Formation of transcobalamin II--vitamin B12 complex by guinea-pig ileal mucosa in organ culture after in vivo incubation with intrinsic factor--vitamin B12.

The in vivo incubation of intrinsic factor--[57Co]vitamin B12 in an ileal loop of a guinea-pig followed by in vitro culturing of segments of the ileum for 180 min has been used to study the transepithelial transport of vitamin B12. Analysis of the solubilized supernate of mucosa following the in vivo phase demonstrated that 44% of the [57Co]vitamin B12 was bound to intrinsic factor (IF), 26% was free, and 16% was bound to transcobalamin II (TCII). Following culture, similar analysis demonstrated that 18% of the vitamin was now bound to IF, 49% was free, and 35% ws bound to TCII. In the culture medium, 54% of the [57Co]vitamin B12 was free and 37% was bound to TCII. The formation of TCII-[57Co]vitamin B12 did not occur if homogenized mucosa was incubated with free[57Co]vitamin B12, but it did form in cultures of ileal segments from animals given an excess of unlabelled vitamin to saturate all circulating TCH, and in the medium containing puromycin. Indirect immunofluorescence using chicken anti-TCII demonstrated that TCII was associated with the mucosal cells of both the ileum and jejunum. These studies demonstrate that following transepithelial flux of vitamin B12 through the ileal mucosa, the vitamin becomes coupled to TCII. This coupling requires a structurally intact mucosa and the source of the TCII appears to be the ileal mucosal cell rather than unsaturated TCII circulating in the blood.