Mice Fed Radiolabeled Protein by Gavage Show Sporadic Passage of Large Quantities of Intact Material Into the Blood, an Artifact not Associated With Voluntary Feeding.

Bovine 2-microglobulin (Bov 2-M) is a small acid- and protease-resistant protein found in cows' milk that has the capacity to interact specifically with molecules of the mouse immune system. We conducted a series of investigations designed to determine whether a portion of orally ingested Bov 2-M could pass intact into the mouse circulatory system. Initial experiments feeding 125I-labeled protein by gavage to nonfed mice indicated that intact Bov 2-M appeared rapidly and at high amounts in the blood (as detected by use of direct sodium dodecyl polyacrylamide gel electrophoresis analysis of a 1-ml sample of plasma and phosphorimaging), but this result was observed sporadically and only in about a third of all animals, with no detectable protein appearing in the plasma of the remaining animals. Identical results were obtained when the mice were lightly anesthetized prior to gavage. When a larger protein, 125I-labeled bovine serum albumin (BSA), was fed by gavage in combination with the labeled Bov 2-M, surprisingly large fragments of the BSA (. 50 kDa) appeared in the plasma whenever the Bov 2-M appeared, again in about a third of the animals. We hypothesized that the passage of large quantities of Bov 2-M into the blood of some mice was due to an intermittent artifact of the gavage feeding (likely spillover into the lungs), and therefore, we developed an alternate approach based on voluntary feeding. Mice were easily taught to drink small samples of liquid voluntarily by removing water for 12 h, then manually generating droplets at the end of a 1-ml syringe. When a mixture containing 125I-labeled Bov 2-M and BSA was fed in this manner, labeled protein could not be detected in any animals by direct analysis of a 1-ml sample of plasma. However, when immunoprecipitation was carried out on a larger volume of plasma (100 ml), intact Bov 2-M could be found in the blood of all animals, whereas similar immunoprecipitation and analysis for BSA did not yield any signal. We conclude that the common method of gavage feeding mice to assess absorption of orally ingested material can lead to artifacts not seen when the same agent is consumed under more natural circumstances. These observations may have relevance for experiments where oral tolerance is assumed to be induced in rodents by repeated gavage feeding, whereas the same effects cannot be obtained in humans by voluntary feeding.