EVALUATION OF THE ISOTOPE-DILUTION TECHNIQUE FOR DETERMINING ILEAL ENDOGENOUS NITROGEN-EXCRETION IN THE RAT

Three body amino acid pools (plasma free, plasma bound and small intestinal tissue) were evaluated as precursors to allow measurement using the isotope dilution technique of endogenous excretion at the terminal ileum of animals. Eighteen 150-g bodyweight rats were given either a protein-free, an enzyme-hydrolysed casein based or a synthetic amino acid based diet, and digesta were collected from the terminal ileum. The animals had been subjected to a constant 8-day infusion of tritiated leucine via subcutaneously implanted osmotic mini pumps. Specific activities (dpm nM-1 leucine) of the ileal digesta and the plasma free, plasma bound and small intestinal tissue pools were determined and the specific activity for the ileal digesta was expressed as a proportion of the respective precursor pool value to give dilution factors for each dietary treatment. For the protein-free diet, where the ileal nitrogenous flow is endogenous by definition, the dilution factor for an appropriate precursor pool would be unity. For the hydrolysed casein and synthetic amino acid diets, in which the peptides and amino acids are expected to bc virtually completely absorbed anterior to the ileum, high dilution factors (close to unity) would be expected. The mean dilution factors based on the plasma free amino acid pool were untenably low (0.2 to 0.3). For the plasma bound amino acid pool mean dilution factors of 1.3 were found for animals given the protein-free and synthetic amino acid diets, while a lower value (0.7) was obtained for the hydrolysed casein treatment. Untenably high factors (1.5) were found with the small intestinal tissue for the protein-free and synthetic amino acid treatments, while the corresponding value for the hydrolysed casein-fed rats was unity. The dilution factor data within treatments were highly variable, and none of the pools examined gave consistently reliable results and could thus be accepted as a valid precursor pool for the endogenous proteins.