Dissociation of Intrinsic Factor from its Antibody: Application to Study of Pernicious Anaemia Gastric Juice Specimens

Intrinsic factor antibody may sometimes be concealed in the gastric secretions of pernicious anaemia subjects, being complexed with residual amounts of intrinsic factor.A method is described for dissociating intrinsic factor from its antibody. Antibody to the vitamin-B₁₂-binding site of intrinsic factor was identified in 16 (57%) out of 28 samples of pernicious anaemia gastric juice after dissociation but in only 10 before dissociation. There was no clear relationship between the incidence of antibody in the serum and in the gastric juice of these patients.     

Isolation of vitamin B-12-binding proteins by combined immuno and affinity chromatography. Comparative studies on the isolated and unisolated proteins

Intrinsic factor or cobalophilin were removed by incubating human gastric juice and pig pyloric extract with purified anti-intrinsic factor and anti-cobalophilin immunoglobulin-G, respectively, covalently coupled to Sepharose. Cobalophilin (transcobalamin I) was also removed from pig serum either by using anti-cobalophilin immunoglobulin-G Sepharose or by Sephadex G-200 chromatography. The one remaining semipurified vitamin B-12-binding protein (intrinsic factor, cobalophilin or transcobalamin II) was then isolated by vitamin B-12-Sepharose affinity chromatography. Intrinsic factors, cobalophilins and transcobalamin II isolated by this two-step procedure were compared by double isotope techniques with the corresponding protein not subjected to affinity chromatography and found to be identical in reaction to antiserum, gel filtration and electrofocusing. The avidity of the isolated and unisolated intrinsic factors for the ileal intrinsic factor receptor was also the same.     

Comparative studies on intrinsic factor and cobalophilin in different parts of the gastrointestinal tract of the pig

The vitamin B₁₂ binders in the pig pyloric mucosa gastric and intestinal juice from the upper gastrointestinal tract were fractionated into only two molecular forms, classified as intrinsic factor and cobalophilin. The unsaturated vitamin B₁₂-binding power due to cobalophilin was lower in the intestinal than in the gastric juice. Electrofocusing revealed that intrinsic factor and cobalophilin in the intestinal juice contained more of the `neutral'-type isoproteins, and the suggestion is made that this is due to enzyme activity. The gastric-juice intrinsic factor contained more acidic isoproteins, which supports the hypothesis that carbohydrate is added on to the polypeptide chain of this protein before it is secreted into gastric juice. The gastric- and intestinal-juice cobalophilins, studied also by electrofocusing, differed from that of pyloric mucosa and they appeared to be of salivary origin. With regard to molecular dimensions there was no significant difference between the intrinsic factors and cobalophilins from all sources studied. All cobalophilins had molecular weights by the formula of Svedberg of approx. 92500, Stokes radii of 4.62nm and sedimentation coefficients of 5.15S. The corresponding values for the intrinsic factors were 63600, 3.57nm and 4.38S. In addition, the intrinsic factors exhibited similar avidities for binding to the solubilized ileal intrinsic-factor receptor. Also the intrinsic factors and cobalophilins, irrespective of their source, bound to the analogous specific xenoantibodies with the same avidity. The present results demonstrate that intrinsic factor remains practically unaltered during its passage through the proximal intestine and render unlikely the speculations made about the presence of an endogenous binder for intrinsic factor as well as the existence of a `pancreatic intrinsic factor'. In addition, they are compatible with the theory that the interference by undegraded cobalophilin may be the reason for the abnormal vitamin B₁₂ absorption observed in patients with pancreatic insufficiency.     

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

Summary 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 pancreas is a major source of intrinsic factor in the dog.     

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.     

Intrinsic-factor Antibody and Absorption of Vitamin B12 in Pernicious Anaemia

The mean urinary excretion in a vitamin-B₁₂ absorption (Schilling) test in control subjects was 19·2% and in pernicious anaemia when given with additional intrinsic factor was as follows: no intrinsic-factor antibodies demonstrable 19·3%, antibodies in serum only 14·4%, antibodies in gastric juice only 11·1%, and antibodies in both serum and gastric juice 8·4%. It is concluded that intrinsic-factor antibody exerts an adverse effect on vitamin-B₁₂ absorption in most patients with pernicious anaemia.     

Purification of rat intestinal receptor for intrinsic factor · vitamin B-12 complex by affinity chromatography

Rat intrinsic factor was bound to vitamin B-12-Sepharose to produce intrinsic factor · vitamin B-12-Sepharose. Intestinal receptor for intrinsic factor · vitamin B-12 complex was purified from rat ileal extract by affinity chromatography using the intrinsic factor · vitamin B-12-Sepharose as an affinity adsorbent with recovery of 48.5% and specific activity increased 335 fold of original sample.     

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.     

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.     

Immunoextracted calcitonin in human gastric secretion

Immunoreactive calcitonin (iCT) has been demonstrated in human gastric juice after immunoextraction with immobilized antibodies and subsequent radioimmunoassay. The basal levels were 4.5 +/- 3.1 (mean +/- SD) pg-Eq/ml gastric juice; range 1.2-9.1 pg-Eq/ml; n = 7, and after stimulatory gastric secretion test with pentagastrin 0.3 +/- 0.2 pg-Eq/ml; range 0.1-0.7 pg-Eq/ml; n = 7 (p less than 0.01). The main fraction of iCT from gastric juice eluted in the same region as synthetic human calcitonin (hCT) on Sephadex G-75 gel chromatography. Reverse phase chromatography in a fast protein liquid chromatography (FPLC) system revealed a slightly less hydrophobic character of the iCT from gastric juice compared to synthetic monomeric hCT. The results were further confirmed by using an additional antiserum. In plasma, the calcitonin (CT) levels were after immunoextraction at the basal state 6.6 +/- 1.7 pg-Eq/ml (mean +/- SD); range 5.1-10.1 pg-Eq/ml; n = 7 and after pentagastrin stimulation 9.4 +/- 5.4 pg-Eq/ml; range 6.3-18.5 pg-Eq/ml; n = 7.     

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.     

Presence of prostaglandins E2 and A2 in canine gastric secretions

The presence of prostaglandins (PGs) was determined in gastric juice obtained from 3 conscious dogs, provided with a chronic gastric fistula. Outputs of acid (mequiv min^?1) and PGs (pg min^?1) were measured in gastric secretions stimulated by pentagastrin (100 or 200 ng kg^?1min^?1). Prostaglandin activity was estimated, after extraction and thin layer chromatography, by radioimmuno-assay of the PGB formed by treatment with alkali. Tritiated PGs were added to gastric juice for the purpose of correcting for PGs recovery. Using this method, the minimum mass of PGB which could be satisfactorily distinguished from zero was 25 pg. Prostaglandins A₂ and E₂ were present in pentagastrin-activated gastric secretions and averaged (mean ± SE, n = 8) 200.7 ± 18.1 and 260.1 ± 18.0 pg min^?1 respectively. The identity of PGA₂ and PGE₂ was confirmed by gas liquid chromatography combined with mass spectrometry. The amount of PGE₂ converted to PGA₂ during extraction, separation and conversion procedures was estimated from the amount of [³H] PGA₂ found when only [³H] PGE₂ had been added to a sample of gastric juice and averaged 14.5% ± 2.0. Our preliminary results support the possibility that PGE₂ and PGA₂ may be of physiological importance in the regulation of canine gastric secretions.     

High Levels of Aromatic Amino Acids in Gastric Juice during the Early Stages of Gastric Cancer Progression

Background

Early-stage gastric cancer is mostly asymptomatic and can easily be missed easily by conventional gastroscopy. Currently, there are no useful biomarkers for the early detection of gastric cancer, and their identification of biomarkers is urgently needed.

Methods

Gastric juice was obtained from 185 subjects that were divided into three groups: non-neoplastic gastric disease (NGD), advanced gastric cancer and early gastric cancer (EGC). The levels of aromatic amino acids in the gastric juice were quantitated using high-performance liquid chromatography.

Results

The median values (25th to 75th percentile) of tyrosine, phenylalanine and tryptophan in the gastric juice were 3.8 (1.7–7.5) µg/ml, 5.3 (2.3–9.9) µg/ml and 1.0 (0.4–2.8) µg/ml in NGD; 19.4 (5.8–72.4) µg/ml, 24.6 (11.5–73.7) µg/ml and 8.3 (2.1–28.0) µg/ml in EGC. Higher levels of tyrosine, phenylalanine and tryptophan in the gastric juice were observed in individuals of EGC groups compared those of the NGD group (NGD vs. EGC, P<0.0001). For the detection of EGC, the areas under the receiver operating characteristic curves (AUCs) of each biomarker were as follows: tyrosine, 0.790 [95% confidence interval (CI), 0.703–0.877]; phenylalanine, 0.831 (95% CI, 0.750–0.911); and tryptophan, 0.819 (95% CI, 0.739–0.900). The sensitivity and specificity of phenylalanine were 75.5% and 81.4%, respectively, for detection of EGC. A multiple logistic regression analysis showed that high levels of aromatic amino acids in the gastric juice were associated with gastric cancer (adjusted β coefficients ranged from 1.801 to 4.414, P<0.001).

Conclusion

Increased levels of tyrosine, phenylalanine and tryptophan in the gastric juice samples were detected in the early phase of gastric carcinogenesis. Thus, tyrosine, phenylalanine and tryptophan in gastric juice could be used as biomarkers for the early detection of gastric cancer. A gastric juice analysis is an efficient, economical and convenient method for screening early gastric cancer development in the general population.     

Rapid purification of cobalamin-binding proteins using immobilized aminopropylcobalamin

Aminopropylcobalamin (AP-Cbl), prepared from 3-chloropropylamine and cob(I)alamin, was immobilized on CNBr-activated Sephacryl beads. The product, Sephacryl-aminopropylcobalamin, contained ca. 1 μmol of AP-Cbl/ml of beads. Cobalamin-binding proteins in biological fluids were adsorbed selectively and quantitatively by Sephacryl-aminopropylcobalamin. After being washed to remove extraneous protein, the beads were photoirradiated to release the cobalamin-binding proteins as their aquacobalamin complexes. The latter could be converted to labeled cyanocobalamin complexes by treatment with [¹⁴C]KCN. The efficacy of this affinity chromatographic method is illustrated by the purification to near homogeneity and in high yield of three representative proteins: transcobalamin II from rabbit serum, intrinsic factor from human gastric juice, and R binder from human saliva.     

Relationship between gastric ulcer and Helicobacter pylori VacA detected in gastric juice using bead-ELISA method

Background. VacA is an important pathogenetic factor produced by Helicobacter pylori. VacA has often been detected in supernatants of liquid cultures or lysates of whole bacterial cells. However, no studies have ever tried to assay VacA produced in the human stomach. We applied a very sensitive and simple method, bead‐ELISA, to detect VacA in gastric juice. Materials and Methods. Forty‐eight H. pylori‐positive patients (16 nonulcer dyspepsia, 16 gastric ulcer, and 16 duodenal ulcer) and four H. pylori‐negative nonulcer dyspepsia patients had endoscopy performed and gastric juice were aspirated. Polystyrene beads coated with the antibody to VacA, were used in this bead‐ELISA method. The nucleotide sequences of vacA in the signal and middle regions were investigated. Results. Of the 48 samples that were positive for H. pylori, 21 [43.8%] were found to be VacA positive in gastric juice. The average and maximum concentrations of detected VacA in gastric juice were 143.2 ± 216.5 and 840 pg/ml, respectively. The average density of VacA from gastric ulcer patients (227.5 ± 276.7 pg/ml) was higher than that found in nonulcer dyspepsia (51.8 ± 39.8 pg/ml) and duodenal ulcer (49.2 ± 21.5 pg/ml) patients. There was no relationship between VacA in gastric juice and vacA genotype. Conclusions. VacA in gastric juice could be directly detected by bead‐ELISA. In this study, the diversity of disease outcome was associated with not the quality but the quantity of VacA. Therefore, not only the quality but also the quantity of VacA is important etiological factors in the pathogenesis of mucosal damage.     

Intragastric generation of antimicrobial nitrogen oxides from saliva--physiological and therapeutic considerations

Salivary nitrite is suggested to enhance the antimicrobial properties of gastric juice by conversion to nitric oxide (NO) and other reactive nitrogen intermediates in the stomach. Intubated patients exhibit extremely low gastric levels of NO, because they do not swallow their saliva. The present investigation was designed to examine the antibacterial effects of human saliva and gastric juice. Furthermore, we studied a new mode of NO delivery, involving formation from acidified nitrite, which could prevent bacterial growth in the gastric juice of intubated patients in intensive care units. The growth of Escherichia coli ATCC 25922 and the formation of NO and nitroso/nitrosyl species were determined after incubation of gastric juice with saliva from healthy volunteers that was rich (nitrate ingestion) or poor (overnight fasting) in nitrite. In a stomach model containing gastric juice from intubated patients, we inserted a catheter with a silicone retention cuff filled with ascorbic acid and nitrite and determined the resulting antibacterial effects on E. coli and Candida albicans. Saliva enhanced the bactericidal effect of gastric juice, especially saliva rich in nitrite. Formation of NO and nitroso/nitrosyl species by nitrite-rich saliva was 10-fold greater than that by saliva poor in nitrite. In our stomach model, E. coli and C. albicans were killed after exposure to ascorbic acid and nitrite. In conclusion, saliva rich in nitrite enhances the bactericidal effects of gastric juice, possibly through the generation of reactive nitrogen intermediates, including NO. Acidified nitrite inside a gas-permeable retention cuff may be useful for restoring gastric NO levels and host defense in critically ill patients.     

The preparation and purification of individual human pepsins by using diethylaminoethyl-cellulose.

A procedure was devised for isolating human pepsins 1, 2, 3 and 5 from gastric juice by repetitive column chromatography on DEAE-cellulose. The combined yields in four different experiments varied from 14% to 90% of the total peptic activity of the starting material. The isolated individual pepsins were shown to behave as single homogeneous proteins on agar-gel electrophoresis at pH 5.0 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. There is preliminary evidence, requiring further study, of two other pepsins, one migrating between pepsins 1 and 2 and the other a pepsin-3 component associating closely with pepsin 5 on chromatography.     

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.     

High concentrations of D-amino acids in human gastric juice

Summary. The concentrations of free D- and L-amino acids were determined in the gastric juice from four groups: patients suffering from early gastric carcinoma with or without Helicobacter pylori infection, and patients without carcinoma but with peptic ulcers, duodenal ulcers or chronic gastritis with or without H. pylori infection. H. pylori is a bacterium associated with gastric inflammation and peptic ulcers and is a risk factor for stomach cancer. The highest D-amino acid ratios (free D-amino acid concentration to the total corresponding free D- and L-amino acid concentration) were 29%, 26%, 18%, 4% and 1% for proline, alanine, serine, aspartate and glutamate, respectively. The gastric juice levels of L-alanine, L-serine, L-proline, L-glutamate and D-alanine in the samples obtained from subjects bearing early gastric carcinoma and H. pylori were significantly higher than in the samples from the other three groups. Except for D-alanine, there was no correlation between the D-amino acid concentrations and presence of carcinoma or H. pylori.     

Size heterogeneity of epidermal growth factor in human body fluids

We measured the concentration of immunoreactive (IR) hEGF in various body fluids by radioimmunoassay (RIA) and evaluated its size heterogeneity by size exclusion high performance liquid chromatography combined with RIA or with time-resolved immunoflurometric assay (TR-IFMA). Mean concentration was 80 ng/ml in urine, 65 ng/ml in milk, 50 ng/ml in seminal plasma, 25 ng/ml in armpit sweat, 1 ng/ml in breast sweat, 0.3 ng/ml in third-trimester amniotic fluid, 3 ng/ml in saliva, 1.5 ng/ml in tears and 0.3 ng/ml in gastric juice.

All the fluids except armpit sweat and gastric juice contained two to five molecular sizes of IR-hEGF. As well as the 6200-dalton (6.2kDa) hEGF we found at least four other different molecular sizes with approximate weights of 300, 150, 70 and 20kDa. The authentic 6.2kDa form made up >90% of the total IR-hEGF in all except the amniotic fluid where its proportion was 71%, and the seminal plasma where the proportion could not be determined.