Effect of divalent cations and pH on intrinsic factor-mediated attachment of vitamin B12 to intestinal microvillous membranes

Abstract

A B S T R A C T Calcium, but not other divalent cations, is required for optimal-uptake of intrinsic factor-bound 57Co-labeled cyanocobalamin (IFB12) by microvillous membranes isolated from hamster ileal-absorptive cells. Chelation of divalent cations by disodium ethylenediaminetetraacetate (EDTA) promptly removes IFB12 previously attached to microvillous membranes. High concentrations of CaC12 or MgCl2 also markedly inhibit membrane uptake of IFBl2 and rapidly remove previously attached IFB12. Similarly, reduction of pH to below 5.4 prevents membrane attachment of IFB,2 and removes virtually all IFB,2 already bound to microvillous membranes. The effects of calcium depletion, increased salt concentrations, and acidification on membrane uptake of IFB, were completely reversible. These findings are consistent with the concept that the formation of calcium salt bridges is essential for attachment of IFB,2 to the ileal-absorptive surface.

Characterization of Ileal Vitamin B12 Binding Using Homogeneous Human and Hog Intrinsic Factors

“…The mechanism of IF-mediated B12 absorption is not well understood, but a number of studies have demonstrated that IF facilitates B12 binding to ileal sacs (1)(2)(3)(4)(5)(6), mucosal homogenates (4,(7)(8)(9)(10), microvillus membrane preparations (11,12), and to a component solubilized from ileal mucosa with Triton X-100 (13). IF-facilitated B12 binding of this type is dependent on pH, is inhibited by EDTA, and is postulated to represent the first step in the complex process by which B12 passes from the intestinal lumen into the portal blood.…”

Intrinsic Factor-mediated Absorption of Cobalamin by Guinea Pig Ileal Cells

“…To be certain that these peaks of 35S radioactivity were not produced by tissue proteases activated during the extraction procedure, we added tissue protease inhibitors throughout one extraction and obtained identical results. (9). Therefore, by incubating isolated ileal cells with radiolabeled IF-bound Cbl and by washing these cells with EDTA or a pH 5.0 buffer, we were able to distinguish radioactivity attached to the cell surface from that which had been internalized.…”

“…Since either EDTA or acidification below pH 5.4 completely dissociates IF-Cbl complex from its receptor on isolated ileal brush borders or microvillus membranes (MVM) (9), surface attachment of labeled Cbl has been distinguished from cellular absorption by measuring radioactivity before and after washing ileal mucosa with EDTA or an acidified buffer. In one recent study (8) (11).…”

Disorders of Cobalamin (Vitamin B12) Absorption and Transport

“…IF-cobalamin complex attaches to surface receptors rapidly. Attachment is neither energy nor temperature dependent, but requires extracellular Ca and a pH greater than 5.4 (22). Subsequent entry of cobalamin into the absorptive cell clearly requires cellular energy (23), but how the vitamin enters the ileal cell and then reaches the portal circulation remains poorly understood.…”