RESUMO
Trypsins have been purified and characterized in a multitude of mammalian and nonmammalian species, but not in the domestic cat (Felis catus). In several species two or more isoforms of trypsin have been isolated. Feline trypsin was purified from feline pancrease by sulfuric acid extraction, ammonium sulfate fractionation, gel filtration and affinity chromatography on a benzamidine-activated sepharose gel. Feline trypsinogen was purified by sulfuric acid extraction, SDS-PAGE and electroelution. Only one isoform of feline trypsin and its zymogen could be demonstrated. Isoelectric focusing on agarose gel revealed an isoelectric point of greater than 10.0 for both feline trypsinogen and trypsin. The molecular weight of feline trysinogen was estimated at 22,600, while the molecular weight of feline trypsin was estimated at 21,000. The N-terminal amino acid sequence of feline trypsinogen was Phe-Pro-Ile-Asp-Asp-Asp-Asp-Lys-Ile-Val-Gly-Gly-Tyr-Thr-Asn-Arg. We conclude that cats either have only one isoform of trypsin or that other isoforms are present in minute quantities, undetectable by commonly used methods. We further conclude that feline trypsinogen and trypsin are cationic. Finally, the N-terminal amino acid sequence of the last 16 amino acid residues of feline trypsinogen is closely related to that of other mammalian species and the final 8 amino acid residues (termed trypsinogen activation peptide) are identical to those of canine cationic trypsinogen.
Assuntos
Pâncreas/enzimologia , Tripsina/química , Tripsina/isolamento & purificação , Sequência de Aminoácidos , Animais , Gatos , Cromatografia de Afinidade , Cromatografia em Gel , Cães , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Dados de Sequência Molecular , Peso Molecular , Pancreatopatias/diagnóstico , Pancreatopatias/veterinária , Análise de Sequência , Homologia de Sequência de Aminoácidos , Tripsinogênio/química , Tripsinogênio/metabolismoRESUMO
OBJECTIVE: To develop and validate a radioimmunoassay for feline trypsin-like immunoreactivity (fTLI). PROCEDURE: A double antibody radioimmunoassay for determination of fTLI was developed, using feline trypsin purified from feline pancreatic tissue antiserum against feline trypsin raised in New Zealand White rabbits, and a commercially available precipitation solution. This assay was validated by determining detection limit, linearity, accuracy, and intra- and interassay variabilities. A control range was determined from samples of 21 clinically normal cats. RESULTS: The detection limit for fTLI in this assay was 1.9 micrograms/L. The linearity of the assay was evaluated by testing dilutional parallelism. Observed/expected values ranged between 95 and 127%. The accuracy of the assay was tested by measuring recovery of added trypsin. Observed/expected values ranged from 99 to 114%. Intra-assay variability ranged between 3.0 and 9.4%, and interassay variability ranged between 4.0 and 8.2%. The control range for serum fTLI was determined to be 17.1 to 48.6 micrograms/L. CONCLUSION: The assay for fTLI described here is sensitive, linear, accurate, precise, and reproducible. CLINICAL RELEVANCE: This assay may be of use for the diagnosis of exocrine pancreatic diseases in cats. Preliminary results of such research have been promising, and further clinical studies are in progress.
Assuntos
Pâncreas/enzimologia , Tripsina/sangue , Animais , Anticorpos , Gatos , Coelhos , Radioimunoensaio/métodos , Valores de Referência , Análise de Regressão , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Three dogs with gastrointestinal tract bleeding of sufficient severity to necessitate blood transfusion were determined to have hypoadrenocorticism on the basis of adrenocortical response to exogenous adrenocorticotropic hormone. All dogs survived the acute crisis and are being managed with fludrocortisone acetate. Hypoadrenocorticism should be considered in the differential diagnosis of acute severe gastrointestinal tract hemorrhage.
