In Vitro Assay for Human Intrinsic Factor*

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

“…Removal of calcium ions from the medium greatly reduces hog IF-mediated uptake of Bt2 by rat liver slices (18) or by everted sacs of rat ileum (8). Similarly, the absence of calcium ions prevents the effect of rat IF on B12 uptake by everted sacs of homogenates of rat ileum (8) as well as the effect of human IF on Bin uptake by everted sacs (9) and homogenates (19) of guinea pig distal bowel. In vivo, chelation of divalent cations by EDTA diminishes Bn absorption in humans (1) and rats (20) and inhibits uptake of the vitamin by isolated, perfused loops of rat ileum (4).…”

“…In vivo, acidification of the perfusion fluid prevents IF-mediated uptake of Bi2 by isolated, perfused loops of rat ileum (4). In vitro, lowering the pH of the incubation medium below pH 5.8 impairs uptake of IFBn by everted sacs (8), homogenates (19), and isolated brush borders (13) of distal small bowel.…”

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.…”

“…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. (2,7,9,11,14,15) or not (3,6,8,16) (17). Binding studies using crude gastric preparations are difficult to interpret because of the presence of B12 binding proteins that lack in vivo IF activity as well as the possible presence of additional gastric inhibitors or stimulators.…”

Studies on Human Gastric Intrinsic Factor

“…But they did not describe the suppressive effect of PAGJ on B12 uptake by the sac. Sullivan et al (14) also assayed the gastric juice from several PA patients by guinea pig intestinal mucosa homogenate system . In their experiment, the gastric juice from one patient with PA resulted in less B12 uptake than that of the saline control.…”