Uptake of vitamin B12 by isolated ileal enterocytes

Isolated ileal enterocytes incubated with intrinsic factor-[57Co]vitamin B12 (IF-B12) at 37 degrees C took up 25 times more B12 than jejunal enterocytes. Uptake of B12 by ileal cells was dependent upon IF and Ca2+. B12 taken up by ileal enterocytes could be separated into two components: 1) B12 which was retained (E component) and 2) released (R-E component) by chelation of divalent cations. The E component could be removed from ileal cells by treatment with Triton X-100. Incubation of ileal enterocytes at 22 degrees C or with 2,4-dinitrophenol reduced incorporation of B12 into the E, but not the R-E, component. Ileal enterocytes were incubated with IF-[57Co]B12 washed and reincubated with unlabeled IF-B12. Reincubation resulted in a decrease in the amount of [57Co]B12 in the R-E component and a concomitant increase of that in the E component indicating that B12 was transferred from the R-E to the E component. Dinitrophenol reduced transfer from R-E to E components. These results suggest that, using isolated enterocytes, two sequential steps in ileal absorption of B12 can be identified: 1) energy-independent binding of IF-B12 to its receptor on the brush border followed by 2) an energy-dependent event which probably represents transfer of B12 from its receptor into the cell.