Absorption and paracellular visualization of fluorescein, a hydrosoluble probe, in intact house sparrows (Passer...

We describe a method to visualize the cellular location of compounds during absorption by the small intestine in intact animals. First, we employed pharmacokinetic methodology to measure the fractional absorption of sodium fluorescein, a small (MW = 376) water-soluble molecule that is widely used as hydrophilic marker molecule for paracellular permeability studies. Based on the hypothesis that the paracellular pathway acts as a sieve, we predicted that fluorescein absorption would be considerable, but less than that of passively absorbed L-glucose which is a smaller molecule (MW = 180). When the two compounds were gavaged into house sparrows simultaneously, the birds absorbed significantly less fluorescein (42 +/- 8%) than L-glucose (82 +/- 7%), as predicted, and absorptions of the two were correlated as one would predict if they shared the same pathway. We removed intestinal tissue 10 min after gavage with sodium fluorescein and determined the cellular location of the compound's fluorescence using confocal laser microscopy. The fluorescent signal was found primarily in the paracellular space. In contrast, in the same type of experiment using instead the similar-sized fluorescent lipophilic compound rhodamine 123 (MW = 381), most fluorescence appeared inside enterocytes, as expected for a compound that diffuses across the apical membrane. Thus, results from all the experiments are consistent with the hypothesis that hydrophilic fluorescein is absorbed primarily via a paracellular pathway. These methods could be applied to visualize absorption pathways of other compounds in other intact animals.