Evaluation of alpha-1-proteinase inhibitor in the diagnosis of protein-losing enteropathy in dogs

Abstract

Canine alpha-l-proteinase (α[1]-PI) inhibitor is a member of the serpin family with a molecular weight of 56,000 daltons. Although the trypsin inhibitory capacities of this protein were described first (thus the alternate name, alpha-1-antitrypsin), it may also inhibit elastase, chymotrypsin, plasmin, kallikrein, and thrombin. Interest in a,-PI first occurred when deficiency of this protease inhibitor was associated with clinical disease in humans. A number of other diseases including neonatal liver disease, adult onset liver disease, and panniculitis have also been associated with a deficiency of this proteinase inhibitor. Although α[1]-PI concentrations have been measured in many animal species, little effort has been directed towards identifying associated clinical syndromes in species other than man. One component of the study reported here was development of a microtiter assay for determination of the presence of trypsin or trypsin-like activity, or trypsin inhibitors, in purification products. Advantages of this assay over many others described in the literature is that it requires only small volumes of unknowns and multiple samples can be assayed quickly and efficiently. Canine PI was purified by a modification of the technique described by Abrams. The purification process involved ammonium sulfate fractionation, gel filtration, ion-exchange chromatography, and affinity chromatography (Concanavalin A-Sepharose column). Antiserum raised against the purified canine α[1]-PI showed immunologic cross-reactivity with human and bovine α[1]-PI. Immunoblotting was investigated as a method of quantitating α[1]-PI for the diagnosis of protein losing enteropathy in dogs. Although α[1]-PI could be detected in the feces of dogs by this method, the specific antibody used recognized other proteins in feces which might be breakdown products of α[1]-PI or albumin, or proteins with common epitopes. Additionally, extraction of α[1]-PI from feces appeared to be highly variable among the samples studied and quantitation of protein content between blots was inconsistent. Given these facts, determination of fecal α[1]-PI in dogs does not presently appear to be a clinically useful tool.