) for the diagnosis of canine proteinlosing enteropathy. J Vet Int Med 1997; 11:115 (abstract)

8.  Sorensen SH, Proud FJ, Adam A, et al. A novel HPLC method for the simultaneous quantification of monosaccharides and disaccharides used in tests of intestinal function and permeability. Clin Chim Acta 1993; 221:115-125.

9.  Steiner JM, Williams DA, Moeller EM. Development and validation of a method for simultaneous separation and quantification of 5 different sugars in canine urine. Can J Vet Res 2000; 64:164-170.

10. Washabau RJ, Strombeck DR, Buffington CA, et al. Evaluation of intestinal carbohydrate malabsorption in the dog by pulmonary hydrogen gas excretion. Am J Vet Res 1986; 47:1402-1406.

11. Melgarejo T, Williams DA, O'Connell NC, et al. Serum unconjugated bile acids as a test for intestinal bacterial overgrowth in dogs. Dig Dis Sci 2000; 45:407-414.

12. Moeller E, Steiner JM, Williams DA, et al. Preliminary studies of a canine 13C-aminopyrine demethylation blood test. Can J Vet Res 2001; 65:45-49.

Speaker Information
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Jörg M. Steiner,, PhD, DACVIM, DECVIM-CA
GI Laboratory, Texas A&M University
College Station, TX, USA

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