Comparison of serum vitamin B12 estimation by saturation analysis with intrinsic factor and with R-protein as binding agents.

It has been reported that serum vitamin B12 levels assayed by saturation analysis methods may give misleadingly high results, so much so that the diagnosis of vitamin B12 deficiency may be obscured. This defect was ascribed largely to assays using a vitamin B12 binder other than pure intrinsic factor. To test out this hypothesis two assays were set up, one using saliva (non-intrinsic factor R-binder) and the other using human gastric (intrinsic factor) as B12-binding agents. Both assays were able to differentiate sera from patients with pernicious anaemia from those from control subjects. Published results accumulated over the past 10 years indicate that properly designed and performed saturation analysis vitamin B12 assays are as reliable as microbiological assay methods for detecting low serum B12 levels. The failure of some methods to do does not appear to be due to the nature of the B12-binding agent.     

The immunoassay of gastric intrinsic factor and the titration of antibody to intrinsic factor

A modification of the charcoal method for the detection and titration of antibody to intrinsic factor in serum and for the assay of intrinsic factor in human gastric juice is described. The antibody was detected in a titre greater than 5 ng units/ml in 52% of the sera of 162 patients with Addisonian pernicious anaemia. False positive results may occur if the serum is withdrawn within 24 hr of the parenteral administration of normal therapeutic doses of vitamin B₁₂.

In the analysis of human gastric juice, the charcoal method of immunoassay clearly distinguishes between intrinsic factor and other vitamin B₁₂ binding substances. A good correlation was observed between the gastric acid secretion, the ability to absorb oral radioactive vitamin B₁₂ in the Schilling test, and the assay of intrinsic factor in the gastric juice. Intrinsic factor as an antigen is stable in acid and alkali media but is destroyed by incubation at 56°C for 30 min. A secretion of less than 100 ng units intrinsic factor in the gastric juice during the post-histamine hour is not compatible with adequate vitamin B₁₂ absorption and is indicative of Addisonian pernicious anaemia.

A good correlation was observed between the immunoassay and the biological activity of preparations of hog intrinsic factor. To correct the absorption of oral vitamin B₁₂ in the Schilling test in patients with pernicious anaemia 300–600 ng units hog intrinsic factor are required. It is suggested that hog intrinsic factor preparations should be defined in terms of nanogram units intrinsic factor per milligram.

     

Standardized annular assay of dual radioisotopes on a well type sodium iodide crystal

A procedure is described for the determination of the separate amounts of two gamma-emitting radioisotopes present simultaneously in large liquid volumes using an annular cell placed over a standard well-type crystal of sodium iodide and a reference source of (137)Cs. This sensitive technique is illustrated with particular reference to the double radioisotope urinary excretion test, using orally administered (57)CoB(12) bound to human gastric juice and (58)CoB(12) simultaneously, for the differentiation between patients with intrinsic factor deficiency and other causes of vitamin B(12) malabsorption.     

Vitamin B12-binders in human body fluids. I. Antigenic and physico-chemical characteristics

Three factors binding vitamin B₁₂ are detected in human gastric juice by autoradiographs of immunoelectrophoresis. Binder 1 is present only in gastric juice and gastric mucosa extract and corresponds to intrinsic factor. Binder 2 is present also in other excretions such as bile, lactoserum, saliva, in serum and leucocyte extracts. Binder 3 present in saliva and gastric juice in lower quantity than binder 2, seems derived from this last one by attachment of sialic acid molecules.

There is no immunological identity between intrinsic factor and the other binders without intrinsic factor activity. Binder 3 is resistant to proteolysis in vivo and in vitro. Binder 2 is modified by peptic digestion in vivo but not in vitro. Binder 1 is insensitive to proteolysis if saturated by vitamin B₁₂, but loses its binding capacity and/or immunological properties if the digestion takes place before fixation of vitamin B₁₂.

The three binders seem to have the same affinity for vitamin B₁₂ as judged by dialysis experiments, and the same molecular weight of 60,000 as judged by volume exclusion on Sephadex G-200.

     

Simultaneous free and bound radioactive vitamin B12 urinary excretion test

The absorption of vitamin B(12) following the simultaneous administration of (58)Co B(12) and a complex of (57)Co B(12) with human gastric juice was assessed by measurement of urinary excretion of radioactivity. Sixteen control subjects, 13 patients with pernicious anaemia, and four who had had total gastrectomy were studied. The method proved a reliable means of detecting those with intrinsic factor deficiency.     

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.     

An immunological and chromatographic comparison of human intrinsic factor and the acid-stable gastric esterase VI A

When examined by immunoelectrophoresis and double-gel diffusion, gastric acid-stable esterase (VI A) and human intrinsic factor (IF) behaved as different substances. The VI A migrated in agar-gel electrophoresis mainly as a β₂/β₁-globulin and IF as β₁/α₂-globulin. Both showed overlapping migration and diffusion near the starting point. On DEAE-chromatography IF was eluted together with VI A at 0·05–0·075 M, pH 7·0. In gel filtration experiments with Sephadex G 100 and G 200, the IF from in vivo neutralized gastric juice (NGJ) and from gastric mucosa (GM) was eluted in a definite range according to its molecular weight of 60,000. No IF was found in acid gastric juice (AGJ).

Two immunologically identical variants of VI A were eluted at two different ranges when NGJ and GM were used for gel filtration. The variant of VI A with the lower molecular weight was eluted together with IF. When AGJ was used, only the VI A with the higher molecular weight was found and in an elution range quite different from that for IF. Of the purified substances, only IF showed vitamin B₁₂ binding, which could be inhibited by serum from a patient with pernicious anaemia, and only heteroimmune serum against this B₁₂-binding fraction contained IF antibodies of the blocking and binding types.

IF and VI A proved to be biochemically and immunologically distinct proteins.

     

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 B₁₂.     

Elimination of the vitamin B12 uptake or synthesis pathway does not diminish the virulence of Escherichia coli K1 or Salmonella typhimurium in three model systems.

The role of iron in infection is of great importance and is well understood. During infection, both the host and the pathogen go through many complicated changes to regulate iron levels. Iron and vitamin B12 share certain features. For example, Escherichia coli has similar transport systems for both nutrients, and binding proteins for both are located in gastric juice, liver, saliva, granulocytes, and milk. It is because of such parallels between iron and B12 that we have explored the role of B12 in virulence. A btuB::Tn10 insertion which disrupts the gene encoding the vitamin B12 receptor from E. coli K-12 was P1 transduced into a virulent E. coli K1 strain. In both an infant-rat model and a chicken embryo model, no difference in virulence between the wild-type and the mutant strains was found. Strains of Salmonella typhimurium with mutations in the cobalamin synthesis pathway (Cob) and in btuB were used in a mouse model of virulence. Mutation of the Cob locus or of btuB does not decrease virulence. Interestingly, the inability to synthesize vitamin B12 actually increases virulence compared with the wild type in the S. typhimurium model. This effect is independent of the B12 intake of the mice.     

Stability, blood partition, and protein binding of NQ12, a new naphthoquinone derivative

The stability of a new phospholipase A2 inhibitor, NQ12, in various pH solutions, human plasma, urine, and gastric juices, and rat liver homogenate, the blood partition of NQ12 between plasma and blood cells of rat blood, and the factors influencing the binding of NQ12 to 4% human serum albumin (HSA) using an equilibrium dialysis technique were evaluated. NQ12 was unstable in various pH solutions, human plasma and urine, and rat liver homogenate when incubated in a water-bath shaker kept at 37 degrees C and at a rate of 50 oscillations per min. However, NQ12 was stable for up to 3-hr incubation in human gastric juice. The plasma-to-blood cell concentration ratios of NQ12 were independent of NQ12 rat blood concentrations when the whole blood was incubated for up to 2-hr; the mean (+/- standard deviation) values were 0.112 +/- 0.0650 and 0.172 +/- 0.105 at initial blood NQ12 concentrations of 10 and 20 microg/ml, respectively. The binding of NQ12 to 4% HSA was considerable (higher than 99%) at NQ12 concentrations ranging from 10 to 100 microg/ml in 4% HSA. The unbound fraction of NQ12 was dependent on NQ12 concentrations, pHs of buffer, and HSA concentrations, but was independent of the concentrations of sulfisoxazole or salicylic acid, incubation temperature, buffers containing various concentrations of chloride ion, and concentrations of heparin and alpha-1-acid glycoprotein.     

Reactions of human and hog intrinsic factors with type I antibody to intrinsic factor

A simple and rapid small-scale gel filtration method was applied in studies of type I antibody to intrinsic factor using radioactive vitamin B₁₂ of high specific activity and purified human and hog intrinsic factor preparations, taking into account the unsaturated B₁₂-binding capacity of the individual pernicious anaemia sera. This procedure allows the use of small amounts of reagents. Evidence was obtained for a close antigenic similarity of determinants of human and hog intrinsic factor. The use of purified intrinsic-factor preparations is important.     

Determination of vitamin B12 absorption by a simple whole body counter

This paper reports the results of estimating vitamin B(12) absorption by whole body counting in patients without known gastrointestinal disorder, and in patients with pernicious anaemia, idiopathic achlorhydria, achlorhydria following gastric operations, and various forms of small intestinal disease. Patients with pernicious anaemia absorbed less than 30% of the test dose; they could be distinguished from patients without gastrointestinal abnormality and from most achlorhydric patients who secreted more than 300 mg units of intrinsic factor in the post-histamine hour. Nevertheless, the wide range of normal absorption and the variable absorption from the normal gastrointestinal tract is emphasized and discussed. There is no relation between histamine-stimulated intrinsic factor production and vitamin B(12) absorption in patients with small intestinal disease.     

Über ein neues Enzym des menschlichen Magensaftes

Zusammenfassung Es wird über ein diagnostisch bedeutsames neues Enzym des normalen menschlichen Magensaftes berichtet. Es handelt sich um eine in den Mikrosomen bestimmter Korpusschleimhautzellen gebildete, in das Lumen sezernierte Carboxylesterase mit einem pH-Optimum von pH 6,6, die in der Agargelelektrophorese in dem elektrophoretischen Bereich der-1/-G-Globuline wandert. Dieses Enzym ist ein Glykoprotein mit besonderen antigen Eigenschaften. Mit heterologen Antiseren kann dieses Enzym, unter Erhaltung seiner enzymatischen Aktivität, in dem Präcipitationskomplex in den üblichen Geldiffusionsverfahren nachgewiesen werden. Diese Magensaftesterase ist mit dem bisher beschriebenen Antigen VI A der Korpusschleimhaut auf Grund des immunologischen, chromatographischen, elektrophoretischen und enzymatischen Verhaltens identisch. Das enzymatisch aktive Glykoprotein ist zu einer Vitamin B 12-Bindung in vitro fähig. Seine Beziehungen zu dem Intrinsicfaktorsystem und den bisher bekannten Autoantigenen in Fällen der perniziösen Anämie und chronisch-atrophischen Gastritis werden diskutiert. In den bisherigen Untersuchungen über carcinomatös veränderten Magenschleimhautabschnitten und über pathologische Magensäfte von Patienten mit perniziöser Anämie, chronisch atrophischer Gastritis und Magencarcinomen wurde die Verminderung dieses Enzyms und Antigens festgestellt. Auf seine mögliche diagnostische Bedeutung wird hingewiesen.

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Summary A new enzyme — a carboxylic esterase — of the normal human gastric juice will be described. This esterase can be found in the microsomial fraction of the fundic mucosa. The pH-Optimum is at pH 6.6, the electrophoretic mobility corresponds to the-1/-Ig G region in agar gel electrophoresis at pH 8.2. This enzyme is a glykoprotein with excellent antigenic properties. By heterologous antisera from rabbits it can be precipitated in agar gel methods without loosing its enzymatic activity. The identity of this secreted enzyme with the formerly described mucosal antigen VI A has been proved by immunological and biochemical methods. This antigenic enzym can bind vitamin B 12 and corresponds to the slow vitamin B 12-binder in gastric juice. Relationships to the intrinsic factor system are postulated. A significant diminuition of this esterase in gastric juice of patients with pernicious anemia, gastric carcinoma and mucosal atrophy has been observed in preliminary experiments.

     

Murine Gc globulin binds to a synthetic sequential polypeptide that is a murine B lymphocyte mitogen

A non-immunoglobulin component in mouse serum was found to compete with antibody for the binding of a polyanionic antigen, poly (Tyr-Glu-Ala-Gly). The percent of the polymer (44 ng/ml) bound at physiological pH by the serum component in normal mouse serum was low (18%), but was much higher at pH 6.0 (85%). As a result, the binding of the polyanionic peptide antigen by antibody in immune serum was considerably lower at pH 6.0 than it was at physiological pH. These results should be taken into account when dealing with similar antigens that have been frequently studied in immunology, such as poly (Glu60 Ala40) and poly (Glu60 Ala30 Tyr10). The non-immunoglobulin serum component has been identified as Gc globulin, a vitamin D-binding protein that has been shown to be present on the surface of B lymphocytes. This binding might be responsible for the murine B cell mitogenicity of poly (Tyr-Glu-Ala-Gly) that has been previously reported.     

Single nucleotide polymorphisms in the transcobalamin gene: relationship with transcobalamin concentrations and risk for neural tube defects

Homocysteine levels are elevated in mothers of neural tube defect (NTD) children, which may be due to a disturbed folate or vitamin B12 metabolism. Vitamin B12 is transported to the tissues by transcobalamin (TC). We previously showed that a low holo-TC/total-TC ratio is a risk factor for NTD, possibly due to an impaired binding of vitamin B12 to TC. The coding region of the TC gene of 12 individuals was analysed for genetic variations responsible for a disturbed vitamin B12 binding. The influence of the genetic variations observed on total-TC, holo-TC, holo-TC/total-TC, erythrocyte vitamin B12, plasma homocysteine concentrations and risk for NTD was explored in 42 mothers of a child with NTD and in 73 female controls. Direct sequencing analyses revealed five single nucleotide polymorphisms (SNPs). Three SNPs affected total-TC concentrations, whereas two SNPs seem to affect the binding of vitamin B12. None of the genotypes defined by the SNPs had a significant effect on homocysteine levels, or was associated with an increased NTD risk. Among the five SNPs observed only P259R could partly explain the reduced proportion of vitamin B12 bound to TC, which has been associated with an increased risk for having a child with NTD. Some of the variants studied affected total-TC and holo-TC/total-TC ratio but a larger study population is required to elucidate whether these SNPs influence delivery of vitamin B12 to the tissue, influence homocysteine levels and whether they are associated with an increased NTD risk.     

Effect of vitamin B12 and folic acid deficiency on small intestinal absorption

Three patients are described, and they provide further evidence that deficiency of folic acid and vitamin B(12) may sometimes affect small intestinal function. Malabsorption of both xylose and vitamin B(12) returned to normal in one patient after treatment of a megaloblastic anaemia due to dietary deficiency of folic acid. Impaired absorption of vitamin B(12) was corrected by vitamin B(12) therapy in the other two patients. The initial cause of the vitamin B(12) deficiency in one patient was not apparent, but she was taking Gynovlar 21, which may have been an aetiological factor. In the third patient the small intestinal defect was secondary to pernicious anaemia, and in a group of 98 other patients with pernicious anaemia intrinsic factor did not improve vitamin B(12) absorption in six, and only partially corrected absorption in 30. The significance of these observations is discussed.     

A case of type IIa early gastric cancer developed in pernicious anemia.

Pernicious anemia is an autoimmune disease characterized by a gastric mucosal defect which results in an insufficiency of intrinsic factor to facilitate the absorption of the physiologic amount of cobalamin. Increased risk of cancers of the stomach has been reported for patients with pernicious anemia. We report here a case of a 65 year old woman who had been diagnosed as having pernicious anemia 16 months previously, was receiving monthly vitamin B12 injections, and developed early gastric cancer type IIa by routine follow-up gastroscopic examination. This patient underwent endoscopic mucosal resection for an early gastric cancer lesion with a free resection margin.     

Secretory IgA does not enhance the bacteriostatic effects of iron-binding or vitamin B12-binding proteins in human colostrum.

Human milk contains an unsaturated iron-binding protein (lactoferrin) and an unsaturated vitamin B12-binding protein. Lactoferrin has bacteriostatic properties, and a bacteriostatic role for the B12-binding protein has been postulated. In this study the bacteriostatic effect of lactoferrin was confirmed for strains of Escherichia coli, Pseudomonas and Proteus. Growth inhibition attributable to the unsaturated B12-binding protein could be demonstrated only with a known vitamin B12-dependent E. coli. It has previously been shown that the bacteriostatic effect of lactoferrin is potentiated by horse IgG antibody, and a similar potentiating effect of secretory IgA antibody in colostrum and milk would have obvious importance. An attempt was therefore made to demonstrate potentiation of bacteriostatic effects by naturally occurring secretory IgA antibody to E. coli. The results obtained indicate that secretory IgA antibody does not enhance the growth-inhibiting effects of either lactoferrin or the vitamin B12-binding protein.