CHARACTERIZATION OF NEUROPEPTIDES EXTRACTED FROM CANINE INTESTINE

Quantitative determination of neuropeptides in biologic tissues by rad ioimmunoassay requires both an efficient extraction of neuropeptides a s well as maintenance of immunochemical reactivity. Vasoactive intesti nal peptide, substance P, and met5-enkephalin were chosen for this stu dy because they are neuropeptides which appear to be involved in multi ple physiologic systems. Since all three neuropeptides have a methioni ne residue within their amino acid sequence, oxidation of methionine t o methionine-sulfoxide during the extraction process could diminish th eir immunochemical reactivity. Multiple factors that might be importan t in extracting these neuropeptides from canine intestine, including p H of the solvent, tissue homogenization, heating, and addition of enzy me inhibitors, were examined. Concentrations of vasoactive intestinal peptide-like immunoreactivity and substance P-like immunoreactivity we re significantly higher in acidic solvents, and tissue homogenization appeared to increase the concentrations of these two neuropeptides. Su bstance P-like immunoreactivity was increased by heating after tissue homogenization, suggesting heat-induced denaturation of tissue enzymes liberated by homogenization. Separation of acidic tissue extracts by high performance liquid chromatography followed by radioimmunoassay fo r all three neuropeptides revealed minor acid-induced oxidation of sub stance P. These results should be useful for planning the extraction o f these three neuropeptides from other tissues.