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Gut: first published as 10.1136/gut.28.3.339 on 1 March 1987. Downloaded from

Glut, 1987, 28, 339-345

Immunohistochemical localisation of B12 R- binder in the human digestive tract

H KUDO, M INADA, G OHSHIO, Y WAKATSUKI, K OGAWA, Y HAMASHIMA, AND T MIYAKE From the Departments ofGeriatric Medicine and Pathology, Kyoto University, Kyoto, Japan

SUMMARY The distribution of R-binder in the human digestive tract was studied using an indirect immunoperoxidase technique. Positive staining for R-binder was found in the mucous cells and ductal epithelial cells of the salivary glands and the oesophageal glands. In normal gastric mucosa, no positive staining for R-binder was found, but in the area with intestinal metaplasia, the columnar epithelial cells and goblet cells showed positive staining. Epithelial cells of the gall bladder, intrahepatic ducts and pancreatic ducts were also positive for R-binder. In the and colon, R-binder was found in the columnar epithelial cells and goblet cells. The measurement of unsaturated vitamin B12 binding capacity and cobalamin content in the extracts from intestinal mucosa also indicated the presence of R-binder in the intestinal mucosa. http://gut.bmj.com/

R-binder, an immunologically distinct vitamin B12 had resections for gastric ulcer, gastric cancer, binding protein, is ubiquitous in human serum, many leiomyoma and perforation of the small intestine, blood cells and various body fluids. In the digestive colon cancer and cholelithiasis. Specimens of the tract, R-binder is present in saliva, gastric juice, bile, major salivary gland, and were and pancreatic juice, but its function is still a matter obtained at necropsy. of speculation. Although the localisation of R-binder Macroscopically normal tissue samples were on September 26, 2021 by guest. Protected copyright. in the salivary gland is studied in one report,' its tissue removed and fixed in 10% formalin and embedded in distribution must be studied in more detail to deter- paraffin. Four micron thick sections were cut and mine its role. deparaffinised in xylene and ethanol series. For We studied the distribution of R-binder in the routine histological examination each section was human digestive tract using the immunoperoxidase stained with haematoxylin-eosin, and histologically technique. In addition, we measured unsaturated normal sections were used to detect R-binder. vitamin B12 binding capacity (UBBC) and endo- genous cobalamin in the extracts from the human ANTISERUM intestinal mucosa to confirm the results of the R-binder was purified from pooled human saliva immunohistochemical study. using affinity chromatography according to the method of Allen et al.3 Purified R-binder showed Methods a single protein band by SDS-polyacrylamide gel electrophoresis. Anti R-binder antiserum was pre- TISSUE PREPARATION pared in . Specificity of the antiserum was Specimens of the and the gall confirmed by Ouchterlony's immunodiffusion bladder were obtained surgically from patients who method and immunoelectrophoresis.

Address for correspondence Dr H Kudo, Dcpartmcnt of Geriitric Medicine. IMMUNOPEROXIDASE TECHNIQUE Fdtculty of Medicine, Kyoto University Sakyo-ku, Kvoto 606, Jlapin Indirect peroxidase technique was used. Deparaffin- Received for publicition 29 June 1986I ised tissue sections were treated with 0/3% hydrogen 339 Gut: first published as 10.1136/gut.28.3.339 on 1 March 1987. Downloaded from

340) Kaido, Inada, Ohshio, Wakatsuki, Ogawa, Hattnashirna, antd Miyake peroxide in methanol for 30 minutes in order to block buffer containing 0()15 M NaCl, pH 7-4, and homo- the endogenous peroxidase activity. Sections were genised for five minutes. The homogenate was centri- precoated with 5%° normal goat serum for 20 minutes. fuged at 15(00 g for 30 minutes and the supernatant and then incubated with anti R-binder antiserum at a was collected. dilution of 1:400 with 0. 1 M phosphate buffered saline (PBS), pH 7-4, containing 0(5% bovine serum ASSAY OF UBBC, TRANSCOBAI,AMIN II (TCII), albumin, at 4°C overnight, followed by peroxidase ENDOGFNOUS COBAILAMIN AND PROTEIN conjugated goat antirabbit IgG antiserum (Cappel, CONTENT IN THFF,XTRACTS USA) at a dilution of 1:80; at room temperature for Unsaturated vitamin B12 binding capacity was 40 minutes. After each application of antiserum, the measured by the technique of Gottlieb et al,4 TcII by sections were washed with PBS three times for 1i) absorption to a microfine precipitate of Silica.' minutes each time. The histochemical reaction for Endogenous cobalamin was assayed by vitamin B12 peroxidase was carried out using 3.3'-Diaminobenzi- [5Col radioassay (Corning, USA, lot 63015). Protein dine (0.05% w/v) and hydrogen peroxide (0.01%) in content was measured using Bio Rad Protein Assay 0(05 M Tris-HCI buffer, pH 7-6. In control prepara- (Bio Rad, USA). UBBC, TcII and cobalamin con- tions, the primary antiserum was replaced by normal tent in the sera of 10 healthy volunteers were also serum or by specific antiserum preincubated assayed for comparison. with an excess of purified R-binder: control stainings were all negative. Results

EXTRACTION OF THE INTESTINAL MUCOSA IMMUNOHISTOCHFMISTRY Samples of the large and small intestine were In all sections, R-binder positive cells were stained obtained from four necropsy cases within two hours with a clear contrast. Gastrointestinal tract tissues after death. In each case, a part of the colon, jejunum examined contained various numbers of polymor- and , which showed macroscopically normal phonuclear leucocytes or histocytes, which served as mucosa, were removed, washed with saline and useful positive controls in judgment. frozen immediately at -70°C. In the salivary glands, R-binder positive reaction After thawing, the mucosa was scraped off, mixed was found in the cytoplasm of mucous cells and with an equal volume of 0()1 M phosphate intercalated duct cells. Myoepithelial cells and serous http://gut.bmj.com/

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Fig. 1 Immunoperoxidase siaitjitigfor R-bitnder in 1the1w .slu ancdibilarglatnd. Posilive staining is folunld in thei mnucous (ell/s a(0( intercalated duct(ell/s. Met/h i greea counltrslainl. Gut: first published as 10.1136/gut.28.3.339 on 1 March 1987. Downloaded from

lItinmuinohistochemical localisation ofvitamin B]2R-binder in the human digestive tract 341 http://gut.bmj.com/ on September 26, 2021 by guest. Protected copyright.

Fig. 2 Immunoperoxidase stainingfor R-binder in the gastric mucosa. (a) Normalfundic mucosa, no positive staining is found. (b) Intestinal metaplastic mucosa, positive staining is found in the columnar epithelial cells and goblet cells. (a) and (b), methyl green counterstain. cells were negative (Fig. 1). Similarly, mucous cells polymorphonuclear leucocytes. In contrast, in the and ductal epithelial cells of the oesophageal glands area with intestinal metaplasia, there was always showed positive staining for R-binder. intense staining for R-binder in both columnar In normal gastric mucosa, no positive staining for epithelial cells and goblet cells (Fig. 2). R-binder was found except for small numbers of Columnar epithelial cells of the gall bladder, Gut: first published as 10.1136/gut.28.3.339 on 1 March 1987. Downloaded from

342 K3Kdo, Inada, Ohshlio, Wakatsluki, Ogawa, Hatnashiima, and MiyVake

Fig. 3 Immunoperoxidase stainlingJor R-bindetr in thlie liver. Positivestaininlg isjountd inlthe epithelial cells ofititrahepatic bile ducts. Metli ill greeni eounterstain. intrahepatic bile ducts and pancreatic ducts were also nique, that R-binder was present both in the mucous positive for R-binder. Hepatocytes and acinar com- secretory acini and in the intercalated ducts of the http://gut.bmj.com/ ponents of the pancreas were negative for R-binder human salivary glands. Although they used anti-hog (Figs 3-5). R-binder antiserum as the primary antiserum, our In the small intestine and colon, R-binder was findings obtained using antihuman R-binder anti- found in the columnar epithelial cells and goblet serum were consistent with theirs. cells. The epithelium of the small intestine was In normal gastric mucosa, R-binder was not found stained weakly (Fig. 6). in the surface epithelium or in the secretory glands,

but in the area with intestinal metaplasia, the colum- on September 26, 2021 by guest. Protected copyright. UBBC TCII AND COBAILAMIN CONTENT IN THE nar epithelial cells and goblet cells showed positive EXTRACT staining for R-binder. Allen et aP reported that The Table shows the UBBC, TcII and cobalamin gastric juice of 11 individuals had ranged from content in the extracts of each case. In all cases there 89-100%, in terms of percentage of inhibition of was detectable UBBC and TcII. The level of total vitamin B12 binding activity by anti- vitamin B 12 binding capacity (UBBC+cobalamin) in antibody. Marcoullis et aF reported that extracts of all extracts was clearly higher than that in the serums. human mucosa contained immunoreactive The colonic extracts showed higher levels of UBBC intrinsic factor and small amounts of R-protein and than those of the small intestine in all cases, and there TcII, which was possibly a contaminant from the was no difference between the levels of ileum and blood. Thus R-binder in the stomach seems to be in jejunum. rather small amounts, if any. R-binder might not be present in normal gastric mucosa and the main source Discussion of R-binder in gastric juice might be derived from swallowed saliva or from secretions of intestinal Our immunohistochemical study revealed that R- metaplastic mucosa which is found frequently in binder was present in almost all digestive organs. In adults. the oral cavity and the oesophagus, saliva and mucus Our results also suggest that the source of R-binder of the oesophagus contain R-binder secreted from in bile and pancreatic juice is from epithelial cells of the salivary glands and oesophageal glands. Nexo et the gall gladder, bile ducts and pancreatic ducts. R- a' showed, using an immunohistochemical tech- binder was present in the epithelium of the small and Gut: first published as 10.1136/gut.28.3.339 on 1 March 1987. Downloaded from

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Fig. 4 Immunoperoxidase 'staliingfor R-binder in the gall bladder. Positive staining is found in the epithelial cell. ofthe gall bldder. Methyl green counterstain. . TcII is reporteds to be present in the human intestinal mucosa. The results clearly showed intestine, whereas the R-binder has not yet been the presence of R-binder in human intestinal mucosa found to be present in the small or large intestinal even if there may be small amounts of contamination

mucosa. To confirm these findings, we measured of blood or R-binder derived from blood cells in the http://gut.bmj.com/ UBBC and cobalamin content in the extracts from mucosa. Staining grades in the immunohistochemical on September 26, 2021 by guest. Protected copyright.

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Fig. 5 Ilntnunoperoxidase stainitgJor R-binder in the panicreas. Positive staininig isfound in the epithelial cells of the pancreatic du ts. Meizthl greeni counterstain. Gut: first published as 10.1136/gut.28.3.339 on 1 March 1987. Downloaded from

344 Kuido, Inada, Ohshio, Wakatsuki, Ogawa, Hamnashima, and Miyake http://gut.bmj.com/ on September 26, 2021 by guest. Protected copyright. ~~in VP20

Fig. 6 Immuinolperoxidase stainitgfor R-binder in the inte.stine. (a) Small intestine, weakly positive staininlg isfounid in the columnar epithelial cells and goblet cells. (b) Large intestine, positive staining isfound in the coluimnar epithelial cells and goblet cells. (a) and (b), Methyl green counterstain.

study, which showed more intense staining for R- heavily in comparison with normal small intestinal binder in the colon than in the small intestine, were tissue. It is speculated that there may be larger paralleled with the data that UBBC and content of amounts of R-binder in the intestinal metaplastic cobalamin in the extracts from the colon were always tissue than in normal small intestinal tissue, however higher than those from the small intestine. In the the reason is uncertain. intestinal metaplastic tissue R-binder was stained A previous study` indicated, using the gel chroma- Gut: first published as 10.1136/gut.28.3.339 on 1 March 1987. Downloaded from

Immunohistochemical localisation ofvitamin B12 R-binder in the human digestive tract 345

Table Unsaturated vitamin B12 binding capacity (UBBC), Tcll and cobalamin content in the extractsfrom the human intestinie and in the sera

Case Age Sex Disease Material Value (pg/mg-protein) Cobalamnitn UBBC TcHl 1 42 Maile rcnal canccr Colon 88X5 122-6 3-9 Ilcum 62-2 11-1 2-9 Jcjunum 79-( 1()() 0- 2 64 Fcmailc utcruscanccr Colon 988X7 235-3 4-1 lcum 696-4 29-1 3-7 Jcjunum 747.7 31-0 2-7 3 54 Fcmalc utcrus canccr Colon 873-7 40-3 2-1 llcum 581X1 13-4 1-3 Jcjunum 182-9 8X2 1-6 4 83 Malc myocardial Colon 280-7 94-5 9-6 infarction Ilcum 291-0 4-5 2-7 Jcjunum 137-5 8-4 4-6 Normal voluntccr (n= It)) Serum 7-7±1-6 15-9±4-5 9-3±1-6

Valucs in scrum arc cxprcssed as mean± I SD. TclI=transcohalamin II. tographic technique for aspirated human intestinal '-related' (Tcl and TcllI) and 'liver-related' fluid, that R-binder of unknown origin was present in (Tcll) B12 binding by instant batch separation using a the intestinal lumen. Our results suggest that intes- microfine precipitate of silica (QUSO G32). J Lab Clin tinal epithelial cells secrete R-binder into the Med 1975; 86: 505-12. 6 Allen RH, Mehlman CS. Isolation of gastric Vitamin lumen. The role of R-binder remains uncertain, but B12-binding proteins using affinity chromatography. 1. it is suggested that R-binder excretes so called Purification and properties of human intrinsic factor. cobalamin analogues from the human body, and J Biol Chem 1973; 248: 3660-9. inhibits bacterial overgrowth.""' Intestinal R-binder 7 Marcoullis G, Grasbeck R. Vitamin B12-binding pro-

may protect the overgrowth of intestinal bacterial teins in human gastric mucosa: general pattern and http://gut.bmj.com/ flora, and at the same time it may protect the demonstration of intrinsic factor isoproteins typical of entrance of non-physiological cobalamin analogues mucosa. ScandJ Clin Lab Invest 1975; 35: 5-11. produced by intestinal microorganisms." 14 8 Chanarin I, Muir M, Hughes A, Hoffbrand AV. Evidence for intestinal origin of transcobalamin II during vitamin B,2 absorption. Br Med J 1978; 1: References 1453-5. 9 Marcoullis G, Nicolas JP, Parmentier Y, Jimenez M, I Ncxo E, Hausen M, Poulsen SS, Olsen PS. Characteri- Gerard P. A derivative of R-type zation and immunohistochemical localization of rat binding proteins in the human intestine. A candidate on September 26, 2021 by guest. Protected copyright. salivary cobalamin-binding protcin and comparison with antibacterial molecule. Biochem Biophys Acta 1980; human salivary . Biochem Biophys Acta 633: 289-94. 1985; 838: 264-69. 10 Allen RH. Human vitamin B1 transport proteins. Prog 2 Allen RH. Majerus PW. Isolation of vitamin B12- Hematol 1975; 9: 57-84. binding proteins using affinity chromatography. 1. 11 Donaldson RM. Intrinsic factor and transport of Preparation and properties of vitamin B112-binding cobalamin. In: Johnson LR, ed. Physiology of the Sepharose. J Biol (Chem 1972; 247: 7695-70(1. gastrointestinal tract. New York: Raven Press, 1981: 3 Burger RL, Allen RH. Characterization of vitamin B12- 64 1-58. binding proteins isolated from human and saliva by 12 Gullberg R. Possible antimicrobiol function of the large affinity chromatography. J Biol Chem 1974; 249: molecular size of Vitamin B ,-binding protein. Scand J 7220-7. Gastroenterol 1974; 9: suppl. 29: 19-21. 4 Gottlieb C, Lau KS, Wasserman LR, Herbert V. Rapid 13 Brandt LJ, Bernstein LH, Wagle A. Production of charcoal assay for intrinsic factor (IF), gastric juice Vitamin B12 analogues in patients with small-bowel unsaturated B12 binding capacity. Blood 1965; 25: bacterial overgrowth. Ann Intern Med 1977; 87: 546-51. 875-83. 14 Albert MJ, Mathan VI, Baker SJ. Vitamin B12 synthesis 5 Jacob E, Hcrbert V. Measurement of unsaturated by human intestinal . Nature 1980; 283: 781-2.