Glut, 1987, 28, 339-345

Immunohistochemical localisation of vitamin 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 of Geriatric Medicine and Pathology, Kyoto University, Kyoto, Japan

SUMMARY The distribution of vitamin B12 R-binder in the human digestive tract was studiedusing an indirect immunoperoxidase technique. Positive staining for R-binder was found in themucous cells and ductal epithelial cells of the salivary glands and the oesophageal glands. In normalgastric mucosa, no positive staining for R-binder was found, but in the area with intestinalmetaplasia, the columnar epithelial cells and goblet cells showed positive staining. Epithelial cellsof the gall bladder, intrahepatic bile ducts and pancreatic ducts were also positive for R-binder. Inthe small intestine 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 theextracts from intestinal mucosa also indicated the presence of R-binder in the intestinal mucosa.

R-binder, an immunologically distinct vitamin B12binding protein, is ubiquitous in human serum, manyblood cells and various body fluids. In the digestivetract, R-binder is present in saliva, gastric juice, bile,and pancreatic juice, but its function is still a matterof speculation. Although the localisation of R-binderin the salivary gland is studied in one report,' its tissuedistribution must be studied in more detail to deter-mine its role.We studied the distribution of R-binder in the

human digestive tract using the immunoperoxidasetechnique. In addition, we measured unsaturatedvitamin B12 binding capacity (UBBC) and endo-genous cobalamin in the extracts from the humanintestinal mucosa to confirm the results of theimmunohistochemical study.

Methods

TISSUE PREPARATION

Specimens of the gastrointestinal tract and the gallbladder were obtained surgically from patients who

Address for correspondence Dr H Kudo, Dcpartmcnt of Geriitric Medicine.Fdtculty of Medicine, Kyoto University Sakyo-ku, Kvoto 606, JlapinReceived for publicition 29 June 1986I

had resections for gastric ulcer, gastric cancer,leiomyoma and perforation of the small intestine,colon cancer and cholelithiasis. Specimens of themajor salivary gland, liver and pancreas wereobtained at necropsy.

Macroscopically normal tissue samples wereremoved and fixed in 10% formalin and embedded inparaffin. Four micron thick sections were cut anddeparaffinised in xylene and ethanol series. Forroutine histological examination each section wasstained with haematoxylin-eosin, and histologicallynormal sections were used to detect R-binder.

ANTISERUMR-binder was purified from pooled human salivausing affinity chromatography according to themethod of Allen et al.3 Purified R-binder showeda single protein band by SDS-polyacrylamide gelelectrophoresis. Anti R-binder antiserum was pre-pared in rabbits. Specificity of the antiserum wasconfirmed by Ouchterlony's immunodiffusionmethod and immunoelectrophoresis.

IMMUNOPEROXIDASE TECHNIQUEIndirect peroxidase technique was used. Deparaffin-ised tissue sections were treated with 0/3% hydrogen

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peroxide in methanol for 30 minutes in order to blockthe endogenous peroxidase activity. Sections wereprecoated with 5%° normal goat serum for 20 minutes.and then incubated with anti R-binder antiserum at adilution of 1:400 with 0. 1 M phosphate bufferedsaline (PBS), pH 7-4, containing 0(5% bovine serumalbumin, at 4°C overnight, followed by peroxidaseconjugated goat antirabbit IgG antiserum (Cappel,USA) at a dilution of 1:80; at room temperature for40 minutes. After each application of antiserum, thesections were washed with PBS three times for 1i)minutes each time. The histochemical reaction forperoxidase was carried out using 3.3'-Diaminobenzi-dine (0.05% w/v) and hydrogen peroxide (0.01%) in0(05 M Tris-HCI buffer, pH 7-6. In control prepara-tions, the primary antiserum was replaced by normalrabbit serum or by specific antiserum preincubatedwith an excess of purified R-binder: control stainingswere all negative.

EXTRACTION OF THE INTESTINAL MUCOSA

Samples of the large and small intestine wereobtained from four necropsy cases within two hoursafter death. In each case, a part of the colon, jejunumand ileum, which showed macroscopically normalmucosa, were removed, washed with saline andfrozen immediately at -70°C.

After thawing, the mucosa was scraped off, mixedwith an equal volume of 0()1 M potassium phosphate

buffer containing 0()15 M NaCl, pH 7-4, and homo-genised for five minutes. The homogenate was centri-fuged at 15 (00 g for 30 minutes and the supernatantwas collected.

ASSAY OF UBBC, TRANSCOBAI,AMIN II (TCII),ENDOGFNOUS COBAILAMIN AND PROTEINCONTENT IN THFF,XTRACTSUnsaturated vitamin B12 binding capacity wasmeasured by the technique of Gottlieb et al,4 TcII byabsorption to a microfine precipitate of Silica.'Endogenous cobalamin was assayed by vitamin B12[5Col radioassay (Corning, USA, lot 63015). Proteincontent was measured using Bio Rad Protein Assay(Bio Rad, USA). UBBC, TcII and cobalamin con-tent in the sera of 10 healthy volunteers were alsoassayed for comparison.

Results

IMMUNOHISTOCHFMISTRYIn all sections, R-binder positive cells were stainedwith a clear contrast. Gastrointestinal tract tissuesexamined contained various numbers of polymor-phonuclear leucocytes or histocytes, which served asuseful positive controls in judgment.

In the salivary glands, R-binder positive reactionwas found in the cytoplasm of mucous cells andintercalated duct cells. Myoepithelial cells and serous

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Fig. 1 Immunoperoxidase siaitjitigfor R-bitnder in 1the1w .slu ancdibilarglatnd. Posilive staining isfolunld in thei mnucous (ell/s a(0( intercalated duct (ell/s. Met/h i greea counltrslainl.

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lItinmuinohistochemical localisation ofvitamin B] 2R-binder in the human digestive tract

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 thecolumnar epithelial cells and goblet cells. (a) and (b), methyl green counterstain.

cells were negative (Fig. 1). Similarly, mucous cellsand ductal epithelial cells of the oesophageal glandsshowed positive staining for R-binder.

In normal gastric mucosa, no positive staining forR-binder was found except for small numbers of

polymorphonuclear leucocytes. In contrast, in thearea with intestinal metaplasia, there was alwaysintense staining for R-binder in both columnarepithelial cells and goblet cells (Fig. 2).Columnar epithelial cells of the gall bladder,

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

Fig. 3 Immunoperoxidase stainling Jor R-bindetr in thlie liver. Positive staininlg isjountd inltheepithelial cells ofititrahepatic bile ducts. Metli ill greeni eounterstain.

intrahepatic bile ducts and pancreatic ducts were alsopositive for R-binder. Hepatocytes and acinar com-ponents of the pancreas were negative for R-binder(Figs 3-5).

In the small intestine and colon, R-binder wasfound in the columnar epithelial cells and gobletcells. The epithelium of the small intestine wasstained weakly (Fig. 6).

UBBC TCII AND COBAILAMIN CONTENT IN THE

EXTRACTThe Table shows the UBBC, TcII and cobalamincontent in the extracts of each case. In all cases therewas detectable UBBC and TcII. The level of totalvitamin B 12 binding capacity (UBBC+cobalamin) inall extracts was clearly higher than that in the serums.The colonic extracts showed higher levels of UBBCthan those of the small intestine in all cases, and therewas no difference between the levels of ileum andjejunum.

Discussion

Our immunohistochemical study revealed that R-binder was present in almost all digestive organs. Inthe oral cavity and the oesophagus, saliva and mucusof the oesophagus contain R-binder secreted fromthe salivary glands and oesophageal glands. Nexo eta' showed, using an immunohistochemical tech-

nique, that R-binder was present both in the mucoussecretory acini and in the intercalated ducts of thehuman salivary glands. Although they used anti-hogR-binder antiserum as the primary antiserum, ourfindings obtained using antihuman R-binder anti-serum were consistent with theirs.

In normal gastric mucosa, R-binder was not foundin the surface epithelium or in the secretory glands,but in the area with intestinal metaplasia, the colum-nar epithelial cells and goblet cells showed positivestaining for R-binder. Allen et aP reported thatgastric juice of 11 individuals had ranged from89-100%, in terms of percentage of inhibition ofvitamin B12 binding activity by anti-intrinsic factorantibody. Marcoullis et aF reported that extracts ofhuman stomach mucosa contained immunoreactiveintrinsic factor and small amounts of R-protein andTcII, which was possibly a contaminant from theblood. Thus R-binder in the stomach seems to be inrather small amounts, if any. R-binder might not bepresent in normal gastric mucosa and the main sourceof R-binder in gastric juice might be derived fromswallowed saliva or from secretions of intestinalmetaplastic mucosa which is found frequently inadults.Our results also suggest that the source of R-binder

in bile and pancreatic juice is from epithelial cells ofthe gall gladder, bile ducts and pancreatic ducts. R-binder was present in the epithelium of the small and

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.)-~~~~~~~~~~~~~~~~~~~~~~~~~~~~M

Fig. 4 Immunoperoxidase 'staliingfor R-binder in the gall bladder. Positive staining is found in theepithelial cell. ofthe gall bldder. Methyl green counterstain.

large intestine. TcII is reporteds to be present in theintestine, whereas the R-binder has not yet beenfound to be present in the small or large intestinalmucosa. To confirm these findings, we measuredUBBC and cobalamin content in the extracts from

human intestinal mucosa. The results clearly showedthe presence of R-binder in human intestinal mucosaeven if there may be small amounts of contaminationof blood or R-binder derived from blood cells in themucosa. Staining grades in the immunohistochemical

11~;2,

Fig. 5 Ilntnunoperoxidase stainitgJor R-binder in the panicreas. Positive staininig isfound in theepithelial cells of the pancreatic du ts. Meizthl greeni counterstain.

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~~in 20VP

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

study, which showed more intense staining for R-binder in the colon than in the small intestine, wereparalleled with the data that UBBC and content ofcobalamin in the extracts from the colon were alwayshigher than those from the small intestine. In theintestinal metaplastic tissue R-binder was stained

heavily in comparison with normal small intestinaltissue. It is speculated that there may be largeramounts of R-binder in the intestinal metaplastictissue than in normal small intestinal tissue, howeverthe reason is uncertain.A previous study` indicated, using the gel chroma-

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

Table Unsaturated vitamin B12 binding capacity (UBBC), Tcll and cobalamin content in the extractsfrom the humanintestinie 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 3-9Ilcum 62-2 11-1 2-9Jcjunum 79-( 1()() 0-

2 64 Fcmailc utcruscanccr Colon 988X7 235-3 4-1lcum 696-4 29-1 3-7Jcjunum 747.7 31-0 2-7

3 54 Fcmalc utcrus canccr Colon 873-7 40-3 2-1llcum 581X1 13-4 1-3Jcjunum 182-9 8X2 1-6

4 83 Malc myocardial Colon 280-7 94-5 9-6infarction Ilcum 291-0 4-5 2-7

Jcjunum 137-5 8-4 4-6Normal 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 intestinalfluid, that R-binder of unknown origin was present inthe intestinal lumen. Our results suggest that intes-tinal epithelial cells secrete R-binder into thelumen. The role of R-binder remains uncertain, butit is suggested that R-binder excretes so calledcobalamin analogues from the human body, andinhibits bacterial overgrowth.""' Intestinal R-bindermay protect the overgrowth of intestinal bacterialflora, and at the same time it may protect theentrance of non-physiological cobalamin analoguesproduced by intestinal microorganisms." 14

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