Human Intrinsic Factor Research Articles

Human intrinsic factor secretion: immunocytochemical demonstration of membrane-associated vesicular transport in parietal cells.

The human gastric parietal cell synthesizes and secretes intrinsic factor (IF) and acid. In contrast to the cellular mechanisms of acid secretion, little is known about the mechanisms of IF secretion. To elucidate these mechanisms we obtained gastric secretions and sequential fundic biopsies from three subjects before and after pentagastrin stimulation (6 microgram/Kg s.c.). IF was localized in the biopsies using an ultrastructural immunoperoxidase technique using a well-characterized, monospecific antibody to human IF. IF output was quantified using a specific radioimmunoassay in concurrently obtained gastric secretions. Before stimulation, IF was associated with tubulovesicles scattered throughout the cytoplasm and with some in rough endoplasmic reticulum (RER). The tubulovesicles associated with IF migrated to the periphery of the secretory canaliculi within 8 min of stimulation. IF was present on secretory microvilli between 8 and 30 min when IF output in gastric juice was at its maximum. The cessation of IF secretion coincided with the depletion of IF associated with tubulovesicles. IF appeared in the perinuclear space and RER as the IF associated with tubulovesicles was secreted. These observations indicate that IF secretion depends upon membrane-associated vesicular transport and provides support for a membrane translocation-fusion hypothesis to explain the morphologic changes that occur in the parietal cell during secretion.

Insolubilized pure human intrinsic factor used for quantitation of cobalamins in serum

The use of insolubilized pure human intrinsic factor as binding protein in an isotope dilution assay for quantitation of serum cobalamins is described. The reference interval was 200-600 pmol/l. The coefficients of variation were 0.05 to 0.13 and the equilibrium constant for association to cyanocobalamin was 2 x 10(11) 1/mol. The major advantage of the presented method is that intrinsic factor is kept saturated with cobalamin and is thereby stable until just prior to use.

New Evidence for Intrinsic Blood Coagulation in Chickens , ,

Coagulation of blood in chickens is considered the result of an extrinsic clotting system initiated, as in mammals, by tissue thromboplastin released from injured tissues. Blood coagulation in mammals depends principally on an intrinsic mechanism in which thromboplastin is generated from blood itself. Only a negligible role, if any, has been ascribed to an intrinsic system in chickens. A reevaluation of intrinsic coagulation in chickens was undertaken in this study. Whole blood of chickens was found to clot over 30% faster when contacted by suitable surface activators such as kaolin or glass than when such contact was omitted. Plasma recalcification times were significantly (P<.02) shortened by contact activators. Clotting functions were measurable both by partial thromboplastin time and activated partial thromboplastin time, tests that bypass extrinsic factors. Intrinsic thromboplastin could be generated from dilute whole chicken blood although at a slower rate than that reported for human blood. Modification of whole blood thromboplastin generation techniques permitted measurement of activities that seem analogous to human intrinsic factors VIII and IX but not XI or XII. These data provide evidence of a functioning intrinsic clotting mechanism in chickens. A complete description and role for this mechanism remains to be defined.

Unreliability of Radiodilution Assays as Screening Tests for Cobalamin (Vitamin B12) Deficiency

Recent studies have demonstrated problems with the radiodilution assay method for measuring serum cobalamin (vitamin B12) levels. We have found the standard commercial radiodilution kit assay to be totally ineffective in screening for cobalamin deficiency. In a four-month period, 352 patients were screened in a routine clinical laboratory using this kit. None of the serum samples yielded a result below the given normal range of 200 to 1,100 pg/mL. However, when 42 patients with low-normal serum cobalamin levels (200 to 500 pg/mL) were retested by a combination of a modified radiodilution kit, a human intrinsic factor assay, and a microbiological (Lactobacillus leichmannii) assay, 16 (36%) were identified as having serum cobalamin levels that were abnormally low. The diagnosis of cobalamin deficiency may have been incorrectly dismissed in many patients on the basis of test results obtained with these kits, which are in widespread use.

Immunocytochemical localization of human intrinsic factor: The nonstimulated stomach

Human intrinsic factor (IF) is found on the surface of parietal cells at the light microscopic level by immunofluorescence, but there is no direct evidence that IF is synthesized in and secreted from this cell. In order to provide this evidence, IF was localized at the ultrastructural level in human stomach, using an immunochemically monospecific antibody against purified human IF. We defined optimal immunocytochemical conditions and identified the subcellular location of IF in fundic biopsies from 4 fasting subjects. Intrinsic factor was found on the perinuclear membrane, on rough endoplasmic reticulum, Golgi apparatus, tubulovesicles, and membranes of the multivesicular body in parietal cells. Intrinsic factor was absent from nuclei, mitochondria, basolateral surface membranes, blunt microvilli, and the cytosol. Thus, we have defined the parietal cell as the site of IF synthesis. The observed distribution of intracellular IF suggested that an interrelationship existed between the stained membranous structures, and that IF secretion may depend upon membrane translocation. Further immunocytochemical and physiologic studies should increase our understanding of the cellular physiology of IF secretion as well as the parietal cell.

Development of a dual label Schilling test for pancreatic exocrine function based on the differential absorption of cobalamin bound to intrinsic factor and R protein

Cobalamin is bound by R protein in gastric juice and is not transferred to intrinsic factor until R protein is partially degraded by pancreatic enzymes. Using this phenomenon, we devised a new dual label Schilling test for pancreatic exocrine function which is based on the relative absorption of R protein-cobalamin and intrinsic factor-cobalamin. The following were given together orally in Schilling tests: (a) 0.2 nmol human intrinsic factor-[57Co]cobalamin; (b) 0.2 nmol hog R protein-[58Co]cobalamin; (c) 0.4 nmol free human intrinsic factor; and (d) 200 nmol cobinamide (a cobalamin analogue that binds only to R protein and prevents endogenous R protein from removing [57Co]cobalamin from intrinsic factor). Values for the ratio R protein-cobalamin/intrinsic factor-cobalamin in 24-hr urines ranged as follows: 26 normals, 0.52–1.00; 15 patients with pancreatic disease and symptoms of pancreatic exocrine insufficiency, 0.02–0.15; and 11 patients with other gastrointestinal diseases, 0.45–0.86. All patients with pancreatic disease and symptoms of pancreatic exocrine insufficiency malabsorbed R protein-cobalamin and had low values for the ratio R protein-cobalamin/intrinsic factor-cobalamin which did not overlap with values obtained with normals or patients with other gastrointestinal diseases. In 18 patients with pancreatic disease without symptoms of pancreatic exocrine insufficiency values ranged from 0.26 to 0.89. Direct comparisons indicated that the test was comparable to the trypsin output test and at least comparable to the fecal fat test in terms of its sensitivity in detecting decreased pancreatic exocrine function. The simultaneous measurement of R protein-cobalamin and intrinsic factor-cobalamin and the use of their ratio obviate the necessity for quantitative urine collections and make the test applicable to patients with decreased renal function and to patients with combined pancreatic and intestinal diseases. The dual label pancreatic Schilling test is superior to the fecal fat test in specificity, speed, and convenience, and to the trypsin output test in cost, convenience, and potential availability. We believe that it will be clinically useful as a diagnostic test for pancreatic exocrine function.

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.

Purification and characterization of rabbit transcobalamin II

Rabbit transcobalamin II has been purified by labile ligand affinity chromatography and G-200 Sephadex gel filtration. Structural studies indicate Stokes' radii of 2.7 nm and 3.0 nm for transcobalamin II saturated and unsaturated with cobalamin. The amino acid content of the protein is very similar to that of human transcobalamin II (Allen, R.H. (1975) Prog. Hematol. 9, 59–84). The aminoterminal sequence for transcobalamin II is reported for the first time: Glu-Ile-Cys-Gly-Val-Pro-Lys-Val-Asp-Ser-Glu-Leu-Val-Glu-Lys-Leu-Gly-Gln-Arg-Leu-Leu-Pro-(Trp)-Met-(Thr). The ultraviolet and circular dichroic spectra of aquo-, hydroxo-, azido- and cyanocobalamin bound to rabbit transcobalamin II are described. On complex formation the molar absorption of the cobalamins increases and the major bands shift to longer wavelengths. The spectra are little affected by change in the fifth ligand, which indicates that the electron density around the cobalt atom is kept fairly constant by the transcobalamin II molecule. This is in contrast to the observations for the same cobalamins attached to human intrinsic factor and human transcobalamin I (Nexø, E. and Olesen, H. (1976) Biochim. Biophys. Acta 446, 143–150).

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 transfer of vitamin B-12 to it from the vitamin B-12-intrinsic factor receptor 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 receptor. 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.

Alteration of human intrinsic factor during affinity chromatography purification using concentrated guanidine.

Alteration of human intrinsic factor during affinity chromatography purification using concentrated guanidine.

An Improved Procedure for Automated Edman Degradation Used for Determination of the N-Terminal Amino Acid Sequence of Human Transcobalamin I and Human Intrinsic Factor

An improved procedure for automated Edman degradation is presented. Three programs are described, one with double cleavage and two with single cleavage. The programs presented are characterized by a reversed delivery scheme for buffer and phenyl isothiocyanate, and by reduced cleavage times. The modified procedures applied on automated Edman degradation of the vitamin B12-binding proteins human transcobalamin I and human intrinsic factor, containing approximately 390 and 350 amino residues respectively, gave the following N-terminal amino acid sequences: Human transcobalamin I Glu-Ile-Cys-Glu-Val-Ser-Glu-Glu-Asn-Tyr-Ile-Arg-Leu-Lys-Pro-Leu-Leu-Asn-Thr-Met-Ile-Gln-Ser-Asn-Tyr-Asn-?-Gly- Human intrinsic factor Ser-Thr-Gln-Thr-Gln-Ser-Ser-Cys-Ser-Val-Pro-Ser-Ala-Gln-Glu-Pro-Leu-Val-Asn-Gly-Ile-Gln-?-Leu-Met-Glu-Thr- The background accumulation seems to be related not only to the length of the polypeptide chain being degraded, but also to the content of serine (and possibly threonine). A possible N leads to O acyl shift during the cleavage is a tentative explanation. The programs here represented lead to a significant reduction in background compared to conventional programs and allowed considerable prolongation of the degradations.

Amino Terminal Sequence of Human Intrinsic Factor

Olesen, H., Nexö, E., Lous, P., Thomsen, J. & Bucher, D. Amino Terminal Sequence of Human Intrinsic Factor. Scand. J. clin. Lab. Invest. 36, 527–529, 1976.The N-terminal sequence of human intrinsic factor is hereby reported as: 1 5 10 Ser-Thr-Gln-Thr-Gln-Ser-Ser-Cys-Ser-Val-Pro-Ser-Ala-Gln-15 20 25 Glu-Pro-Leu-Val-Asn-Gly-Ile-Gln- -Leu-Met-Gul-Thr-It is concluded that the cysteine at position 8 is involved in a disulfide bridge in the native protein.

Changes in the ultraviolet and circular dichroism spectra of aquo-, hydroxy-, azido-, and cyanocobalamin when bound to human intrinsic factor or human transcobalamin I

The ultraviolet and the circular dichroic spectra of aquo-, hydroxy-, azido-, and cyanocobalamin free or bound to purified human intrinsic factor or purified human transcobalamin I were recorded. Except for azidocobalamin-transcobalamin I, the molar absorption for the γ 1-band in the ultraviolet spectra increased with a factor between 1.2 and 1.4 when the cobalamins were bound to protein. The ultraviolet spectrum of azidocobalamin-transcobalamin I changed so that the γ 2-band became the most intense. Minor changes were observed in the other ultraviolet spectra. The most prominent change in the CD-spectra of protein bound cobalamins was the increase in negative elipticity above 400 nm. This increase was most intense for cyano- and azidocobalamin-transcobalamin I. The study emphasizes that the mechanism by which human intrinsic factor and human transcobalamin I bind cobalamins differ.

Amino terminal sequence of human intrinsic factor.

Olesen, H., Nexö, E., Lous, P., Thomsen, J. & Bucher, D. Amino Terminal Sequence of Human Intrinsic Factor. Scand. J. clin. Lab. Invest. 36, 527–529, 1976.The N-terminal sequence of human intrinsic factor is hereby reported as: 1 5 10 Ser-Thr-Gln-Thr-Gln-Ser-Ser-Cys-Ser-Val-Pro-Ser-Ala-Gln-15 20 25 Glu-Pro-Leu-Val-Asn-Gly-Ile-Gln- -Leu-Met-Gul-Thr-It is concluded that the cysteine at position 8 is involved in a disulfide bridge in the native protein.

Effect of Prolonged Administration of Homologous and Heterologous Intrinsic Factor Antibodies on the Parietal and Peptic Cell Masses and the Secretory Function of the Rat Gastric Mucosa

In order to determine the possible effects of the circulating intrinsic factor antibodies (IFA) on gastric morphology and secretory function, four groups of 12 rats each received intravenous injection daily for 8 to 12 weeks of immunoglobulin G (IgG) fractions separated on DEAE-cellulose columns from various sources: (1) sera of patients with pernicious anemia, containing both IFA and parietal cell antibodies (PCA), (2) sera from patients with atrophic gastritis, containing parietal cell antibody only, and (3) and (4) sera of rabbits immunized with semipurified human or rat intrinsic factor (IF). In addition three control groups of 12 rats each received intravenous injections daily for 8 to 12 weeks of either (5) saline or (6) and (7) IgG processed from human or rabbit normal sera. Still another group of 12 rats (8) did not receive any injections whatsoever for the same duration of time. One-third of the rats were intubated biweekly after histamine stimulation and the hourly outputs of CHl pepsin, and IF were determined. At conclusion of the experiments, rats were killed, the mucosal surface and thickness of the mucosa were measured, and parietal cell and peptic cell masses were counted. The control groups showed either progressive growth of the cellular mass in gastric mucosa and increase of the HCl, pepsin, and IF outputs, or no significant changes. In contrast, rats injected with IgG containing IFA to human or rat IF showed a statistically significant thinning of the gastric mucosa, reduction of peptic cells, which are known to secret IF in this species, and corresponding decreases in the ouputs of pepsin and IF. These became reduced by about 50% from initial values, and by 62 or 75%, respectively, when compared to rats injected with normal human or rabbit IgG's. In rats injected with IgG's from pernicious anemia sera, which contained both IFA and PCA, the outputs of IF, pepsin, and HCl decreased signigicantly, as well as the peptic and parietal cell masses. The rats injected with PCA only demonstrated thinning of the gastric mucosa, reduction of parietal cell mass, and a significant decrease of the HCl output. These findings imply an active role of the circulating gastric.

Isolation and characterization of an abnormal human intrinsic factor.

A patient has been described previously who presented at age 13 with vitamin B(12) (B(12)) deficiency secondary to a functionally abnormal intrinsic factor (IF). IF has now been isolated from the gastric juice of the patient, his sister, and both parents, who are first cousins, by using affinity chromatography on B(12)-Sepharose. Patient IF appeared normal in terms of (a) B(12) binding, (b) mol wt, (c) total amino acid and carbohydrate composition, and (d) immunodiffusion with rabbit anti-patient and anti-normal IF sera. After adsorption with normal IF, however, anti-patient IF serum precipitated the various IFs as follows: patient IF (> 95%); mother, father, and sister IF (50%); and normal IF (< 10%). Additional adsorption with mother, father, or sister IF completely inhibited the precipitation of patient IF. The association constant determined for patient IF-B(12) and human ileal mucosal homogenates (0.1 x 10(9) M(-1)) was 60-fold lower than that determined with normal IF-B(12) (6.0 x 10(9) M(-1)). Intermediate amounts of ileal IF-B(12) binding were observed with mother, father, and sister IF-B(12). These in vitro studies were supported by multiple Schilling tests, performed with a totally gastrectomized volunteer, that gave the following mean urinary excretions of [(57)Co]B(12): free B(12) (0.5%); + patient gastric juice (2.6%); + mother or father gastric juice (17%); and + normal gastric juice (26%). These studies demonstrate that the patient is homozygous and that the mother, father, and sister are heterozygous for a structurally abnormal IF that has a markedly decreased, but not absent, affinity for ileal IF-B(12) receptors. These studies also indicate that the B(12) and ileal binding sites are located on different portions of the IF molecule.

Human intrinsic factor, isolation by improved conventional methods and properties of the preparation

Intrinsic factor possessing high biological activity was isolated as its cyanocobalamin complex from gastric juice. The purification method consisted of ammonium sulphate precipitations, ion exchange and gel filtration steps and had a yield of about 42%. The vitamin B 12-protein complex was in monomeric form with a molecular weight of approximately 63 000. The amino acid composition resembled that of hog intrinsic factor. Six different carbohydrates were found by gas chromatography, the total content being 8.3%. Spectra in water and alkali indicated that in binding to protein the absorbance of vitamin B 12 was increased over a wide wavelength range. In strong alkali the spectrum approached that of the free vitamin. Accordingly, previous spectrophotometric estimates of the vitamin B 12 content of cyanocobalamin-protein complexes need revision. In isoelectric focusing and gel filtration, no indications were found that the isolated protein differed from that in unfractioned gastric juice.

Isolation of Gastric Vitamin B12-binding Proteins Using Affinity Chromatography: I. PURIFICATION AND PROPERTIES OF HUMAN INTRINSIC FACTOR

Abstract Human intrinsic factor has been isolated from gastric juice. The protein was purified 853-fold with a yield of 85% utilizing affinity chromatography on vitamin B12-Sepharose as the sole purification technique. The final preparation was homogeneous based on polyacrylamide gel electrophoresis and sedimentation equilibrium ultracentrifugation. The isolated protein (20 µg) corrected vitamin B12 malabsorption when given to a patient with pernicious anemia in a standard Schilling test. Human intrinsic factor binds 30.1 µg of vitamin B12 per mg of protein and contains a single vitamin B12-binding site with an association constant for vitamin B12 of 1.5 x 1010 m-1. The molecular weight determined by sedimentation equilibrium ultracentrifugation was 45,200 to 47,700 while that determined by amino acid and carbohydrate analyses was 44,200. The protein contains 15.0% carbohydrate which accounts for the elevated molecular weight values (59,000 to 66,000) obtained using sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. In the presence of vitamin B12, human intrinsic factor aggregates to form dimers and higher molecular weight oligomers. When vitamin B12 binds to human intrinsic factor, the spectral maximum for vitamin B12 shifts from 361 nm to 362 nm and the absolute absorbance at 361 nm increases by 32%.

Purification of human intrinsic factor by affinity chromatography

Hydroxocobalamin was insolubilized by covalent coupling to albumin which in turn was coupled to bromoacetyl-activated cellulose. This product allowed specific adsorption of cobalamin-binding proteins from human gastric juice. The cobalamin-binding proteins were eluted by increase of temperature and addition of cyanocobalamin. After further chromatographic purification the net result of pure intrinsic factor was about 5% or some 75 mg from about 500 1 of gastric juice. The biological activity was proven by the Schilling test. It was homogeneous by sodium dodecyl sulphate gel electrophoresis and immunoelectrophoresis. Stokes radius (3.3 nm) was the same as the one of native intrinsic factor in gastric juice. The partial specific volume determined by ultracentrifugation in water and in deuterated solvent was 0.743 cm 3/g. The amino acid content was similar to the one of hog intrinsic factor. Sedimentation equilibrium ultracentrifugation indicated the presence of one or two protomers with a mol.wt of 30 000, unable to bind cyanocobalamin, and a monomer of mol.wt 60 000 binding one molecule of vitamin B 12.

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

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 .