Evidence for the absorption of immunoreactive intrinsic factor into the intestinal epithelial cell during vitamin B 12 absorption.

Abstract

Abstract To investigate the distribution and nature of vitamin B 12 in the subcellular fractions of the ileal epithelial cells during the process of absorption, 57 CoB 12 bound to human intrinsic factor (IF) was incubated in isolated ileal loops of anesthetized guinea pigs and periodically for 120 minutes the animals were killed and the homogenate of the mucosa separated by differential centrifugation into (1) cell debris fraction containing large fragments of partially disrupted surface structures, nuclei, and other cell constituents, including some mitochondria, (2) mitochondrial fraction, (3) microsomes, and (4) cytosol. The particulate composition of each fraction was confirmed using electron microscopy. Radioactivity accumulated continuously during the observation period in the cell debris and mitochondria-rich fractions. Lesser amounts were found with the microsomes and in the cytosol. The nature of the 57 CoB 17 in the cell debris and mitochondrial fractions was further studied by sonicating each fraction to solubilize the radioactivity and then removing the insoluble particulate by centrifugation at 10,000 g. Eighty to 94 per cent and 57 to 78 per cent of the solubilized radioactivity in the supernates of the cell debris and mitochondrial fractions, respectively, could not be extracted by albumin-coated charcoal, indicating that a major fraction of the 57 CoB 12 was bound to a macromolecule. When these supernates were then incubated with antihuman IF antibody, 71 to 84 per cent and 54 to 69 per cent of the radioactivity in the cell debris and mitochondrial fractions, respectively, precipitated with the immunoglobins at 15 per cent Na 2 SO 4 indicating that a major fraction of the absorbed 57 CoB 12 was bound to a large molecule with the immunologic identity of IF. Although significantly less radioactivity in the supernates of these sonicated fractions precipitated at 15 per cent Na 2 SO 4 in the absence of anti-IF antibody, it was still significantly more than precipitated when IF- 57 CoB 12 in gastric juice was subjected to this salt concentration. This finding suggests that either a small fraction of the absorbed immunoreactive IF-B 12 complex has been altered in some way, or that some of the absorbed 57 CoB 12 becomes bound to a different protein which is insoluble at 15 per cent Na 2 SO 4 .