Antibodies, intrinsic factor

Pernicious anemia arises when vitamin B,2 deficiency blocks the metabohsm of folic acid, leading to functional folate deficiency. This impairs erythropoiesis, causing immature precursors of erythrocytes to be released into the circulation (megaloblastic anemia). The commonest cause of pernicious anemia is failure of the absorption of vitamin B,2 rather than dietary deficiency. This can be due to failure of intrinsic factor secretion caused by autoimmune disease of parietal cells or to generation of anti-intrinsic factor antibodies. 

This case prompted a report of 10 metformin- associated patients with cobalamin deficiency among 162 patients with vitamin Bi2 concentrations below 200 pg/ml (91). They had taken a mean dose of metformin of 2015 mg/ day for an average of 8.9 years. The mean vitamin B12 concentration was 140 pg/ml. All had normal serum folate and creatinine concentrations and no antibodies to intrinsic factor. In one patient there was malabsorption. 

M4. Moestrup, S. K., Kozyraki, R., Kristiansen, M., Kaysen, J. H., Rasmussen, H. H., Brault, D., Pontillon, F., Goda, F. O., Christensen, E. I., Hammond, T. G., and Verroust, P. J., The intrinsic factor-vitamin B12 receptor and target of teratogenic antibodies is a megalin-binding peripheral membrane protein with homology to developmental proteins. J. Biol. Chem. 273, 5235—5242 (1998). 

Although measurement of the concentration of vitamin B12 in serum is the most widely used test for the investigation of a possible deficiency, there are a number of other tests used to measure absorption, the integrity of the methylmalonyl metabolic pathway, and the presence of antibodies to intrinsic factor. These additional tests are also described in this section. 

Examination of the bone marrow, although important, will only confirm that the hemopoiesis is megaloblastic. A deficiency of folic acid will also cause a megaloblastic anemia and it is not possible to identify the cause on the basis of morphology. A serum assay of both vitamins will usually indicate which is responsible. If the patient is vitamin B12 deficient, the next step is to carry out a vitamin B12 absorption test to confirm that the deficiency is due to a lack of intrinsic factor. Preferably this should not be done until the patient s vitamin B12 and hemoglobin levels have returned to normal, since the gastric and intestinal cells are also affected by a lack of vitamin B12 aborption may be less than optimal if it is attempted too early. Patients with pernicious anemia also have a histamine-fast achlorhydria and gastric atrophy. The disease appears to have an autoimmune basis and antibodies to intrinsic factor can be demonstrated in the serum of more than half of affected patients. 

This condition has often been referred to in the past as juvenile pernicious anemia but it appears to be a quite separate entity. Confusion probably arose because there is a deficiency of intrinsic factor resulting in vitamin B12 malabsorption in both conditions. However, it differs from the disease in adults in that free acid is present in the gastric secretion (A8,L3,M5), the gastric mucosa is usually normal, and antibodies to intrinsic factor are not a feature. Megaloblastic anemia usually develops during the first 2 years of life but this depends on the amount of residual intrinsic factor available, and... 

Patients with severe atrophic gastritis may have impaired absorption of vitamin B12 and a reduced serum level of the vitamin, but this is not accompanied by either megaloblastic anemia or neuropathy. Parietal-cell antibodies have been found in 33% of patients with gastritis, none of these patients having pernicious anemia (12, V4). Intrinsic factor antibodies were not found, and this was not surprising since it is rare to find antibodies to intrinsic factor in the absence of pernicious anemia. Patients with superficial gastritis usually have normal vitamin B12 absorption and normal serum levels of the vitamin. 

G6. Gottlieb, C., Lau, K. S., Wasserman, L. R., and Herbert, V., Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated B12 binding capacity, antibody to IF, and serum unsaturated B12 binding capacity. Blood 25, 875-883 (1965). 

Isokoski, M., Krohn, K., Varis, K., and Siurala, M., Parietal cell and intrinsic factor antibodies in a Finnish rural population sample. Scand. ]. Gastroenterol. 4, 521-527... 

Schwartz, M., Intrinsic factor antibody in serum from patients with pernicious anaemia. Lancet 2, 1263-1267 (1960). 

Schilling s test assesses the oral absorption of vitamin B12 and is used to diagnose pernicious anaemia. The patient is injected intramuscularly with non-labelled vitamin B12, to saturate body stores. An oral dose of vitamin B12 labelled with cobalt-58 is administered, followed by a second dose labelled with cobalt-57 bound to intrinsic factor. Prior saturation of body stores ensures any absorbed radiolabelled vitamin B12 is rapidly excreted in the urine. Urinary excretion of orally administered vitamin B12 is low in patients with pernicious anaemia due to poor absorption. Absorption is increased when it is administered with intrinsic factor. The ratio of cobalt-57 to cobalt-58 is thus raised in patients with pernicious anaemia. Intrinsic factor antibody testing is now generally used to diagnose pernicious anaemia, though the Schilling s test may occasionally be used. 

Failure of intrinsic factor secretion is commonly a result of autoimmune disease 90% of patients with pernicious anemia have complement-fixing antibodies in the cytosol of the gastric parietal cells. Similar autoantibodies are found in 30% of the relatives of pernicious anemia patients, suggesting that there is a genetic basis for the condition. 

About 70% of patients also have antiintrinsic factor antibodies in plasma, saliva, and gastric juice. These canbe either blocking antibodies, which prevent the binding of vitamin B12 to intrinsic factor, or precipitating antibodies, which precipitate both free intrinsic factor and intrinsic factor-vitamin B12 complex. Some patients have both types of antiintrinsic factor antibody. Although the oral administration of partially purified preparations of intrinsic factor will restore the absorption of vitamin B12 in many patients withpernicious anemia, this can eventually result in the production of antiintrinsic factor antibodies, so parenteral administration of vitamin B12 is the preferred means of treatment. 

For patients who secrete antiintrinsic factor antibodies in the saliva or gastric juice, oral intrinsic factor will be of no benefit. 

A number of methods have been developed to permit assessment of folate and vitamin B12 nutritional status and to differentiate between deficiency of the vitamins as a cause of megaloblastic anemia. Obviously, detection of antibodies to intrinsic factor or gastric parietal cells will confirm autoimmune pernicious anemia rather than nutritional deficiency of either vitamin. 

The absorption of vitamin B12 can be determined by the Schilling test. An oral dose of [ Co] or [ Co]vitamin B12 is given with a parenteral flushing dose of 1 mg of nonradioactive vitamin, and the urinary excretion of radioactivity is followed as an index of absorption of the oral material. Normal subjects excrete 16% to 45% of the radioactivity over 24 hours, whereas patients lacking intrinsic factor or with antiintrinsic factor antibodies excrete less than 5%. 

The test can be repeated, giving intrinsic factor orally together with the radioactive vitamin B12 - if the impaired absorption was because of a simple lack of intrinsic factor, and not to antiintrinsic factor antibodies in saliva or gastric juice, then a normal amount of the radioactive material should be absorbed and excreted. 

The vitamin B12 receptor which facilitates uptake of the vitamin-intrinsic factor vitamin-binding protein complex has been used to enhance oral delivery and gastrointestinal uptake of peptides and proteins as their vitamin B12 conjugates (37). Commercial efforts are under way to exploit this receptor as well as the fetal Pc receptor which facilitates intestinal uptake of antibodies from colostrum/milk (38) and the polymeric immunoglobulin receptor which facilitates the serosal to mucosal transport of IgA and IgM (39). 

Antibodies to intrinsic factor-active materials from human and hog stomach have been demonstrated in the sera of pernicious anemia patients by Taylor et al. (T9-T12) and Schwartz (S12, S13), and of rabbits immunized with these materials by Lowenstein et al. (L13). Antibody formation in rabbits is species specific, although antibodies have shown some cross-reactions. An antigenic factor common to human and hog material containing intrinsic factor probably produces formation of these antibodies (G75-G75b). 

Circulating antibodies to hog intrinsic factor were also demonstrated in two pernicious anemia patients immunized with purified hog intrinsic factor concentrate (K5). Antibodies developed in their sera, which precipitated hog intrinsic factor in various immune reactions, immobilized it on electrophoresis, and depressed or abolished its enhancing effect upon vitamin B12 absorption in the intestine of other pernicious anemia patients. 

In addition to these, another antibody to intrinsic factor may develop in a certain percentage of pernicious anemia patients, orally treated with hog intrinsic factor concentrate. As shown by Schwartz (S12, S13), this antibody is directed exclusively toward hog intrinsic factor concentrate and not human gastric juice or intrinsic factor derived from the human stomach. Apparently, it causes a topical antibody reaction at the mucosal level, hlocking the promoting action of hog intrinsic factor concentrate on intestinal absorption of vitamin B12 in these pernicious anemia patients. This produces refractoriness to oral treatment with vitamin B12 and hog intrinsic factor concentrate in many pernicious anemia patients (S15). 

A complement-fixing antibody in the serum of pernicious anemia patients, diabetics, and those widi iron-deficiency anemia has been detected against mucosal extracts of the fundus and body of the hmnan stomach and the microsomal fraction of gastric mucosal extracts (D8, 14, M8, M48, RIO, TI3). Immunofluorescent studies have shown that activity is directed against gastric parietal cell cytoplasm (15, RIO, T13) and probably represents another type of antibody, which develops about twice as frequently as the one directed against intrinsic factor in pernicious anemia serum (Fig. 27). 

Thus, in addition to an antibody to the human intrinsic factor or the Bi2-binding principle, another antibody forms in pernicious anemia. 

Fig. 27. Correlation of results of tests for parietal cell and IF antibodies in pernicious anemia patients, showing their independence. The parietal cell antibodies were tested by complement fixation test (CF) while the IF antibodies were assayed by electrophoretic retention test, i.e., immobilization of the intrinsic factor related Bj2 binder on electrophoresis. From Taylor et al. (T13).

Al. Abels, J., Bouma, W., Jansz, A., Woldring, M. G., Bakker, A., and Nieweg, H. O., Experiments on the intrinsic factor antibody in serum from patients with pernicious anemia. /. Lab, Clin. Med. 61, 893-906 (1963). 

A9a. Ardeman, S., and Chanarin, I., A method for the assay of human gastric intrinsic factor and for the detection and titration of antibodies against intrinsic factor. Lancet 2, 1350-1354 (1963). 

G75. Gullberg, R., and Kistner, S., A study of precipitating antibodies against human intrinsic factor. Acta Med. Scand. 172, 385-388 (1962). 

J7. Jeffries, C. H., Hoskins, D. W., and Sleisenger, M. H., Antibody to intrinsic factor in serum from patients with pernicious anemia. J. Clin. Invest. 41, 1106-1115 (1962). 

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