Intrinsic factor assay

Radioisotope dilution assays are based on the principle of competition between radioactive labeled ( Co) vitamin B 2 and cobalamins extracted from matrices for binding sites on the intrinsic factor (a glycoprotein). Binding is in proportion to the concentration of the radioactive and nonradio active B 2 with the concentration of intrinsic factor as the limiting factor. Free cobalamins are separated from those bound on the intrinsic factor by absorption... 

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. 

E4. Ellenbogen, L., and Williams, W. L., Improved urinary excretion test for assay of intrinsic factor. II. Sensitive counting technic. Proc. Soc. Exp. Biol. Med. 91, 617-619 (1956). 

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). 

Castro-Curel, in our laboratory, using an in vitro assay of intrinsic factor activity on guinea pig intestinal mucosal homogenates, demonstrated almost complete loss of intrinsic factor, however, by acid human gastric juices if stored for over 2 weeks, even in a frozen state (CI2, C13). 

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).

Ala. Abels, J., Bouma, W., and Nieweg, H. O., Assays of intrinsic factor with anti-intrinsic factor serum in vitro. Biochim. Biophys. Acta 71, 227-229 (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). 

C12. Castro-Curel, Z., and Glass, G. B. J., Assay of intrinsic factor activity on guinea pig intestinal mucosa homogenate. Proc. Soc. Exptl. Biol. Med. 112, 715-723 (1963). 

E3. Ellenbogen, L., Purification, properties and assay of intrinsic factor. Vitamin Bj2 und Intrinsic Factor, 2. Europ. Symp., Hamburg, 1961 pp. 443-456. Enke, Stuttgart, 1962. 

One of the most common explanations for unresponsiveness of apparently estrogen receptor positive tumors to hormonal therapy is tumor heterogeneity. We found that 38% of the results change when comparing the estrogen receptor proteins from the primary tissue with those from the metastatic tissue. Although there are extrinsic factors, as already mentioned, there could be intrinsic factors that alter the receptor measurement. These findings underscore the need to conduct biopsies and assay readily accessible recurrences rather than to rely on the results of prior specimens. Variation of results also occurred with metastatic tissue from some patients, obtained usually after a period of 19 months (R4). 

Fbr the radioassay of vitamin B-12 a cobalt-57 tag is usually used. Most vitamin B-12 assays are not immunoassays a thermolabile glucoprotein, intrinsic factor, is used as a binder rather then an antibody. Such assays are therefore called "radioassays" but not "radioimmunoassays". Ting and Helman (1972c) describe an assay of this type. Gutcho et al (1973) also describe a vitamin B-12 assay and also discuss a method for optimizing a liquid scintillation counter for the measurement of cobalt 57. 

Major vitamin Bi2-dependent metabolic processes include the formation of methionine from homocysteine, and the formation of succinyl coenzyme A from methylmalonyl coenzyme A. Thus, apart from directly determining vitamin B12 concentration in serum, elevated levels of both methylmalonic acid and homocysteine may indicate a vitamin B12 deficiency. Serum cobalamine concentration is often determined by automated immunoassays using an intrinsic factor as binding agent. These assays have mainly replaced the microbiological methods. Literature data about vitamin B12 concentration in serum varies. Values <110-150pmoll are considered to reflect deficiency, whereas values >150-200pmoll represents an adequate status. 

Cell fractionation studies of homogenates of the gastric mucosa have demonstrated that the intrinsic factor is found both in mitochondria and the supernatant fluid. The factor remains difficult to assay a variety of methods have been used, but none is entirely satisfactory. These methods include (1) administration of intrinsic factor preparation to gastrectomized animals (rats or hogs) (2) measurement of vitamin 6 2 binding capacity of the preparation (3) measurement of vitamin Bi2 uptake in tissue and (4) evaluation of the inhibition of cobamide coenzyme activity in the glutamate isomerase reaction. 

In humans, the two principal methods used for assaying the intrinsic factor are the reticulocyte response after administration of the intrinsic factor to pernicious anemia patients, and studies of the effect of intrinsic factor on urinary or fecal excretion, on hepatic uptake, on blood plasma radioactivity, and total body radioactivity after the administration of labeled vitamin B12. 

Bouma W, Nieweg HO. Assay of intrinsic factor with anti-intrinsic factor serum in vitro. 

To our knowledge, all commercially available assays today employ intrinsic factor as the binding protein. The challenge with this is that the most commonly used source of intrinsic factor is the porcine stomach, a source that contains a substantial amount of haptocorrin in addition to intrinsic factor. Because of this, care is needed to remove haptocorrin prior to using porcine gastric intrinsic factor. It seems obvious that, in order to standardize assays for cobalamin across the analytical platforms, the use of recombinant intrinsic factor is relevant. Recombinant human intrinsic factor is available and has... 

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