Identification of a macromolecular factor in the ileum which binds intrinsic factor and immunologic identification of intrinsic factor in ileal extracts

A iB S T R A C T The effect of partial pancreatectomy(80-90%,) on vitamin Bn absorption was studied in the rat. The absorption of 5 ng of^'Co-labeled vitamin B1u was significantly reduced from 70 ±2.5% (mean +SE) in control and sham-operated rats to 32 ±2.6% in partially pancreatectomized rats. Hog pancreatic extract (0.17 g/kg) improved vitamin B12 absorption from 30.0 to 61.0% in partially pancreatectomized rats but did not alter vitamin B,2 absorption in control rats. Chloramphenicol did not enhance vitamin B12 absorption in partially pancreatectomized rats with pancreatic extractimproved vitamin B12 malabsorption. The partially pancreatectomized rats with pancreatic extract-improved vitamin B12 malabsorption were sacrificed and the stomach and small bowel studied in vitro to further define the pathogenesis of the vitamin B12 malabsorption. Rat gastric intrinsic factor stimulated vitamin B12 uptake by intestinal sacs prepared from partially pancreatectomized rats 3.1-fold. Gastric intrinsic factor prepared from partially pancreatectomized rats was as effective in promoting vitamin B12 uptake by rat intestinal sacs as intrinsic factor prepared from control rats. These data indicate that partially pancreatectomized rats develop an abnormality in the absorption of labeled vitamin B12 which can be corected by pancreatic extract. The vitamin B12 malabsorption is due to neither an alteration in gastric intrinsic factor activity nor an impairment of the intrinsic factor-vitamin B12 receptor in the intestine. It is suggested that in the partially pancreatectomized rats the intrinsic factor-vitamin B12 complex exists in a form which is not available for absorption. INTRODUCTIONAlthough several patients with pancreatic exocrine insufficiency have been noted to poorly absorb vitamin B,2 the importance of normal pancreatic function for optimal vitamin Bi absorption is generally not appreciated 1-5). Furthermore, the pathogenesis of the observed vitamin B12 absorptive defect has not been clarified.The relationship between the pancreas and the intestinal absorption of vitamin B12 was studied by measuring the absorption of this vitamin in rats subjected to partial pancreatectomy. The results demonstrate (a) that the partially pancreatectomized rat has a defect in vitamin B12 absorption, (b) that the administration of hog pancreatic extract restores vitamin B12 absorption to normal, (c) that intrinsic factor extracted from the gastric mucosa -of partially pancreatectomized rats stimulates vitamin Bn absorption in vitro, and (d) that gastric intrinsic factor stimulates vitamin B12 uptake in gut sacs prepared from partially pancreatectomized rats. These data indicate that partially pancreatectomized rats develop a defect in vitamin B12 absorption which is due to neither an alteration in gastric intrinsic factor activity nor to an impairment of the intrinsic factor-vitamin B12 receptor in the intestine.

Section: Discussionmentioning

confidence: 99%

The Role of the Pancreas in Vitamin B12 Absorption: Studies of Vitamin B12 Absorption in Partially Pancreatectomized Rats

Toskes¹,

Deren²

1972

“…Centrifugation at 54,500 g of a similar preparation resulted in the loss of this activity. Rothenberg similarly reported the presence of a binder of IF-B12 in the supernate obtained when guinea pig mucosal membranes were centrifuged at 30,000 g (9). Several authors have suggested that Rothenberg may not have been sedimenting tiny particles of membrane and that the binder he studied may actually have been particulate (10,25).…”

Section: Methodsmentioning

confidence: 95%

“…Several attempts to isolate this receptor have been reported (5,9), but uncertainty exists about the validity of the claims of success (10).…”

mentioning

confidence: 99%

Solubilized Receptor for Intrinsic Factor-Vitamin B12 Complex from Guinea Pig Intestinal Mucosa

Katz¹,

Cooper²

1974

A B S T R A C T The absorption of vitamin B12 in many animals requires its prior association with intrinsic factor (IF) and attachment to a specific receptor in the intestine. Employing Triton X-100, we have solubilized from guinea pig ileum a factor that binds intrinsic factor-vitamin B12 complex (IF-B12). This binding factor was soluble to the extent that it was not sedimented by centrifugation at 100,000 g for 1 h and was small enough to enter the included volume of a Sepharose 4-B column.Furthermore, the ileal extract contained no microfine particles of membrane upon electron microscopic search. When a portion of the extract was incubated with a mixture of gastric juice and 6"Co-labeled vitamin B12, a portion of the radioactivity was excluded from a Sephadex G-200 column. When gastric juice from a patient with a congenital abnormality of IF that prevented its binding to intestine was substituted for normal human gastric juice, radioactivity was not excluded from the gel, indicating failure of this abnormal IF-B12 to bind to the intestinal extract. These data suggested the presence of a specific binder of IF-B12 in the ileal mucosal extract. The reactions of normal IF-B12 with the solubilized binding factor and with the membrane-bound "receptor" had several characteristics in common, including calcium dependence, temperature independence, and pH optimum near neutral. Extracts from the distal intestine showed more activity than did those from the proximal. The solubilized binding facter seemed specific for IF-B12 in that it was not blocked by prior incubation with excesses of either free vitamin B12 or IF. Binding activity of the extract was decreased by incubation at pH 2.0, by heating to 560C, and by incubation with chymotrypsin and dithiothreitol. Incubation with trypsin, neuraminidase, and sulphydryl blockers did not affect it.The Triton X-100 extract of guinea pig ileal mucosa contains a specific binding factor that probably is the

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Hooper¹,

Alpers²,

Burger³

et al. 1973