The effect of pH and ionic strength on the association kinetics of gastric intrinsic factor and intrinsic factor blocking antibody: a soluble system.
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Antibodies↗
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[Juvenile pernicious anemia with congenital intrinsic factor deficiency and intermediate intrinsic factor secretion in the parents].
A 11 1/2-year-old girl of Turkish origin showed symptoms and signs of pernicious anaemia with low B12 level in blood but normal gastric acid secretion. There were no antibodies to mucosal cells or intrinsic factor. Quantitative determination of intrinsic factor in gastric juice, done by a RIA method, proved absence of the factor. Both parents had normal values for haemoglobin, normal B12-absorption tests and slightly decreased B12 blood levels. The gastric acid secretion was normal in both, but intrinsic factor in the gastric juice was decreased markedly. These findings support the hypothesis that congenital pernicious anaemia, e.g. congenital absence of intrinsic factor, is transmitted by an autosomal recessive trait.
Anemia, Pernicious↗
The reaction kinetics of gastric intrinsic factor and IgG intrinsic factor blocking antibody: a soluble system.
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Ammonium Sulfate↗
A radioisotope dilution assay for unlabelled vitamin B12-intrinsic factor complex employing the binding intrinsic factor antibody: probable evidence for two types of binding antibody.
A new radioisotope dilution assay for vitamin B12-intrinsic factor complex is described. The method is based on the use of the binding type intrinsic factor antibody (the binding reagent), which when combined with the intrinsic factor-vitamin B12 complex (labelled ligand), is quantitatively adsorbed onto zirconium phosphate gel at pH 6.25. The new assay has been shown to provide a measure of intrinsic factor comparable with other intrinsic factor assays, but it has the important advantage of being able to measure the unlabelled vitamin B12-intrinsic factor complex (unlabelled ligand), and will, therefore, be valuable in the study of physiological events in the gastrointestinal tract. During the study, it was found that there is some evidence for a least two types of binding intrinsic factor antibody: One which combines preferentially with the intrinsic factor-vitamin B12 complex and one which combines equally well with this complex or with free intrinsic factor.
Antibodies↗
Binding assay and physicochemical characteristics of solubilized intrinsic factor receptor in ileal mucosal homogenates using phenyl-Sepharose to separate the saturated receptor from free intrinsic factor.
A radioisotopic assay was set to determine the physicochemical properties of the solubilized intrinsic factor receptor in pig mucosal extracts. In this assay, phenyl-Sepharose was used to separate the receptor-intrinsic factor-labelled cobalamin complex from the free saturated intrinsic factor. The association constant (at pH 7.4) of the receptor-intrinsic factor complex was estimated at 3.4 +/- 0.3 nM-1. Adsorption of the apo-receptor to phenyl-Sepharose allowed its binding site to be made accessible to intrinsic factor with an association constant in order of 6 nM-1. The receptor binding activity obtained with five mucosal extracts was closely correlated with that obtained by gel filtration of the intrinsic factor-receptor complex (r = 0.99). The radioisotope assay was used to detect the unsaturated receptor (apo-receptor) in sucrose density ultracentrifugation and in superose 6 gel filtration. The sedimentation coefficient was 9.5 s. The apo-receptor was eluted in three peaks in gel filtration, corresponding to the formation of oligomers. The peak of the monomer was increased in presence of EDTA. Its molecular mass was estimated at 270 kDa and its Stokes radius at 5.9 nm. It was concluded that calcium is involved in the oligomerisation of the apo-receptor.
Adsorption↗
Amino terminal sequence of hog intrinsic factor.
1. Hog intrinsic factor has been purified from gastric mucosa by labile ligand affinity chromatography. 2. The following N-terminal amino acid sequence has been determined: Thr-Arg-Ser-Ser-Cys-Ser-Val-Pro-Ser-Ala-Glu-Gln-Pro-Leu-Val-Asn-Gly-Ile- Gln-Val - Leu-Met. 3. However, approx 60% of the preparation lacked the first two residues. 4. A high degree of homology between the amino terminal sequence of human and hog intrinsic factor exists.
Amino Acid Sequence↗
In vitro expression and secretion of functional mammalian intrinsic factor using recombinant baculovirus.
Intrinsic factor was produced at levels of 1-2 mg per 1 (0.25 micrograms per 10(6) cells) by growth of recombinant baculovirus-infected Sf9 cells in spinner culture. The recombinant IF showed a binding affinity for cobalamin (2.6.10(-10) M) and for the intrinsic factor-cobalamin receptor (3.5.10(-10) M) nearly identical with native IF. Purification of the recombinant intrinsic factor could be accomplished by affinity chromatography, but final purification by gel chromatography (FPLC) was necessary to separate intrinsic factor from a 62 kDa protein secreted from uninfected Sf9 cells. This protein binds selectively to the cobalamin-Sepharose column, but demonstrates no cobalamin binding activity after elution. Microgram quantities of radiolabelled protein could be produced for metabolic and autoradiographic studies. The stability of intrinsic factor to pancreatic proteinases was nearly identical with human gastric intrinsic factor, both native and recombinant as produced in mammalian cells. Glycosylation of the intrinsic factor was demonstrated by lectin binding to the recombinant protein separated on SDS-PAGE, and by a shift in apparent molecular mass from 47 kDa to 43 kDa following treatment of Sf9 cells with tunicamycin. Most of the recombinant IF was produced by Sf9 cells in the first 48 h post infection.
Animals↗
Solubilization and preliminary characterization of the human ileal vitamin B12-intrinsic factor receptor.
The human ileal intrinsic factor receptor was solubilized with Triton X-100 using an improved method originally devised for the porcine receptor. At pH 7.4 and in the presence of Ca2+ the receptor bound the vitamin B12 complexes of normal human and pig intrinsic factor but not that of an abnormal biologically inert human intrinsic factor. EGTA dissociated vitamin B12-intrinsic factor from the receptor complexes. The solubilized vitamin B12-intrinsic factor receptor complex consisted of three to four molecular species termed HC-L, HC-20 S, HC-12 S and HC-8.5 S (the three last-mentioned referring to sedimentation coefficients). Of these HC-20 S was the dominating component and had a Stokes radius of 18 nm. Radioactive calcium was shown to be bound to vitamin B12-intrinsic factor and to be contained in its complex with the receptor.
Animals↗
Cellular immunity to intrinsic factor in pernicious anemia.
Cellular immunity to hog intrinsic factor was detected by a modified agarose-leukocyte migration test in 18 patients with pernicious amemia. Lymphocytes from 17 out of 18 patients with pernicious anemia gave positive responses to a concentrate of hog intrinsic factor; the intrinsic factor present in 1 mg. of this concentrate bound 128 ng. of vatamin B12. Six patients with atrophic gastritis, 7 with regional enteritis, and 9 out of 10 healthy adults did not respond to this preparation. No correlation existed between the presence of serum autoantibodies to intrinsic factor and in vitro lymphocyte responsiveness to intrinsic factor. The results demonstrate that cellular immunity to intrinsic factor concentrates is present in the majority of patients with pernicious anemia.
Adult↗
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↗
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↗
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↗
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↗
[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↗
Solubilised intrinsic factor receptor from pig ileum and its characteristics.
The vitamin B-12-intrinsic factor receptor was shown to be present in pig ileum and localized on the brush borders of the enterocytes, and to be solubilisable with Triton X-100. At neutral pH and in the presence of Ca2+ it bound the vitamin B-12-intrinsic factor but not the vitamin B-12-cobalophilin complex. The solubilised vitamin B-12-intrinsic factor-receptor complex consisted of two molecular species with clear Stokes radii (13.11 and 33.53 nm), sedimentation coefficients (15.1 and 45.1 S) and molecular weights (1 600 000 and 12 000 000). A third smaller macromolecule possibly also represented the receptor. Some receptor activity was present in extracts prepared with buffers lacking detergent. There was evidence that the receptor is a membrane lipoprotein from which the lipids reversibly dissociate. Free intrinsic factor also bound to the solubilized receptor and its vitamin B-12-binding site seemed not to be involved in the attachment to the receptor. A small portion of the vitamin B-12-intrinsic factor spontaneously dissociated from the receptor and nearly all dissociated in the presence of Na2-EDTA. TheStokes radius of the dissociated vitamin B-12-intrinsic factor complex was 0.17 nm smaller than before binding to the receptor. Intrinsic factor and cobalophilin were present in ileal extract and observations were made on their molecular characteristics. These proteins, polymers of the vitamin B-12-intrinsic factor complex and binding of vitamin B-12 and its protein complexes to detergent micelles may give spurious receptor-like effects which must be properly controlled.
Animals↗
Purification of the intestinal receptor for intrinsic factor by affinity chromatography.
The intestinal receptor for the intrinsic factor vitamin B-12 complex has been solubilized and then purified from the guinea pig ileum using a double structured affinity resin comprised of intrinsic factor coupled to vitamin B-12 which, in turn, was covalently linked to Sepharose 4B. The receptor purified approximately 57 000-fold from the crude homogenate, appears to be a homogenous protein which may be composed of two subunits which separated when the preparation was subjected to polyacrylamide disc gel electrophoresis. Ethylenediaminetetraacetic acid induced dissociation of the complex between the purified receptor and intrinsic factor-B-12 could not be reversed by the addition of excess Ca2+, unlike the effect of EDTA with semipurified receptor or crude ileal homogenates. Calcium reversed the EDTA effect only after the mixture was subjected to extensive dialysis suggesting that the chelating agent interacts directly with the receptor protein. Intrinsic factor-vitamin B-12 competively inhibited the binding of intrinsic factor-[57Co] vitamin B-12 to the purified receptor whereas vitamin B-12 free intrinsic factor did not, even at a 100-fold greater concentration.
Animals↗
Studies on the solubilized porcine ileal intrinsic factor receptor and on a 340 000-dalton component binding vitamin B-12.
A 340 000-dalton component "C-III" was found when Triton X-100-containing extracts of ileal mucosa were incubated with human or porcine intrinsic factor vitamin B-12 preparations. It was not formed when abnormal human intrinsic factor, unable to attach to the intrinsic factor receptor, was used. Prolonged storage promoted the trnasfer of vitamin B-12 to it from the vitamin B-12-intrinsic factor recptor species C-I and C-II. The component was also present in ileal extracts prepared with or without detergent and it bound vitamin B-12 directly. Immunologically and by electrofocusing it could be classified as a cobalophilin but its molecular dimensions were larger than described for cobalophilin. It thus represents a novel vitamin B-12 binding protein, possibly a macromolecular acceptor of vitamin B-12 which accepts vitamin B-12 bound via intrinsic factor to the ileal intrinsic factor recptor. In the presence of EDTA or at low pH, vitamin B-12-intrinsic factor did not bind to any of the receptor species and under the same conditions it could all be dissociated from the receptor complexes but not from C-III. The dissociated receptor was able to recombine with vitamin B-12-intrinsic factor and it appeared to bind free and vitamin B-12-bound intrinsic factor in vivo.
Animals↗
Primary structure of human intrinsic factor: progress report on cyanogen bromide fragmentation.
Human intrinsic factor purified by labile ligand affinity chromatography was cleaved with cyanogen bromide and fractionated by gel filtration. Four of the fragments were purified and sequenced to a total of eighty-four amino acid residues. Including the N-terminal amino acids this amounts to one third of the total amino acid sequence of human intrinsic factor. One of the fragments contained a tyrosine labelled only on iodination of intrinsic factor devoid of cobalamin.
Amino Acid Sequence↗