Jörg M. Steiner, DrMedVet, PhD, DACVIM, DECVIM-CA, AGAF
Diseases of the gastrointestinal tract are a common cause for dogs and cats to be presented to a veterinarian. In order to arrive at the most appropriate diagnosis, veterinarians need to employ diagnostic tests. While no diagnostic test is always positive in a patient with a certain disease, and no diagnostic test is always negative in a patient without the disease, the clinician needs to strive to use those tests with the highest clinical accuracy possible. In addition to having a high accuracy, diagnostic tests should also be economical and as minimally invasive as possible. In general, the clinician is guided by the golden rule to only perform a diagnostic test when the outcome of the test will have an impact on the management of the patient. This golden rule can be expressed more scientifically by stating that a diagnostic test should only be performed if the post-test probability for a disease is either significantly smaller or significantly larger than the pre-test probability. This means that the probability of a certain disease either needs to increase or decrease after the results of a diagnostic test have been received and interpreted.
While some diagnostic tests are either negative or positive, most diagnostic tests will yield a quantitative result that needs to be interpreted. This is achieved by comparison of the result found in a diseased animal to that found in a healthy animal (reference interval, control interval, or normal range). However, a result outside the reference interval does not necessarily indicate disease, and cut-off values need to be determined that afford the best test characteristics for the diagnosis of a disease. This optimal cut-off value can be determined by use of a receiver-operator statistic. However, different investigators use this type of statistic in a different manner. While some investigators determine the overall optimal performance of an test, by determining the maximum area under the curve, it may be diagnostically more meaningful to set minimum requirements for either sensitivity (e.g., in case a test is intended to be used as a screening test) or specificity (e.g., in case a test is intended to be used for arriving at a definitive diagnosis) and then determine the optimal cut-off value. It is important to note that a result outside the reference interval may have different implications for different tests. For example, if the upper limit of the reference interval for serum creatinine concentration is 1.5 mg/dL, a result of 1.7 mg/dL may very well be significant, while a serum ALT activity of 76 U/L in a dog may be of no significance when the reference interval is 0–65 U/L. Therefore, a cut-off value for each diagnostic test must be identified, that determines whether an abnormality is significant enough to warrant further clinical work-up or even a specific diagnosis of a disease.
Serum Folate Concentration
Folate is a water-soluble B vitamin (vitamin B9) that is plentiful in most commercial pet foods. However, folate in the diet is mostly supplied as folate polyglutamate, which cannot be readily absorbed. In the proximal small intestine, folate polyglutamate is deconjugated by folate deconjugase and the resulting folate monoglutamate is absorbed by specific folate carriers in the proximal small intestine.
In patients with proximal small intestinal disease, both folate deconjugase and folate carriers can be destroyed. If the disease process is severe enough, either folate polyglutamate is no longer deconjugated or folate monoglutamate is no longer absorbed, leading to folate malabsorption. If the condition continues for a significant period of time, folate body stores are depleted and serum folate concentration decreases. The same is true in patients with diffuse small intestinal disease, as long as the proximal small intestine is involved in the disease process. Many bacterial species synthesize folate, and it is believed that an increased number of bacterial species (i.e., small intestinal dysbiosis) can lead to significant increases in serum folate concentrations.
Serum Cobalamin Concentration
Cobalamin (vitamin B12) is also a water-soluble vitamin that is plentiful in most commercial pet foods. Dietary deficiencies are thus not very common. However, owners who feed their pets exclusively vegetarian or even vegan diets may inadvertently cause cobalamin deficiency in their pets, unless the diet is sufficiently fortified with cobalamin. Dietary cobalamin is bound to animal-based dietary protein and cannot be absorbed in this form. In the stomach, dietary protein is digested by pepsin and HCl and cobalamin is being released. The free cobalamin is immediately bound by R-protein, which is a cobalamin transporter protein that is synthesized in the stomach. In the small intestine, R-protein is digested by pancreatic proteases, and the free cobalamin is bound by intrinsic factor. In dogs and cats, the vast majority of intrinsic factor is secreted by the exocrine pancreas. This is different from humans where the majority of intrinsic factor is secreted by the stomach. Intrinsic factor/cobalamin complexes are absorbed by specific receptors in the ileum.
Distal small intestinal disease, if severe, will lead to destruction of cobalamin receptors in the ileum, leading to cobalamin malabsorption. Cobalamin malabsorption will ultimately lead to depletion of cobalamin body stores and cobalamin deficiency. Diffuse small intestinal disease can also lead to cobalamin malabsorption as long as the ileum is involved in the disease process. Exocrine pancreatic insufficiency also commonly leads to cobalamin deficiency. Finally, an increased number of bacterial organisms in the small intestine will lead to competition for the available cobalamin and may also lead to cobalamin deficiency.
Fecal α1-Proteinase Inhibitor Concentration
Many gastrointestinal disorders, if severe, can be associated with gastrointestinal protein loss. Traditionally, the gold standard for evaluation of gastrointestinal protein loss is 51Cr-albumin excretion, but this diagnostic test is labor- and time-intensive and is also associated with exposure of the patient and personnel to radioactivity. Recently, assays for the measurement of canine and feline α1-proteinase inhibitor (α1-PI) concentration in feces have been developed and analytically validated. Alpha1-proteinase inhibitor is synthesized in the liver and inhibits a variety of different proteinases. Alpha1-proteinase inhibitor has a molecular mass of approximately 60,000 Da, which is similar to that of albumin. Thus, when gastrointestinal disease is severe enough to be associated with gastrointestinal loss of albumin, α1-PI is lost as well. In contrast to albumin, α1-PI is not hydrolyzed by digestive and bacterial proteinases in the gastrointestinal lumen. This is due to the fact that α1-PI is a proteinase inhibitor. Therefore, fecal α1-PI concentration can be used as an estimate for gastrointestinal protein-loss. Currently, assays for measurement of both canine and feline α1-PI are only available through the gastrointestinal laboratory at Texas A&M University (www.cvm.tamu.edu/gilab).
Clinically, fecal α1-PI concentration should be evaluated in dogs and cats with hypoalbuminemia that do not have clinical signs of gastrointestinal disease and where an extragastrointestinal source of protein loss cannot be identified. Also, dogs belonging to a breed that is associated with a high prevalence of protein-losing enteropathy (e.g., Norwegian Lundehund, Soft-Coated Wheaten Terrier, Yorkshire Terrier) that don't have any clinical signs of gastrointestinal disease, but are intended for breeding, should be tested.
Serum C-Reactive Protein Concentration
Canine C-reactive protein (CRP) is a systemic marker of inflammation that is receiving increasing interest for the assessment of inflammatory diseases in dogs. C-reactive protein is a member of the acute phase reactant family of proteins in the dog. Synthesis of this group of proteins is dramatically increased during inflammatory disease.
C-reactive protein concentrations in serum have been reported to be increased in dogs with inflammatory bowel disease, and have also been shown to correlate well with the IBD clinical disease activity index. Thus, the greatest clinical utility of this assay is for monitoring of response to treatment in dogs with inflammatory bowel disease, as effective dietary or medical therapy will be associated with a decrease in serum CRP concentration.
Concentrations of S100 Proteins in Feces
S100 proteins are a group of Ca-binding proteins that are extremely important in maintaining inflammation. At the moment, there are two assays for the measurement of S100 proteins in dogs - one assay measures calprotectin, while the other one measures S100A12. Both proteins can be measured in serum or feces and have been shown to be increased in either sample type with a variety of acute and chronic gastrointestinal diseases. Similar to measurement of CRP, the greatest clinical utility of these assays is for monitoring of response to treatment in dogs with inflammatory bowel disease. However, further research will need to be conducted to elucidate the clinical utility of these assays for the assessment of dogs and cats with signs of gastrointestinal disease.
There have been conflicting results regarding the importance of mast cells in dogs and cats with idiopathic inflammatory bowel disease. Mast cells do release histamine upon degranulation, but histamine itself is rather unstable. In contrast, N-methylhistamine is a stable histamine metabolite and thus can be used as a marker for mast cell degranulation. In a recent study, approximately 50% of dogs with chronic enteropathy showed increases in fecal N-methylhistamine concentrations, suggesting that at least in a subgroup of dogs with chronic enteropathies, the disease is associated with an increase in mast cell degranulation.