Radioiodination of human intrinsic factor.

Human intrinsic factor (IF) saturated with (60)Co-labeled cyanocobalamin ((60)CoB(12)) was purified and then iodinated with (125)I to yield (125)I-labeled IF-(60)CoB(12) preparations of high specific activity. Sephadex G200 and DEAE-cellulose chromatography of the iodinated IF-(60)CoB(12) complex showed coincidence of the major (125)I and the (60)Co radioactivity peaks. During starch-gel electrophoresis (60)Co radioactivity from noniodinated and iodinated complexes migrated to the same extent while (125)I radioactivity from the iodinated complex migrated slightly further anodally than did the (60)Co radioactivity. After the iodinated complex was mixed with antibody to the IF-B(12) complex (antibody II) the (125)I and (60)Co radioactivity were: (a) precipitated in similar amounts by antiglobulin serum. (b) eluted coincidentally in the 19S region on Sephadex G200, and (c) excluded to the same extent from starch gel during electrophoresis. After equilibrium exchange of IF "blocking" antibody (antibody I) for (60)Co-vitamin B(12) on (125)I-labeled IF. (125)I radioactivity from the IF-antibody I complex: (a) was precipitated by antiglobulin serum, (b) was eluated in the 19S region on Sephadex G200 gel filtration, and (c) migrated slowly towards the anode on starch-gel electrophoresis. Urinary excretion of (60)Co radioactivity in pernicious anemia patients after oral administration of (60)Co-vitamin B(12) bound to freshly prepared (125)I-labeled IF was similar to that obtained with noniodinated intrinsic factor. These results show that iodination of IF-(60)CoB(12) complex does not markedly alter the chromatographic, electrophoretic, antigenic, or absorption-promoting properties of IF.

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

The precipitate which resulted when (57)CoB(12) bound to normal human gastric juice was subjected to a 15% concentration of Na(2)SO(4) contained virtually no radioactivity. However, after in vivo incubation of the gastric juice-(57)CoB(12) mixture in the distal ileum of the guinea pig, the dialyzed extract of the washed mucosa contained a fraction of (57)CoB(12) which was precipitated at 15% Na(2)SO(4). In addition, in vitro incubation of gastric juice-(57)CoB(12) with an extract of the ileal mucosa or brush border membranes also resulted in the formation of a 15% Na(2)SO(4)-insoluble fraction which contained (57)CoB(12). The formation of this (57)CoB(12)-containing insoluble fraction did not occur or was diminished by (a) addition of an excess of B(12)-free normal human gastric juice. (b) reducing the incubation pH to 2, (c) incubating the mixture at 4 degrees C, (d) pretreating the ileal extract at 56 degrees C for 30 min, (e) incubating the reaction in sodium EDTA but not calcium EDTA, (f) incubating gastric juice-(57)CoB(12) with an extract of jejunal mucosa. Sephadex gel filtration was used to demonstrate that the factor in the ileal extract which reacted with the gastric juice-(57)CoB(12) filtered through G-100 and G-200 columns in the excluded volume. When the ileal extract obtained after in vivo incubation with gastric juice-(57)CoB(12) was subjected to starch gel electrophoresis one peak of radioactivity remained at the origin and another moved anodally. Eluates of each peak reacted with anti-intrinsic factor antibody indicating that at least the immunologically reacting portion of the intrinsic factor molecule was present in two fractions with different electrophoretic mobility.These studies indicate that immunologically intact intrinsic factor can be extracted from the ileum after in vivo incubation with gastric juice-(57)CoB(12), and that a macromolecular factor is present in the distal ileal mucosa which binds intrinsic factor both in vitro and in vivo, changing its solubility and electrophoretic properties. It is suggested that this ileal binding factor is the previously postulated intestinal receptor for intrinsic factor.

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.

[Pernicious anemia: a teenager with an unusual cause of iron-deficiency anemia]. / La maladie de Biermer: une cause possible d'anémie microcytaire chez l'adolescent.

A teenager was admitted with an iron-deficiency anemia. The gastroscopy found an atrophic body gastritis, which revealed a pernicious anemia. This diagnosis is rare in paediatric patients, the frequency of pernicious anemia increasing with age. Iron-deficiency anemia is mainly described in young people.

Investigations proposed to accurately classify chronic gastritis.

Patients of chronic gastritis should be investigated with gastric mucosal biopsy, parietal cell antibody, intrinsic factor antibody, Helicobacter pylori antibody, urea breath test or faecal antigen test for Helicobacter pylori, to accurately classify them. The results of these tests will indicate Helicobacter pylori infection (present or past), the role of hereditary factor (intrinsic factor antibody present or absent) and the success or failure of Helicobacter pylori eradication treatment.

Structural homologies of cobalamin-binding proteins. Tryptic peptide mapping of intrinsic factor, transcobalamin and haptocorrin from man, hog and rabbit.

We have explored the structural features of cobalamin binding proteins by peptide mapping. The present report is a comparison of the radioiodinated tryptic peptides of intrinsic factor, transcobalamin and haptocorrin from man, hog and rabbit. The results show that the homology between analogous proteins from different species is close for intrinsic factor and transcobalamin and weaker for haptocorrin. The results also suggest the existence of one or more regions which, with minor changes, are conserved among all proteins investigated. This implies a common evolutionary origin for all the cobalamin binding proteins studied.

Identification and characterization of intrinsic factor and cobalophilin from pig ileal and pyloric mucosa.

Pig ileal mucosa was found to bind about 240 ng vitamin B12/g and to contain two vitamin B12-binding proteins. One was highly active in the Schilling test, behaved immunologically as intrinsic factor and was responsible for about half of the total vitamin B12-binding capacity. The other binder was identified as cobalophilin (R-protein). Immunochemical purification of these proteins from pig ileum and pylorus was performed and the molecular characteristics (sedimentation and diffusion coefficients, Stokes radii, frictional ratios and molecular weights) of their vitamin B12 complexes were estimated. Isoelectric focusing revealed differences between the ileal and pyloric intrinsic factors but not between the cobalophilins. The mean isoelectric points of the pyloric and ileal intrinsic factors were pH 5979 and 5.30, respectively, whereas the corresponding figures for the cobalophilins were 4.13 and 4.10.

Purification of human intrinsic factor using high-performance ion-exchange chromatography as the final step.

Human intrinsic factor was purified 1430-fold from gastric juice with a yield of 75% using two steps: labile ligand affinity chromatography and high-performance ion-exchange chromatography. Intrinsic factor precipitated in the presence of specific autoantibodies and 15% sodium sulfate, had an estimated Mr of 59 000 in 5% SDS electrophoresis and could bind to the specific ileal receptor in vitro. Its carbohydrate composition could be related to N-lactosaminic and O-glycosidic chains. High-performance ion-exchange chromatography was a mild, rapid and efficient procedure to separate completely intrinsic factor from haptocorrin (another glycoprotein of gastric juice which binds cobalamin) and from other contaminating proteins.

Use of radioisotope techniques in the clinical evaluation of patients with megaloblastic anemia.

Because virtually all cases of vitamin B12 deficiency seen in this country are due to malabsorption, the availability of radioactive vitamin B12 for direct measurement of absorption of this essential nutrient has proved to be of great clinical value. These tests are useful not only in demonstrating vitamin B12 malabsorption but also often in defining the pathophysiological mechanism responsible for this abnormality. The urinary excretion test of Schilling remains the most useful test for vitamin B12 absorption. Minor precautions and modifications in technique make the test results more reliable and easier to interpret. The 8-hr plasma test for vitamin B12 absorption can no longer be considered acceptable. Some patients with vitamin B12 malabsorption have results in the normal range when studied by this method. Serum vitamin B12 assays utilizing radioactive vitamin B12 and the isotope dilution principle are not widely used and are useful screening tests. Low normal or borderline results observed in patients with clinical evidence suggestive of vitamin B12 deficiency should be interpreted with caution or confirmed by radioactive vitamin B12 absorption studies. Radioactive vitamin B12 can also be used for rapid, reliable assay of gastric intrinsic factor, antibody to intrinsic factor and unsaturated vitamin B12 serum. Methods using radioactive folate compounds for similar in vivo and in vitro studies are not yet applicable for routine use in nuclear medicine laboratories.

Intrinsic factor, free of R proteins, can be prepared from mouse stomach and used in a ligand assay specific for "true" cobalamin.

A ligand assay specific for cobalamin that uses mouse stomach as the source of intrinsic factor has been developed. When mouse stomach extract incubated with radiocobalamin is fractionated by gel chromatography, the radioactive complex elutes as a single peak with apparent molecular weight of 54,900. Formation of the complex is greater than 98% inhibited by human anti-intrinsic factor antibody. When the equivalent of 10,000 pg/ml of cobinamide is added to serum, the apparent cobalamin concentration detected averages 8.5 pg/ml. Correlation with the Lactobacillus leichmannii microbiologic assay results in the regression equation y = 0.97x + 20. In six patients who had megaloblastic anemia the serum cobalamin by the mouse intrinsic factor ligand assay ranged from 0 to 9 pg/ml. Because the primary source of intrinsic factor is free of R proteins, there is no need for extensive purification of the extract. The assay is sensitive, precise, and accurate, and no more difficult to perform than other conventional ligand assay procedures.

Effect of Berkefeld filtration on the binding activity of human gastric juice.

The intrinsic factor content of Berkefeld-filtered human gastric juice has been studied. This appears to vary with the pH at which filtration is carried out and also between individual filters. Significant losses of intrinsic factor may result from filtration and complete loss when filtration is carried out at a low pH. The most suitable pH for filtration appears to be in the range pH 7 to 8.

A comparison of two methods for the immunoassay of intrinsic factor.

The intrinsic factor content of 263 samples of gastric juice was determined by immunoassay using charcoal absorption and by immunoelectrophoresis on acrylamide gel. There was good correlation between the results of the two techniques and, with a few exceptions, both made it possible to predict which patients would have malabsorption of radioactive vitamin B(12). Of the two methods, immunoassay using charcoal absorption generally gave the higher readings. Further study showed that this could not be attributed to the recognized difference in the principles of the two methods but that it was more likely to be due to the dissimilarity of the experimental conditions.

Rapid protein A assay for intrinsic factor and its binding antibody.

A simple and rapid method for the measurement of cobalamin bound intrinsic factor (Cbl-IF) complex and intrinsic factor binding antibody is described. The method is based on the principle of affinity chromatography and adapted to a batch separation technique. A specific ligand staphylococcal protein A was coupled to Sepharose to form a convenient solid phase matrix. The intrinsic factor binding antibody in patients with pernicious anaemia was used to form an immune complex with Cbl-IF. This complex was adsorbed on to staphylococcal protein A. Gastric juice from control subjects and patients with pernicious anaemia was assayed for intrinsic factor activity and the results correlated very closely with two other established methods. Sera from 30 control subjects were assayed for binding intrinsic factor antibody and all were found to be negative; of 15 patients with pernicious anaemia, six were positive. These patients were selected with blocking antibody. The method does not require technologically advanced protein separation techniques and could therefore be applied in any clinical laboratory using radioisotopes. It could also be adapted to assay cobalamin in body fluids or in food.

A simple assay of intrinsic factor-vitamin B12 complex employing the binding intrinsic factor antibody.

The reaction between binding intrinsic factor antibody and intrinsic factor-vitamin B(12) complex has been studied. Initially in the zone of antibody excess, the relationship between the amount of antigen present and the amount of antigen-antibody complex adsorbed onto zirconium phosphate gel was linear. With increasing amounts of antigen, the curve departed from linearity and reached a plateau. The linear portion of the reaction forms the basis of a simple and reproducible assay for quantitating intrinsic factor to which vitamin B(12) has already been bound. The assay provides a method for studying the fate of intrinsic factor-vitamin B(12) complex during digestion and absorption. In two normal subjects given radioactive vitamin B(12) orally, aspiration of ileal contents showed that only 50 to 70% of the radioactivity was bound to intrinsic factor at that level.

Estimation of intrinsic factor and detection of intrinsic factor antibodies using non-radioactive cyanocobalamin and microbiological assay.

A method employing non-radioactive vitamin B(12) and microbiological assay is described for estimating intrinsic factor in gastric juice and for detecting antibody to intrinsic factor in serum. Satisfactory agreement was obtained between the results by this method and by a modification of the method of Ardeman and Chanarin (1963). During the first hour after gastric stimulation 11 patients with pernicious anaemia secreted between 0 and 240 units of intrinsic factor compared with between 1,600 and 39,000 units in 21 patients with other conditions. The results in three out of four patients with gastric atrophy were higher than those in pernicious anaemia but lower than in other conditions.

Concentration and physicochemical characterisation of unsaturated cobalamin binding proteins in amniotic fluid.

Indirect evidence for the presence of intrinsic factor in amniotic fluid has been provided recently, using either a radioisotope binding assay or a radioimmunoassay. We have determined the unsaturated cobalamin binding capacity and the physicochemical properties of the 3 cobalamin binding proteins in 59 amniotic fluids using radioisotope binding assay, gel filtration and isoelectrofocussing. A good correlation with gestational age was found for the total unsaturated Cbl binding capacity (r = 0.735) and for the concentration of unsaturated haptocorrin (r = 0.746), but not for the concentration of unsaturated intrinsic factor (r = 0.003). When their binding capacities were expressed as a percentage of the total unsaturated Cbl binding capacity, the percentage of intrinsic factor, transcobalamin II and transcobalamin III (the less acidic fraction of haptocorrin) decreased and the percentage of haptocorrin increased in function of gestational age. The physicochemical properties of intrinsic factor in amniotic fluid were close to those in gastric juice: the molecular mass was estimated to 49,200 +/- 4,900 Da (n = 24) in Sephacryl S 300 gel filtration, the cobalamin-protein complex was resolved in 2-6 isoproteins isoelectric at a pH range of 4.6-5.8 and with a mean isoelectric point of 5.18 +/- 0.16 (n = 5) in isoelectrofocusing and it crossreacted with anti-intrinsic factor autoantibodies (from a Biermer anaemia serum). Amniotic fluid collected at 13 wk of gestational age was found to contain intrinsic factor and haptocorrin with less acidic isoproteins than those usually observed in gastric juice and serum. This could indicate that sialic acid is less involved in the composition of the carbohydrate core of cobalamin binding glycoproteins in this period of the gestational age than later on and that cobalamin binding proteins have mainly a foetal origin.

The preparation of a sensitive partial thromboplastin reagent from bovine brain.

Commercial partial thromboplastin reagents markedly differ in their sensitivity to factor deficiency, heparin, or the lupus anticoagulant. These differences may be partly due to the variable phospholipid content of different commercially available reagents. For over 15 years, we have routinely used a partial thromboplastin prepared from human brain. In the past four years, we have been using a similarly prepared bovine partial thromboplastin reagent. This report describes the preparation of our partial thromboplastin reagent, as well as an analysis of the phospholipid composition of both the human and bovine thromboplastin reagents. Four separate brain preparations produced consistent percentages of the anionic phospholipids, phosphatidylserine, and phosphatidylinositol. The bovine reagent was also compared with commercial partial thromboplastin reagents in the detection of coagulation factor deficiency, heparin, and the lupus anticoagulant.

Regional variation in tensile properties and biochemical composition of the human lumbar anulus fibrosus.

STUDY DESIGN: The structure-function relationship of anulus fibrosus of nondegenerate lumbar intervertebral discs was investigated. OBJECTIVES: The tensile properties and biochemical composition of single lamella specimens from human anulus fibrosus and their variations with anatomic region were determined. SUMMARY OF BACKGROUND DATA: Regional differences in composition and ultrastructure suggest differences in tensile properties. METHODS: Single lamella specimens were isolated from the anulus, equilibrated in 0.15 mol/L NaCl and tested in uniaxial tension using a slow strain-rate protocol. Adjacent specimens were used to determine biochemical composition (including hydration, collagen, proteoglycan, and hydroxypyridinium crosslink density). Tensile properties, biochemical composition, and anatomic location were compared. RESULTS: Significant radial and circumferential variations in tensile properties of anulus were detected, with the anterior being stiffer than the posterolateral regions, and the outer being stiffer than the inner regions. CONCLUSIONS: The regional differences in tensile properties may result predominantly from structural rather than compositional variations and may contribute to the clinical frequency of anulus failure in the posterolateral region.

Location and secretion of gastric intrinsic factor in the sheep.

The site of production and secretion of intrinsic factor (IF) in the sheep has been studied using a human auto-antibody directed against IF. Immunofluorescent studies indicated the abomasal parietal cell was the source of IF in the sheep. Concentrations of IF in pure gastric secretion of sheep were relatively stable at 3 to 4 iu/ml and it was estimated that the total abomasal output of IF was 10,000 to 23,500 iu per 24 hours.

Immunologic tests of value in diagnosis. 1. Acute phase reactants and autoantibodies.

Acute phase reactants are nonspecific indicators of tissue necrosis and/or inflammation but may be helpful in determining activity of disease. Rheumatoid factor is likewise rather nonspecific, but its presence is helpful in predicting the course, severity, and complications of rheumatoid arthritis. Numerous antinuclear antibodies have been identified in collagen vascular diseases; perhaps the most specific association is between anti-Sm antibody and systemic lupus erythematosus. Anti-smooth-muscle and antimitochondrial antibodies can aid in differential diagnosis of liver disease, while antithyroid antibodies can perform a similar function in diffuse goiter. Anti-parietal-cell and anti-intrinsic-factor antibodies are quite specific for pernicious anemia.