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PubMed1982

Functional activity of intrinsic factor measured by using solubilized receptor protein.

The traditional radioimmunoassay for gastric intrinsic factor, in which this protein is measured on the basis of immunoreactivity rather than function, is of no value for identifying intrinsic factor that binds cobalamin but does not bind to the ileal receptor site, or for detecting animal intrinsic factor, which does not cross react with human intrinsic factor. Accordingly, we have applied a radioassay for the intrinsic factor receptor protein to measure the functional activity of intrinsic factor in gastric juice. The receptor protein reagent was partly purified from guniea pig ilea and its interaction with intrinsic factor--CN[57Co]-cobalamin was determined by precipitation with sodium sulfate at a final concentration of 150 g/L. Results of this assay were comparable with results obtained for intrinsic factor by radioimmunoassay. The receptor protein did not bind immunoreactive intrinsic factor that was functionally abnormal. This functional radioassay for intrinsic factor is not species specific and will be of value when specific antiserum to intrinsic factor is not available and when cobalamin malabsorption is to be evaluated in patients who are secreting normal amounts of immunoreactive intrinsic factor.

Gastric Juice↗
PubMed1986

Rapid collection of human intrinsic factor uncontaminated with cobalophilin (R binder).

Intrinsic factor and R binder cobalamin binding capacity (CblBC) were measured in gastric juices obtained through feeding gastrostomy tubes (FG-GJ) from six patients with relatively complete obstruction to swallowing because of surgical resections for oropharyngeal or laryngeal carcinomas. Controls were six nonpernicious anemia gastric juices. In five of the six FG-GJ 97% of CblBC was intrinsic factor. In the sixth FG-GJ, with GJ pH = 7.2, in addition to intrinsic factor there was 22% nonintrinsic factor cobalamin-specific binder, which presumably represented partially degraded R binder refluxed from the duodenum. These data demonstrate that relatively "pure" intrinsic factor, i.e., uncontaminated with other cobalamin binders, can easily be obtained by simple drainage of FG-GJ. The study also supports evidence that gastric juice R binder is either swallowed or refluxed from the duodenum, and does not originate in the stomach.

Gastric Juice↗
PubMed1976

Solubilization, partial purification and radioassay for the intrinsic factor receptor from the ileal mucosa.

A macromolecule which binds intrinsic factor saturated with vitamin B12 has been solubilized from the guinea-pig ileum by homogenization followed by mechanical disruption without organic solvents or detergents. This intrinsic factor 'receptor' was further purified by precipitation with 30% saturated ammonium sulphate, centrifugation at 105000 g, and filtration through Sephadex G-200. Failure to precipitate the receptor following centrifugation at 105000 g for 3 h and filtration of the receptor with the included volumes through Sepharose 4B and 6B was evidence that it was solubilized. The purification of the receptor was monitored by a radiometric assay where the intrinsic factor-[57Co]vitamin-B12 complex coupled to the solubilized receptor precipitated at 15% sodium sulphate while intrinsic factor-[57Co]B12 alone remained soluble at this salt concentration. This radioassay also permitted the in vitro study of the interaction of the solubilized receptor and intrinsic factor saturated with [57Co]B12. The receptor did not bind intrinsic factor-[57Co]B12 below pH 5 while binding was observed to pH 9.0. Binding was equivalent at 37 degrees C and 25 degrees C, but was markedly reduced at 4 degrees C and 56 degrees C and was destroyed at 100 degrees C. The receptor resisted 60 min of digestion by trypsin, chymotrypsin, pronase and subtilisin. After 180 min digestion, pronase and subtilisin inactivated 90% and 41% of the receptor respectively, whereas trypsin and chymotrypsin inactivated only 21% and 23%. Trisodium EDTA inhibited the binding of intrinsic factor-[57Co]B12 to the receptor and this inhibition could be reversed by the addition of excess Ca2+. Mg2+ and Mn2+ were less effective than Ca2+ for the activity of the receptor. Kinetic analysis of the reaction indicated a maximum velocity of 0.083 nmole IF bound B12/min with a Km of 1.36 x 10(-10) M. The solubilized receptor had a greater affinity for intrinsic factor bound to vitamin B12 than for intrinsic factor free of vitamin B12. The solubilization of this intrinsic factor receptor without chemicals suggests that it is not an integral component of the microvillus membranes hydrophobically bonded to the lipid matrix, but rather a peripheral protein weakly associated with the membrane by non-covalent interaction.

Animals↗
PubMed1977

Evidence that pancreatic proteases enhance vitamin B12 absorption by acting on curde preparations of hog gastric intrinsic factor and human gastric juice.

Crude preparations of hog gastric intrinsic factor or their own previously collected gastric juices administered with labeled vitamin B12 did not enhance vitamin B12 absorption in patients with vitamin B12 malabsorption secondary to pancreatic insufficiency. However, when these sources of gastric intrinsic factor were incubated with three times crystallized preparations of insolubilized bovine trypsin or chymotrypsin, the proteolytic enzymes were removed by centrifugation, and the preparations of gastric intrinsic factor were readministered to these patients, the absorption of vitamin B12 was markedly enhanced. Studies of hog gastric intrinsic factor before and after exposure to proteolytic enzymes failed to show any difference on Sephadex chromatography or polyacrylamide gel electrophoresis or on its affinity for vitamin B12 or the ileal receptor in guinea pigs. These investigations demonstrate that: (1) gastric intrinsic factor as secreted by subjects with pancreatic insufficiency or obtained from hog pyloric mucosal extracts is ineffective in promoting vitamin B12 absorption in patients with pancreatic insufficiency, (2) incubation of crude preparations of gastric intrinsic factor with insolubilized pancreatic proteases modified these preparations of gastric intrinsic factor in an as yet undefined manner, allowing them to enhance vitamin B12 absorption, and (3) in vitro studies using gut sacs or brush border preparations do not reflect the abnormality in vitamin B12 absorption associated with pancreatic dysfunction.

Anemia, Pernicious↗
PubMed1983

Cellular origin and release of intrinsic factor from isolated rat gastric mucosal cells.

The cellular content and secretion of intrinsic factor was measured by [57Co]cyanocobalamin binding using isolated rat gastric mucosal cells. The intrinsic factor/R-protein ratio was above 9:1 as evaluated by specific anti-intrinsic factor antibodies. In unfractionized cells with 23 +/- 1.3% parietal cells the intrinsic factor content of 148 +/- 47 fmol/10(6) cells remained almost unchanged over 3 h, whereas basal secretion rose up to 57 +/- 10. In fractionized cells (Percoll) with 3-85% parietal cells most intrinsic factor was found in the parietal cell-depleted fraction (content: 441 +/- 30, secretion/3 h: 139 +/- 16, mean formation/h: 50 +/- 12 fmol/10(6) cells). The intrinsic factor content of the different cell fractions correlated with that of pepsin. [14C]Aminopyrine uptake, an indirect measure of parietal cell H+ production, was inversely related. Carbachol (1 X 10(-6)-10(-3) mol/l) stimulated intrinsic factor secretion, 1 X 10(-3) mol/l being maximally effective (90 +/- 8% above basal). This response was inhibited by atropine and pirenzepine, but not by prostaglandin E2 (PGE2) and somatostatin. Dibutyryl cyclic adenosine monophosphate (dibutyryl cAMP, 43 +/- 7%) and hexoprenaline (24 +/- 5%) enhanced intrinsic factor secretion less effectively and pentagastrin like histamine lacked any stimulatory effect. We conclude that in the rat intrinsic factor is produced and released from chief cells mainly under cholinergic control.

Aminopyrine↗
PubMed1991

Intrinsic factor secretion and cobalamin absorption. Physiology and pathophysiology in the gastrointestinal tract.

Intrinsic factor is produced by the gastric parietal cell. Its secretion is stimulated via all pathways known to stimulate gastric acid secretion: histamine, gastrin, and acetylcholine. There is, however, a different mode of secretion for both substances: atropine, vagotomy, and H2 receptor antagonists inhibit both intrinsic factor and acid secretion, but secretin and the hydrogen-potassium ATPase antagonist omeprazole have no effect on intrinsic factor while substantially reducing acid secretion. Cobalamin in food is bound to animal protein. Cobalamin deficiency due to inadequate dietary intake is rarely seen in extreme vegetarians (vegans). In the stomach cobalamin is liberated from its protein binding by peptic digestion and bound to R-proteins. Hypochlorhydria or achlorhydria, whether medically induced or not, may impair cobalamin uptake. The cobalamin-R-protein complex is split by pancreatic enzymes in the duodenum, where cobalamin is bound to intrinsic factor. Pancreatic insufficiency may lead to cobalamin deficiency. Lack of intrinsic factor is the commonest cause of cobalamin deficiency; very rarely, aberrant forms of intrinsic factor are produced, but the clinical syndrome is similar. Gram-negative anaerobe bacteria bind the cobalamin-intrinsic factor complex, and bacterial overgrowth of the small intestine diminishes cobalamin resorption. Parasitic infections with fish tape-worm and Giardia lamblia are also associated with cobalamin malabsorption. The cobalamin-intrinsic factor complex binds to the ileal receptors in the terminal ileum. Cobalamin absorption may be impaired after resection or by diseases affecting more than 50 cm of the terminal ileum, such as Crohn's disease, coeliac disease, tuberculosis, lymphoma or radiation. There is clearly a wide diversity in the aetiology of cobalamin deficiency, which requires a versatile diagnostic approach.

Humans↗
PubMed1996

Etiology of dental erosion--intrinsic factors.

Dental erosion due to intrinsic factors is caused by gastric acid reaching the oral cavity and the teeth as a result of vomiting or gastroesophageal reflux. Since clinical manifestation of dental erosion does not occur until gastric acid has acted on the dental hard tissues regularly over a period of several years, dental erosion caused by intrinsic factors has been observed only in those diseases which are associated with chronic vomiting or persistent gastroesophageal reflux over a long period. Examples of such conditions include disorders of the upper alimentary tract, specific metabolic and endocrine disorders, cases of medication side-effects and drug abuse, and certain psychosomatic disorders, e.g. stress-induced psychosomatic vomiting, anorexia and bulimia nervosa or rumination. Based on a review of the medical and dental literature, the main symptoms of all disorders which must be taken into account as possible intrinsic etiological factors of dental erosion are thoroughly discussed with respect to the clinical picture, prevalence and risk of erosion.

Drug-Related Side Effects and Adverse Reactions↗
PubMed1977

Intrinsic factor secretion after vagotomy.

The intrinsic factor (IF) output during basal and Histalog-stimulated gastric secretion has been estimated in two series of patients with chronic duodenal ulcer before and 3 months or more after treatment by either highly selective vagotomy or truncal vagotomy and pyloroplasty. The effects of the two different vagotomy operations appear to be virtually identical and each produced significant reductions in intrinsic factor secretion after Histalog stimulation. This confirms the view expressed by previous workers that it is the vagotomy as such which is responsible, excluding the drainage procedure from any possible role. Furthermore, as these results were demonstrated 3 months after operation, it is likely that the depressed IF secretion is a permanent feature and one which, it is postulated, may become progressively more severe. In both series there is a marked reduction in IFoutput during the second hour of stimulated gastric secretion, indicating an early wash-out of preformed IF. This persists after vagotomy.

Gastric Juice↗
PubMed1976

[Blocking and binding antibodies to intrinsic factor and their behaviour during oral therapy with intrinsic factor-vitamin B 12 complex (author's transl)].

13 patients with pernicious anaemia and 2 patients who had been subjected to total gastrectomy were treated orally with a hog instrinsic factor preparation (IF) for 1 to 4.5 years. During this therapy 11 of the patients with pernicious anaemia and both gastrectomized patients developed blocking and binding antibodies to IF. Antibodies already present before the commencement of therapy showed an increase in titre. All patients remained in complete haematological remission. The conclusion is drawn that circulating antibodies to IF do not play any significant role in the absorption of the hog IF-B 12 complex.

Administration, Oral↗
PubMed2000

Expression of intrinsic factor in rat and murine gastric mucosal cell lineages is modified by inflammation.

Intrinsic factor is produced primarily by chief cells in rat and mouse, but 4 to 11% of isolated rat parietal cells also contain intrinsic factor. To test whether local conditions could alter the distribution of intrinsic factor expression, two rodent models of chronic lymphocytic gastric inflammation were examined. Immunocytochemistry was performed using antiserum against human intrinsic factor and H/K ATPase (a parietal cell marker), counting the percent of intrinsic factor-positive parietal cells. HLA-B27 transgenic rats develop chronic gastritis at age 3 months. Congenic controls expressed intrinsic factor in 8.9 +/- 3.8% (mean +/- SD) of parietal cells; in inflamed areas of transgenic rats 21 +/- 5.2% (P < 0.0001) of parietal cells were positive. In adjacent areas without inflammatory infiltrate 16 +/- 3.6% of parietal cells contained intrinsic factor. C57BL/6 mice inoculated with Helicobacter felis develop gastritis by 4 weeks. After 4 and 8 weeks of infection, intrinsic factor-positive parietal cells increased from 7.8 +/- 2.8% in the congenic controls to 17.6 +/- 4.1% in the inflamed gastric body (P < 0.0001). Isolated rat parietal cells incubated with interleukin-1beta demonstrated a twofold increase in intrinsic factor-positive parietal cells. These studies are consistent with the concept that intrinsic factor expression is both predetermined in chief cells and can be expressed in parietal cells in response to local inflammatory factors. The differences between inflamed and adjacent noninflamed areas in the rat model suggest a tissue gradient of soluble inducer(s), possibly cytokines.

Animals↗
PubMed1967

Studies on antibody to intrinsic factor.

Sera from a group of 79 patients with pernicious anemia were studied for the presence of antibody to intrinsic factor. Two general types of antibody activity were found, and it was possible to distinguish three groups of pernicious anemia sera on the basis of their content of these types. Type I antibody blocks the binding of radioactive vitamin B(12) to intrinsic factor when added to intrinsic factor before the B(12); it is not detected on intrinsic factor when added after B(12). This antibody blocks intrinsic factor-mediated B(12) absorption in vivo when mixed in the sequence intrinsic factor + antibody I + B(12), but not when mixed in the sequence intrinsic factor + B(12) + antibody I. Type II antibody reacts with intrinsic factor when B(12) is attached. This antibody prevents the absorption of B(12) from intrinsic factor in pernicious anemia patients when mixed in the sequence intrinsic factor + B(12) + antibody II, and is thereby distinguished from antibody I.

Anemia, Pernicious↗
PubMed1990

Cellular localization of intrinsic factor in pancreas and stomach of the dog.

A cobalamin (vitamin B12)-binding protein has recently been identified in canine pancreatic juice which is biochemically, immunochemically and functionally similar to canine gastric intrinsic factor. However, the cellular sources of both this pancreatic intrinsic factor and gastric intrinsic factor in the dog are not known. Antisera raised against canine gastric intrinsic factor have been used to examine the distribution of intrinsic factors in the canine pancreas and stomach. Immunoreactivity was demonstrated in duct cells but not acinar or endocrine cells in the pancreas, and in fundic peptic and pyloric gastric pit cells in stomach. All immunostaining was abolished by preabsorption of the antisera with purified canine gastric and pancreatic intrinsic factors. A cellular source of pancreatic intrinsic factor has not been previously described, and the demonstration of intrinsic factor-like immunoreactivity in two cell types in the canine stomach contrasts with its localization in a single cell type in the gastric mucosa of other mammalian species. Furthermore, immunoreactivity in pancreatic duct cells was detected at much higher dilutions of antisera than those required for staining of peptic and gastric pit cells. This suggests a higher concentration of antigen, and supports previous evidence that the pancrease is a major source of intrinsic factor in the dog.

Animals↗
PubMed1996

Model for the factor VIIIa-dependent decay of the intrinsic factor Xase. Role of subunit dissociation and factor IXa-catalyzed proteolysis.

The intrinsic factor Xase complex (FXase) is comprised of a serine protease, FIXa, and a protein cofactor, FVIIIa, assembled on a phospholipid surface. Activity of FXase decays with time and reflects the lability of FVIIIa. Two mechanisms potentially contribute to this decay: (i) a weak affinity interaction between the FVIIIa A2 subunit and Al/A3-Cl-C2 dimer and (ii) FVIIIa inactivation resulting from FIXa-catalyzed proteolysis of the Al subunit. At low reactant concentrations (0.5 nm FVIIIa; 5 nm FIXa), FXase decay is governed by the inter-FVIIIa subunit affinity and residual activity approaches a value consistent with this equilibrium, as judged by reactions containing exogenous A2 subunit. Analysis using a mutant form of FVIII (FVIIIR336I) possessing an altered FIXa cleavage site, showed similar rates of FXase decay (0.12 min(-1)) and confirmed the lack of contribution of proteolysis under these conditions. When the concentration of FIXa was increased 10-fold, the initial rate of decay of FXase containing native FVIIIa increased (0.82 min(-1)) and paralleled the rate of proteolysis of Al subunit. However, the rate of decay of FXase containing the FVIIIaR336I was reduced (0.048 min(-1)) consistent with the elevated concentration of FIXa stabilizing the labile subunit structure of the cofactor. Reconstitution of FVIII with FIXa-cleaved light chain showed that cleavage at the alternate FIXa site (A3 domain) was not inhibitory to FXase. The presence of substrate FX resulted in a 10-fold reduction in the rate of FIXa-catalyzed proteolysis of FVIIIa. These results suggest a model whereby decay of FXase results from both FVIIIa subunit dissociation and FIXa-catalyzed cleavage, dependent upon the relative concentration of reactants, with greater contribution of the former at low values and, in the absence of substrate, greater contribution of the latter at high values.

Amino Acid Sequence↗
PubMed1990

Physicochemical characterization and biological activity of intrinsic factor in cystic fibrosis.

Absorption of crystalline labeled cobalamin is strongly decreased in cases of cystic fibrosis. In order to determine if this is due to an alteration or a lack of activation of intrinsic factor by proteases, the physicochemical properties and biological activity of intrinsic factor have been studied. Intrinsic factor was purified 800-fold from stimulated gastric juice of cystic fibrosis patients with a yield of 64.2%. Cystic fibrosis intrinsic factor had an estimated Mr of 57,000 in SDS-polyacrylamide gel electrophoresis. Its carbohydrate content resembled that of normal human intrinsic factor, except that the ratio fucose/sialic acid was higher (6.1 and 1.6, respectively) and that the content in N-acetylgalactosamine was decreased. The same alterations in carbohydrate composition were observed for Hc purified from cystic fibrosis saliva. Purified intrinsic factor from cystic fibrosis gastric juice was biologically active in vitro in the presence of ileal solubilized receptor as well as in vivo (Schilling test). The fate of iodinated cystic fibrosis intrinsic factor in guinea pig ileum studied by high-resolution radioautography was similar to that of normal intrinsic factor. In conclusion, despite modifications of the carbohydrate content of the molecule, the biological activity of intrinsic factor is not altered in cases of cystic fibrosis. The malassimilation of crystalline cobalamin observed in cystic fibrosis is due to a mechanism independent from intrinsic factor secretion.

Animals↗
PubMed1996

Human gastric intrinsic factor expression is not restricted to parietal cells.

Gastric parietal cells have been accepted as the only site of intrinsic factor production in the human stomach. In animals, however, intrinsic factor has been localised to various other cell types of foregut origin, including chief and enteroendocrine cells in gastric mucosa, and duct cells from salivary glands and pancreas. The availability of recombinant human intrinsic factor has led to production of high titre, monospecific antiserum which was used to reexamine the distribution and subcellular localisation of intrinsic factor in the human stomach. Immunolight microscopy revealed that most positively stained cells were gastric parietal cells, but at the margins of the anatomical regions (e.g. cardia/fundus, body/antrum) clusters of gastric chief cells and individual enteroendocrine cells were found to contain intrinsic factor. Immunoelectron microscopy demonstrated the highest antigen density on endocytic and apical membranes of parietal cells. Exocrine secretory granules of a subpopulation of chief cells, the secretory granules of some enteroendocrine cells, and the plasma membranes and smooth vesicles of endothelial cells of the lamina propria capillaries underlying enteroendocrine cells were also positive for the antigen. Labelling in all cells was specific, as it was abolished by preabsorption of the antisera with purified recombinant human intrinsic factor. These findings demonstrate a potential for cellular expression of human intrinsic factor in nonparietal cells. Because such expression occurs normally at the margins of anatomical gastric regions, it suggests that local factors may influence expression of intrinsic factor.

Adult↗
PubMed1981

Intrinsic factor deficiency in adults with normal hydrochloric acid production.

Two cases of intrinsic factor deficiency with normal hydrochloric acid production are presented. Both patients had markedly reduced intrinsic factor secretion and substantial titers of type I (blocking) intrinsic factor antibodies. No parietal cell antibodies were recognized by immunofluorescence in either case. Three hypotheses are offered to explain these findings: (a) These patients have congenital intrinsic factor deficiency that remained in the preanemic phase until adulthood; (b) they have pernicious anemia detected at a stage before gastric mucosal atrophy and achlorhydria have occurred; or (c) they represent cases of a new syndrome characterized by isolated intrinsic factor deficiency.

Anemia, Macrocytic↗
PubMed1997

Autoantibodies in pernicious anemia type I patients recognize sequence 251-256 in human intrinsic factor.

Pernicious anemia is an organ-specific autoimmune disease characterized by cobalamin deficiency, megaloblastic anemia, neuropathy, and autoimmune gastritis with anti-intrinsic factor autoantibodies. Type 1 anti-intrinsic factor autoantibodies block the cobalamin binding site of the intrinsic factor, a gastric protein required for the assimilation of cobalamin. The aim of our study was to identify the epitope domain of type 1 antibodies. Different series of peptides derived from the intrinsic factor sequence were synthesized and tested for antibody binding in enzyme-linked immunosorbent assay, radioisotope assay, gel filtration, and SDS-PAGE autoradiography. One of these peptides, named IF-R7 (the intrinsic factor aminoacid sequence 251-265), showed a type 1 antibody binding activity and inhibited, in vitro, their blocking activity with Ki at 2.3 microM. The cross-linking of IF-R7 to beta-lactoglobulin produced type 1 anti-intrinsic factor antibodies in immunized sheep. In vivo Schilling tests performed on guinea pigs also revealed IF-R7 peptide inhibition of type 1 antibody blocking activity. 256Ser, 258Lys, 262Tyr and 265Val of the IF-R7 were essential for the epitope recognition. Reactivity with type 1 antibodies was found in IF-R7 homologous peptides from herpesvirus Saimiri and from pathogenic Escherichia coli. In conclusion, the epitope of type 1 anti-intrinsic factor autoantibodies is located in the 251-265 amino acid sequence of the protein. The identification of this epitope will enable the definition of an experimental animal model of anti-IF autoimmunity in order to study the pathogenesis of pernicious anemia.

Amino Acid Sequence↗
PubMed1988

Effect of glycosidases and proteinases on cobalamin binding and physicochemical properties of purified saturated haptocorrin and intrinsic factor.

The effect of exoglycosidase, N-glycanase, trypsin and chymotrypsin was studied on the binding capacity and physicochemical properties of intrinsic factor and of haptocorrin using Superose 6 gel filtration. Intrinsic factor was purified as recently described by us. Haptocorrin was purified 6000-fold from human saliva using thermolabile affinity chromatography and high-performance cationic exchange chromatography with a specific activity of 20.6 nmol of cobalamin (Cbl) per mg protein and a yield of 44.7%. Exoglycosidases provoked a decrease of 54.3 and 78.2% of the Cbl binding capacity of haptocorrin and intrinsic factor, respectively. The sequential incubation of haptocorrin and intrinsic factor wit exoglycosidases and proteinases provoked a decrease of, respectively, 100 and 92.7% of their Cbl binding capacity, whereas the incubation with proteinase decreased the Cbl binding capacity of, respectively, 67.9 and 7.9%. The result of the incubation of [3H]intrinsic factor or [3H]haptocorrin with chymotrypsin and trypsin gave, respectively, no change in the elution position and a shift corresponding to a decrease of 50% of the estimated molecular mass. The estimated molecular mass of Cbl-intrinsic factor and of Cbl-haptocorrin decreased, respectively, to 57.1 kDa and to 88.1 kDa after incubation with exoglycosidases. It was concluded that (1) the carbohydrate core of intrinsic factor protects the whole protein whereas the carbohydrate core of haptocorrin protects only half part of the protein and (2) the carbohydrates are implicated in the formation of the cobalamin binding site of haptocorrin and intrinsic factor.

Chemical Phenomena↗