Clinical Techniques in Small Animal Practice
Volume 18, Issue 4 , Pages 203-210, November 2003

Laboratory assessment of gastrointestinal function

  • Jan S Suchodolski, med vet, Dr med vet

      Affiliations

    • Corresponding Author InformationAddress reprint requests to Jan S. Suchodolski, Dr med vet, Gastrointestinal Laboratory, Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, TXM 77843-4474, USA
    • Gastrointestinal Laboratory, Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA
    • ,
  • Jörg M Steiner, med vet, Dr med vet, PhD, Diplomate ACVIM, Diplomate ECVIM-CA

      Affiliations

    • Gastrointestinal Laboratory, Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA

Article Outline

Abstract 

Over the past decade, several laboratory tests have been introduced to veterinary medicine that allow the minimally invasive assessment of diseases of the gastrointestinal tract, liver, and pancreas. Although some of those tests have limited clinical use in a practice setting and have more use as research tools, other tests, such as serum cobalamin and folate concentrations, find wide application in everyday veterinary practice. In some cases, definitive diagnosis requires invasive or expensive procedures, but these new laboratory tests have greatly aided veterinarians in diagnosing gastrointestinal diseases. This discussion provides an overview of new diagnostic tests that will allow the minimally invasive assessment of gastrointestinal function.

 

Endoscopy has been viewed as the most useful tool in the diagnosis of gastric and intestinal disease, allowing direct visualization of the gastric and intestinal mucosa, and also the collection of biopsy samples. Disadvantages of endoscopy include the need for expensive equipment, expertise in handling the endoscope, experience in obtaining biopsy specimens, and the potential risks of general anesthesia.1 Limitations of histopathology in the assessment of gastrointestinal biopsies have become apparent recently.2 Over the past decade, new, minimally invasive markers for the diagnosis of gastric, pancreatic, and intestinal disease have been evaluated for use in veterinary medicine.

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Laboratory assessment of gastric function and pathology 

Gastric permeability testing 

Sucrose is a disaccharide that is large enough to be excluded from passive permeation by the intact gastric and intestinal epithelia. As sucrose is rapidly digested within the proximal small intestine, absorption of the intact sucrose molecule and recovery in serum or urine implies damage to the mucosa before this site.3 Sucrose permeability has increased in laboratory dogs with experimentally induced gastropathy.4 Results of this study suggest that this technique is more sensitive for generalized mucosal damage than for the presence of discrete, endoscopically visible ulceration. Because sucrose testing is often performed in combination with other markers that assess intestinal permeability, this technique is discussed in more detail later (see Gastrointestinal Permeability and Intestinal Absorptive Function).

Laboratory assessment of helicobacter pylori infection 

H pylori infection is now accepted as the most common cause of chronic gastritis and duodenal ulceration in human beings. Other Helicobacter spp have been linked to gastritis in dogs and cats.5 Although H pylori has so far only been identified in a colony of laboratory cats, other Helicobacter spp, including H felis, H bizzozeronii, H salomonis, and Flexispira rappini, have been isolated from the stomach of dogs and cats. Many investigators report a high prevalence of infection, up to 100%.6 Helicobacter spp are able to disrupt the gastric mucosal barrier through the secretion of phospholipases and disruption of gastric secretory activity.7 Because Helicobacter spp have been found in clinically healthy animals, the pathogenic role of Helicobacter spp in canine and feline gastritis is unclear.5 Nevertheless, a variety of tests have been developed to detect Helicobacter spp infection in dogs and cats. Most of these tests detect the presence of Helicobacter spp but do not reveal whether the infection is leading to pathology.

Most of the diagnostic tests for the diagnosis of Helicobacter spp infection require gastroscopy. Gastroscopy may reveal superficial nodules that suggest lymphoid follicle hyperplasia.5 Other endoscopic findings include diffuse gastric rugal thickening, mucosal flattening, punctate hemorrhages, and erosions. Histopathology of biopsy samples using special stains, such as Warthin Starry and modified Steiner, allows visualization of the spiral-shaped organisms and is considered to be the most sensitive test. A commercially available, in-house rapid-urease test (eg, CLO or Campylobacter-like organism test) can be performed on gastric biopsy specimens obtained during gastroscopy.8 Polymerase chain reaction of deoxyribonucleic acid (DNA) extracted from biopsy specimens or from gastric juice is also a highly sensitive test. Some animals have a patchy distribution of Helicobacter spp infestation throughout the stomach. Thus, it is crucial to take several biopsy samples to increase the diagnostic sensitivity of tests that use gastric biopsies. During gastroscopy, impression smears can be taken from the gastric mucosa using a cytology brush. The brush is then rolled across a microscope slide and the slide stained either with May-Grünwald-Giemsa, Diff-Quik, or Gram stain.8 This test shows excellent sensitivity and can easily be performed in a practice setting.

Bacterial culture to detect Helicobacter spp requires special protocols and media, yet has a low success rate. Serologic testing measures circulating antibodies directed against Helicobacter spp in serum. Although serologic tests have a relatively high sensitivity, they cannot distinguish between infections with different Helicobacter spp. In addition, serologic tests cannot be used to monitor eradication of Helicobacter spp infections because serum IgG concentrations remain increased up to 6 months after successful eradication of an infection.

Another noninvasive test to diagnose Helicobacter spp infection is based on detection of the metabolic activity of Helicobacter spp using the 13C-urea breath or blood test (13C-UBBT). Helicobacter spp produce the enzyme urease, which catalyzes the metabolism of orally administered 13C-urea. The 13C is released from the urea molecule and is incorporated into 13CO2, which can be quantified in either breath or blood samples.9 The major advantages of the 13C-UBBT are that it does not require endoscopy, and it measures the metabolic activity of Helicobacter spp, making it useful for monitoring the eradication of infection.

Laboratory assessment of gastric emptying 

Although scintigraphy is considered the gold standard for the assessment of gastric emptying, radiation safety concerns and the need for expensive equipment limit this technique to a small number of institutions. Alternatively, radiopaque markers can be used to assess gastric emptying.10 Depending on their size, radiopaque markers mimic gastric emptying of either solid or liquid food, but they do not mimic gastric emptying of a more complex meal. Recently, a 13C-octanoic acid breath test has been introduced to assess gastric emptying time in horses, dogs, and cats.11, 12, 13 It showed good correlation with scintigraphy.13 Octanoic acid is a medium chain length fatty acid that is completely absorbed in the duodenum and oxidized in the liver, where 13C is incorporated into 13CO2. The 13CO2 can be quantified in breath or blood samples. The major advantage of this test is that it is practical to mix 13C-octanoic acid into many kinds of food. This test is not yet available for routine use but shows promise for the near future.

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Assessment of intestinal function and pathology 

Gastrointestinal permeability and intestinal absorptive function 

Marker molecules used for permeability testing or absorptive function penetrate the gastrointestinal mucosa in a known fashion. Depending on the marker molecule, a decrease or increase in the urinary concentration of the administered marker molecules is observed in gastrointestinal disease. Sucrose, a disaccharide, has been used as a marker of gastric permeability in dogs. Sucrose is too large to penetrate the intact gastric mucosa. Unabsorbed sucrose that reaches the small intestine is rapidly hydrolyzed to glucose and fructose by the brush border enzyme sucrase. Thus, permeability towards intact sucrose is low in clinically healthy individuals. Increased permeability towards sucrose has been associated with gastric mucosal damage in dogs.4

The inert sugars rhamnose and lactulose are commonly used as markers for intestinal permeability (Fig 1). Rhamnose, a monosaccharide, penetrates the intestinal mucosa through small aqueous pores that are believed to be located in the cell walls (transcellular uptake). The frequency of those pores is relatively high. In contrast, the disaccharide lactulose can only penetrate the intestinal mucosa through larger pores that are located near the tight junctions of the intestinal cells (paracellular uptake). These larger pores are less frequent than the smaller pores that allow penetration of monosaccharides. Therefore, in animals with an intact intestinal epithelium, relatively less lactulose than rhamnose will penetrate the intestinal mucosa and can be recovered in urine. This recovery is expressed as the lactulose/rhamnose (L/R) ratio. Intestinal disease often leads to a decrease in the total surface area of the intestinal epithelium and, subsequently, to a change in the number of available pores (Fig 1). The number of small pores that usually facilitate permeation of small molecules, such as rhamnose, decreases with decreased epithelial surface area, while the number of large pores increases. Therefore, the urinary recovery of lactulose increases, while the recovery of rhamnose decreases. Increased intestinal permeability thus leads to an increase in urinary L/R ratio.

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  • Fig 1. 

    The assessment of intestinal permeability and mucosal absorptive function. A mixture of 2 sugar probes, rhamnose and lactulose, is given orally and quantified in urine. (A) Rhamnose (R), a monosaccharide, penetrates the intestinal mucosa through aqueous pores (transcellular uptake). The larger molecule, lactulose (L), penetrates the intestinal mucosa through pores near the tight junctions between cells (paracellular uptake). These pores are infrequent. Therefore, in animals with an intact epithelium, relatively more rhamnose than lactulose will penetrate the mucosa and will be recovered in urine (low urinary lactulose/rhamnose [L/R] ratio). (B) Gastrointestinal disease leads to a decrease in the mucosal surface area (ie, decrease in the number of aqueous pores) and an increase in the number of larger pores. Relatively more lactulose can be recovered in urine, leading to an increase in the urinary L/R ratio in intestinal disease.

Mucosal absorptive function testing involves administration of markers that are absorbed via a carrier-mediated transport process. The monosaccharides xylose (X) and methylglucose (M) are commonly used to evaluate small intestinal absorption. Xylose is absorbed by fructose carriers, and methylglucose is absorbed by glucose carriers in the small intestine. The use of 2 different markers increases the sensitivity for intestinal damage that leads to impaired intestinal absorption.14 Urinary recovery of both sugars is reported as X/M ratio.

Methodology 

The sugars can be ordered from the Gastrointestinal Laboratory at Texas A&M University. Depending on the suspected localization and type of gastrointestinal disease, a combination of different sugars can be administered (2-sugars: lactulose and L-rhamnose; 4-sugar: 2 sugar solution plus methylglucose and D-xylose; 5-sugars: 4 sugar solution plus sucrose). Food is withheld overnight. Before the test procedure, the bladder is emptied completely by catheterization. The sugars are then dissolved in water and administered by gavage using a feeding tube. Over a period of 6 hours, all urine is collected and the volume recorded. After the 6 hours, the bladder is catheterized again and the volume present added to any collected during the 6-hour period. Complete urine collection is important for accurate calculations of the amount of the sugars that have permeated the intestinal mucosa or have been absorbed and subsequently excreted in the urine. The collected urine is mixed thoroughly, and a frozen aliquot, together with the record of total urine volume collected, is submitted to the laboratory. The use of serum sugar ratios to assess gastrointestinal permeability and function is under investigation. This serum test will avoid the need for catheterization and collection of urine.

Serum cobalamin and folate 

Cobalamin (vitamin B12) and folate uptake from the small intestine can be affected by several factors, and serum concentrations of these vitamins can, therefore, be used as an indirect marker for gastrointestinal disease. Small intestinal inflammation, exocrine pancreatic insufficiency (EPI), and small intestinal bacterial overgrowth (SIBO) can all lead to changes in serum cobalamin and/or folate concentrations. In addition, the measurement of serum cobalamin and folate may yield information on the site of intestinal disease. Table 1 outlines the interpretation of combinations of serum concentrations of cobalamin and folate in the assessment of gastrointestinal disease, and their potential use to localize a disease process within the gastrointestinal tract.

TABLE 1. Alterations in Serum Concentrations of TLI, Cobalamin and Folate in Dogs and Cats with Gastrointestinal Disease
EPISIBOProximal Small Intestinal DiseaseDistal Small Intestinal Disease
TLIdecreasedN/AN/A in dogs; may be elevated in catsN/A in dogs; may be elevated in cats
Serum Cobalaminmay be low in dogs; almost always low in catsmay be low in dogs and catsno changemay be low in dogs and cats
Serum Folatemay be elevated in dogs and cats with secondary SIBOmay be elevated in dogs; SIBO is rare in catsmay be low in dogs and catsN/A
Cobalamin 

Cobalamin is an essential water-soluble vitamin and an important cofactor for a variety of biochemical reactions. Cobalamin is usually abundant in canine and feline diets, making a dietary deficiency unlikely. The mechanisms that lead to absorption of cobalamin are very complex and depend on a correctly functioning digestive system (Fig 2). In the diet, cobalamin is initially bound to dietary protein. After digestion of these proteins in the stomach by pepsin and hydrochloric acid, cobalamin is released and immediately bound to R-protein, secreted in saliva and gastric juice. Pancreatic enzymes (ie, trypsin and chymotrypsin) digest the R-protein, again releasing cobalamin. The intrinsic factor, produced in the stomach and pancreas, binds to cobalamin and serves as a transporter to the distal small intestine where the cobalamin/intrinsic factor complexes are absorbed by specific receptors located in the ileal mucosa.

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  • Fig 2. 

    Normal mechanism of cobalamin absorption by cobalamin carriers in the distal small intestine. Cobalamin enters the gastrointestinal tract bound to dietary protein. (A) In the stomach, pepsin and hydrocholoric acid degrade the dietary protein, releasing the cobalamin. The cobalamin is immediately bound by R-protein, which is produced in the stomach mucosa, and is transported in this form to the duodenum. In the duodenum, pancreatic proteinases digest the R-protein, releasing the cobalamin. (B) Free cobalamin in the duodenum is bound yet again by intrinsic factor for transport to the distal small intestine. In dogs and humans, intrinsic factor is produced by both the stomach and the pancreas. In the cat, only the pancreas produces intrinsic factor. (C) Cobalamin remains bound to intrinsic factor during passage through the cranial small intestine. (D) In the distal small intestine, the cobalamin/intrinsic factor complexes are taken up by specific receptors found only on enterocytes in the ileum. The enterocytes process the intrinsic factor/cobalamin complex and release cobalamin into circulation where a final set of binding proteins (ie, transcobalamins) bind the vitamin and carry it to the cells.

Folate 

Folate is a water-soluble vitamin that, similar to cobalamin, is abundant in canine and feline diets, making a nutritional deficiency unlikely. Dietary folate is usually present in the poorly absorbable polyglutamate form (Fig 3). Folate deconjugase, a brush border enzyme produced in the jejunum, removes all but one glutamate residue from the molecule. Specific carriers for folate monoglutamate in the upper small intestine promote folate uptake. Disease processes in the proximal small intestine can lead to the damage of folate carriers, resulting in a decreased serum folate concentration. Folate uptake in the jejunum is optimal at a mildly acidic pH. A decrease in luminal pH increases folate uptake. Erythrocytes contain high levels of folate, and hemolysis of red blood cells may lead to falsely increased folate concentrations. Blood should be redrawn for folate determination if hemolytic serum is obtained.

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  • Fig 3. 

    Normal mechanism of folate absorption by folate carriers in the proximal small intestine. Dietary folates enter the gastrointestinal tract predominantly in the polyglutamate form. Folate deconjugase, a brush border enzyme in the cranial small intestine, deconjugates the folate polyglutamate to form folate monoglutamate. Specific folate carriers present on enterocytes located in the cranial small intestine subsequently absorb folate monoglutamate. Both the deconjugase and folate carrier molecules are restricted to the cranial small intestine. There is no appreciable absorption of folate in the distal small intestine or colon.

Serum cobalamin and folate concentrations in patients with small intestinal disease 

Changes in serum cobalamin and folate commonly occur with disorders of the exocrine pancreas, and both vitamins are often abnormal in animals with EPI. Therefore, it is important first to assess exocrine pancreatic function. If serum trypsin-like immunoreactivity (TLI) is normal, then alterations in serum cobalamin and folate are highly suggestive of small intestinal disease.15 As mentioned previously, both vitamins are taken up by specific receptors located in different areas in the small intestine. Gastrointestinal disease, such as chronic inflammation, may damage these receptors. The loss of receptors may lead, depending on the localization of inflammation, to subnormal serum concentrations of one or both vitamins.16 Not all intestinal disorders are sufficiently severe or long-standing to deplete body stores of each vitamin. Also, not all animals with intestinal disease have morphologic abnormalities of the receptors.

Serum cobalamin and folate concentrations in patients with EPI 

Abnormalities of both vitamins are common in dogs, and are especially common in cats with EPI. Serum cobalamin must bind to intrinsic factor to be absorbed by the receptors located in the distal ileum. The sole source of intrinsic factor in cats, and the major source in dogs, is the pancreas. EPI will lead to decreased secretion of intrinsic factor and, therefore, decreased uptake of cobalamin. Cats with EPI almost always have subnormal serum cobalamin concentrations.15 Similar, subnormal cobalamin concentrations can be observed in dogs with EPI. In dogs, intrinsic factor is also secreted by the gastric mucosa. Thus, dogs with EPI less frequently have low serum cobalamin concentrations than cats. Cats with EPI will also often have subnormal serum folate. Cats with EPI often have concurrent inflammatory bowel disease that may damage folate receptors in the proximal small intestine.

In contrast to cats, dogs with EPI often have increased folate concentrations. The pancreatic juice contains antibacterial substances that help suppress bacterial colonization within the small intestine. In EPI, secretion of these substances is reduced, which may lead to SIBO. Bacteria present in the proximal small intestine can produce folate, which is absorbed by folate specific receptors located in the proximal small intestine. In contrast to dogs, SIBO is an uncommon disorder in cats, and they rarely present with increased folate concentrations.

Serum cobalamin and folate concentrations in patients with SIBO 

Measurement of serum cobalamin and folate is the most helpful aid in the diagnosis of SIBO, although the 2 have relatively poor sensitivity and specificity.17 Serum cobalamin may be decreased, and serum folate may be increased in animals with SIBO. If both vitamins are altered, this is highly suggestive of SIBO. Changes may occur only in long-standing disease Fig 2, Fig 3.

Aberrations in the small intestinal microflora may lead to increased competition for cobalamin, resulting in decreased absorption by the host. Bacteria in the gut may compete with receptors located in the distal small intestine and, therefore, prevent cobalamin uptake by enterocytes. Bacteroides spp are believed to be the principal organisms involved because they can bind the cobalamin-intrinsic factor complex, while other bacteria can only bind the free cobalamin that is present in lower concentrations in the gut. The reported sensitivity of serum cobalamin for the diagnosis of SIBO ranges between 25% and 55%.17

Bacteria present in the distal small intestine and large intestine produce large quantities of folate, which is subsequently excreted in feces. The folate carriers that promote folate uptake are located exclusively in the proximal small intestine, and, thus, folate produced in distal sections of the intestine will not typically be absorbed. If folate producing bacteria migrate upward into the proximal small intestine, this folate can be absorbed by the host, resulting in increased serum folate concentrations. The reported sensitivity of serum folate for the diagnosis of SIBO in dogs ranges from 50% to 66%.17 As mentioned previously, dogs with EPI have a decreased pancreatic secretion of antibacterial products, resulting in a predisposition for SIBO. Dogs with EPI often have increased serum folate concentrations. Cats rarely have SIBO, even in EPI. Therefore, increased serum folate concentrations are rarely observed. Cats with EPI often have rather moderate-to-severe subnormal folate concentrations.

Serum unconjugated bile acids in dogs with SIBO 

The measurement of the serum concentration of unconjugated bile acids (SUBA) allows an indirect assessment of the metabolic activity of the small intestinal microflora. Some bacteria present in the lumen of the intestine can deconjugate bile acids through the action of deconjugase enzymes, a feature unique to bacterial cells.18 Unconjugated bile acids are readily absorbed by passive diffusion and transported to the liver.19 Hepatic clearance of unconjugated bile acids is less efficient than for conjugated bile acids, and unconjugated bile acids are bound strongly to albumin, promoting spill-over from the portal to the systemic circulation. Because the deconjugation of bile acids in the small intestine is unique to bacterial cells, the measurement of unconjugated bile acid in serum is considered an index of bacterial activity in the small intestine.20 This test has been validated for use in dogs with a reported sensitivity of 80%.20 However, in recent studies, the sensitivity and specificity of SUBA for dogs suspected of having SIBO was low.17 SUBA show a marked postprandial increase.21 Therefore, a long fasting period, at least 12 hours and preferably longer, is necessary to avoid measuring increased concentrations that originate from cholecystokinin mediated bile acid release rather than reflecting a pathologic state.

Fecal α1-proteinase inhibitor for the diagnosis of protein-losing enteropathy in dogs 

Protein-losing enteropathies (PLE) are defined as a heterogeneous group of diseases in which plasma proteins are lost into the gastrointestinal lumen.22 Gastrointestinal diseases that may cause PLE include inflammatory enteropathies (eg, mucosal erosion and ulceration), lymphatic obstruction, neoplasia, foreign bodies, intussusception, SIBO, parasitic and fungal enteropathies, and immune-mediated disorders.23 Traditionally, enteric protein loss has been measured by determining the fecal loss of radiolabeled albumin (51Cr-albumin).24 Although this test is considered the gold standard for the diagnosis of PLE in dogs, obvious disadvantages, such as the use of radioactivity, the requirement for approved facilities, and the collection of fecal samples for at least 5 days, have made this test impractical for routine clinical testing. Recently, an enzyme-linked immunosorbent assay (ELISA) for the measurement of α1-proteinase inhibitor (α1-PI) has been validated for the diagnosis of PLE in dogs.25 Alpha1-PI is a specific proteinase inhibitor that is present in the lumen of the gastrointestinal tract only in trace amounts. Because α1-PI has a similar molecular mass to albumin, it is considered a good indicator of the loss of plasma proteins into the gastrointestinal tract. A major advantage of α1-PI, when compared with albumin, is its resistance to proteolytic enzymes and, therefore, its ability to be recovered in feces as an intact molecule. Fecal α1-PI may be increased before protein loss is severe enough to notice hypoalbuminemia. The ELISA for measuring fecal α1-PI is species specific and has been validated for dogs only.

Sample preparation for the measurement of fecal α1-PI 

It is important to submit 1 g of feces per sample to allow for accurate determination of fecal α1-PI in the sample. Therefore, special, preweighed containers are provided by the Gastrointestinal Laboratory at Texas A&M University, which is currently the only laboratory that provides this clinical test. To correct for intersample variations, fecal samples (1 g each) are collected from either 3 different bowel movements from the same day or 3 different bowel movements collected on 3 different days. Samples collected from spontaneous defecation show a lower interday variation than samples collected via rectal palpation.26 Samples should be frozen immediately to prevent oxidation of fecal α1-PI. A fecal α1-PI concentration higher than 15 μg/g of feces in any of the 3 fecal samples is suggestive of an excessive protein loss into the gastrointestinal tract. An average fecal α1-PI concentration higher than 9.4 μg/g of feces is considered diagnostic for PLE.

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Assessment of hepatic function and pathology 

Bile acids-fasting and postprandial 

Bile acids are exclusively synthesized in the liver. Following release into the duodenum with feeding, bile acids undergo enterohepatic circulation. After bile acids are excreted, 95% are reabsorbed in the ileum and return to the liver through portal circulation. A normal liver is capable of rapidly clearing these circulating bile acids from the portal circulation, resulting in low fasting serum bile acids and a slight increase after feeding. Total serum bile acids are typically measured after a 12-hour fast (preprandial). After collection of a blood sample, a test meal is fed to the animal, and a second blood sample is collected 2 hours after feeding (postprandial). Studies have shown that the analysis of fasting and postprandial total serum bile acids is useful for the diagnosis of hepatobiliary disease in dogs and cats.27 Several factors can influence the circulation of bile acids, which can, therefore, interfere with individual and total serum bile acid concentrations. Those factors include the rate of bile acid synthesis, synchronization and completeness of gall bladder contractions, gastric emptying, the efficacy of ileal bile acid reabsorption, the integrity of the portal circulation, the function of the biliary system, and enterohepatic cycling frequency. In addition, the type of test meal administered can influence postprandial bile acid concentrations. Insufficient food intake, or variations in amino acid or fat content can result in the failure of cholecystokinin stimulation and gall bladder emptying.

13C-aminopyrine demethylation test 

The 13C-aminopyrine demethylation test (ADT) is a noninvasive and specific test to assess liver function and has shown good correlation with the severity of hepatic disease in human beings.28 Exogenously administered, aminopyrine is exclusively demethylated by hepatic microsomal enzymes. The ADT is based on the administration of a labeled form of aminopyrine and measuring the rate of demethylation. After intravenous administration of aminopyrine, labeled with 13C, a stable isotope of carbon, the compound is transported to the liver via the hepatic and portal circulation, and is demethylated, forming 13CO2. The 13CO2 then diffuses into the vascular space, and the amount of 13CO2 present can be determined as a percent dose of 13C-aminopyrine in blood samples.29 Reduced hepatic function is associated with reduced demethylation of the 13C-aminopyrine. The ADT has been validated for use in dogs and cats, and the diagnostic performance of this test is under active investigation in veterinary species.

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The assessment of exocrine pancreatic insufficiency 

This section only discusses the laboratory assessment of canine and feline exocrine pancreatic insufficiency (EPI). For a more detailed discussion on the diagnosis of pancreatitis see Ruaux: Diagnostic Approaches to Acute Pancreatitis, pp 245–249. EPI is caused by a loss of pancreatic acinar cells, resulting in decreased production of pancreatic digestive enzymes, which leads to a malabsorption syndrome.30 In dogs, this disease process is most commonly caused by idiopathic pancreatic acinar cell atrophy. In contrast to dogs, EPI in cats is caused most commonly by chronic pancreatitis.15 Clinical signs of EPI are due to a malabsorption and malassimilation syndrome resulting from insufficient digestion of dietary components. Patients show weight loss with normal to increased appetite, increased fecal volume, and continuous or intermittent soft stools. In rare cases, polyuria/polydipsia has been reported in animals with concurrent EPI and diabetes mellitus secondary to chronic pancreatitis.

Diagnosis of EPI 

Serum lipase and serum amylase activity do not exclusively originate from the pancreas, and significant serum lipase and amylase activities remain in pancreatectomized dogs or dogs with EPI.31 Thus, serum amylase and lipase activities are of no diagnostic value in the diagnosis of EPI in dogs and cats.15 Trypsin-like immunoreactivity (TLI) is a pancreas specific marker. Trypsinogen is synthesized and stored exclusively by the acinar cells of the pancreas. Under physiologic conditions, small quantities of trypsinogen are released into the vascular space and can be measured by immunoassay.15 Measuring serum TLI provides an excellent indirect assessment of pancreatic function, and is the test of choice to establish the diagnosis of EPI in both dogs and cats. The test principle is based on measuring circulating trypsinogen and trypsin by immunoassay.32 A loss of acinar cells, either through pancreatic acinar atrophy or chronic pancreatitis, will result in decreased trypsinogen production and subsequently subnormal TLI concentrations in serum. The decreased TLI concentrations, in combination with clinical signs consistent with EPI, are a result of long-standing disease when most of the acinar cells are already destroyed.33 In rare cases, serum TLI concentrations can be subnormal, even before clinical signs of EPI are seen.15

Serum canine TLI (cTLI) and serum feline TLI (fTLI) concentrations have been highly sensitive and specific for EPI in dogs and cats, respectively.34 The reference range for serum cTLI concentration is 5 to 35 μg/L. A cTLI concentration ≤2.5 μg/L is diagnostic for EPI. A serum TLI concentration in the questionable range (2.5 to 5.0 μg/L) can be seen in patients with early disease, may be due to failure to withhold food before sampling, or may be seen in patients with chronic small intestinal disease.15 In these cases, retesting a properly fasted serum sample 6 to 8 weeks later yields more definitive results in most cases. Some dogs with repeatedly decreased serum cTLI concentrations in the questionable range may not show any clinical signs of EPI. It has been speculated that the severely decreased serum TLI concentration may reflect continuous destruction of pancreatic acinar cells, and clinical signs consistent of EPI will eventually develop in those dogs.35

EPI is less common in cats than in dogs. The reference range for fTLI is 12 to 82 μg/L. A fTLI concentration less than 8 μg/L is diagnostic for EPI in cats. A fTLI concentration between 8 and 12 μg/L is in the questionable range. Those cats should be retested after 6 to 8 weeks. Similar to serum TLI concentration, canine serum pancreatic lipase immunoreactivity (cPLI) is highly specific for exocrine pancreatic function and could be used for the diagnosis of EPI.36 The immunoassay for cPLI specifically measures the mass concentration of pancreatic lipase in the serum rather than its kinetic activity. However, there is a small degree of overlap in serum cPLI concentrations between normal dogs and dogs with EPI, making the measurement for cPLI slightly inferior to TLI for a definitive diagnosis of EPI. The clinical use of serum PLI concentration for the diagnosis of feline EPI has not been evaluated yet.

It is also recommended to assay serum cobalamin and serum folate whenever serum TLI is assayed. Abnormal serum concentrations of these vitamins are common in dogs and are especially common in cats with EPI. Therapeutic supplementation with cobalamin and folate may be essential before an optimal response to pancreatic enzyme supplementation is obtained.

Other laboratory tests for EPI 

Other tests for the diagnosis of EPI, such as microscopic evaluation of fecal samples for nondigested food particles, detection of steatorrhea using Sudan-III stain, fecal proteolytic activity, the N-benzoyl-l-tyrosyl-p-aminobenzoic acid (BT-PABNA) test, and measuring canine pancreatic elastase in fecal samples by ELISA, have been used to diagnose EPI. All of these tests have lower sensitivities and specificities for EPI compared with the TLI test and are not recommended for clinical use.

Handling and storage of serum samples 

Table 2 outlines clinical tests available for the assessment of gastrointestinal function in dogs and cats, and the specific areas of sample handling that need to be observed for each test. The TLI and PLI tests are species specific, distinct immunoassays have been validated for use in dogs and cats. Therapeutic substitutions of pancreatic enzymes from commercial sources (usually manufactured from porcine pancreata) do not interfere with the immunoassay. Therefore, it is not necessary to interrupt treatment before drawing a serum sample. Feeding may lead to a postprandial increase in serum TLI in dogs and a slight increase in cats.37, 38 It is suggested that food should be withheld overnight before drawing a blood sample.

TABLE 2. Clinical Tests Used in the Assessment of Gastrointestinal Function in Dogs and Cats
Serum Bile AcidsCanine TLI (cTLI)Feline TLI (fTLI)Serum CobalaminSerum FolateCanine PLI (cPLI)Feline (fPLI)SUBAFecal α1PIInert Sugar Probes
Stable at RTyesyesyesyesyesyesyesnonoyes
HemolysisyesnononoyesnononoN/A?
LipemiayesyesyesnononononoN/A?
Bilirubinemia?nononononononoN/A?
Food intakeyes∗yesyesnonono?yesnoyes
Pancreatic enzyme supplementationN/Ano∗∗no∗∗N/AN/AN/AN/AN/AN/AN/A
Renal insufficiency?yes‡yes‡??nononoN/A?
Any drugs known to interferenone knownnonoB12 injectionsfolic acidnonone knownursodiolnono
Species specificnoyesyesnonoyesyesnoyesno
Validated for use in dogsyesyesnoyesyesyesyesyesyesyes
Validated for use in catsyesnoyesyesyesyesyesnonoyes

∗ Test requires measurement of 12-hour fasting and 2-hour postprandial sample.

∗∗ Therapeutic supplements are not detected, and do not need to be withdrawn before testing.

‡ Severe renal failure only.

Cobalamin and folate are measured by a variety of automated assay systems. These tests are not species specific, and the vitamins can be measured by any laboratory that has the appropriate equipment. However, it is important to ensure that the laboratory performing these tests has validated the assays for use in dogs and cats, and has established reference ranges for both species.

TLI, cobalamin, and folate are stable in serum at room temperature for several days. Erythrocytes contain high levels of folate, and hemolysis of red blood cells may lead to falsely increased folate concentrations, resulting in a false clinical interpretation of the test result. Blood should be redrawn when hemolytic serum was obtained. Severe lipemia in serum will interfere in the radioimmunoassays commonly used for the measurement of TLI. Blood should be redrawn to obtain a serum sample free of lipemia.

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References 

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PII: S1096-2867(03)00075-6

doi:10.1016/S1096-2867(03)00075-6

Clinical Techniques in Small Animal Practice
Volume 18, Issue 4 , Pages 203-210, November 2003

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