Chemical characterization of circulating porcine ileal polypeptide in plasma from normal adult pigs.

Antisera against porcine ileal polypeptide (PIP) were raised in New Zealand rabbits and tested in a double antibody immunoassay system. All reactants were diluted in Veronal buffer, pH 8.6, and benzamidine hydrochloride (BzCl) was added to all tubes to a concentration of 1 mM. [125I]PIP was prepared by the lactoperoxidase method, and bound and free were separated by the addition of a second antibody, goat anti-rabbit immunoglobulin G. Antisera A-2 was chosen for use in the immunoassay at a final dilution of 1:64,000. The assay is sensitive to 0.5 ng/ml and has a detection limit of 0.1 ng/ml. Blood samples were collected from the ear vein of conscious adult Yucatan pigs and from commercial pigs at the time of slaughter, and BzCL and EDTA were added. Plasma was diluted 1:10 for assay and was found to have 32.5 +/- 5.5 and 50.6 +/- 6.6 ng/ml immunoreactive PIP, respectively. Fractionation of plasma on Sephadex G-50 F demonstrated a single peak of immunoreactive PIP eluting coincident with the [125I]PIP marker run with the sample. This peak was dialyzed and electrophoresed on acid gels at pH 2.5 in 2 M urea, and the gels were sliced and eluted for assay. A single narrow band of immunoreactivity migrated identically with PIP run on a parallel gel. Intramuscular injection of 1 microgram PIP every hour for 3 h in conscious rats with a ligated pylorus stimulated both volume and acid secretion by the stomach (P less than 0.0005): PIP (n = 25) 286 +/- 23 mumol H+, and 3.23 +/- 0.23 ml fluid vs. control (n = 27) 237 +/- 20 mumol H+ and 2.61 +/- 0.15 ml fluid. It is concluded that PIP is secreted into the circulation in normal pigs and causes an increase in gastric secretion of acid and fluid volume in vivo as well as the previously observed action on the gastric mucosa in vitro.