Improved method for measuring vitamin B₁₂in serum using intrinsic factor,⁵⁷CoB₁₂, and coated charcoal

Abstract

SYNOPSiS An improved and simplified method is described for the measurement of vitamin B12 in serum using intrinsic factor, 57CoB12, and coated charcoal. The extraction of serum in the presence of cyanide and the incorporation of B12-deficient serum into the intrinsic factor control has increased the accuracy of the method for both sera and crystalline B12 solutions. There are interesting differences between the results obtained for some sera by the isotope and L. leichmannii methods and the reasons for these differences are discussed.A method for the measurement of vitamin B12 (B12) in serum by radioisotope dilution using intrinsic factor, 57CoB12, and coated charcoal was described be Lau, Gottlieb, Wasserman, and Herbert (1965). With some modifications this method appeared to be satisfactory for the routine assay of B12 in serum (Raven, Walker, and Barkhan, 1966) but further experience with the method in the assay of many hundreds of sera has brought to light several discrepancies. It was found (1) that for occasional sera, irrespective of their B12 values, the isotope method gave falsely low values and in the case of some B12-deficient sera even negative values; (2) sera with B12 levels greater than 500 to 600 pg/ml gave lower values in the isotope method than in the microbiological (L. leichmannii) method; (3) falsely low values were obtained when crystalline B12 solutions were assayed.We studied the reasons for these anomalous results and found that there were two factors affecting the accuracy of the method. The first was that the use of cyanide in the extraction process resulted in higher values for those sera with B12 levels over 500 pg/ml (Raven, Walker, and Barkhan, 1967;Raven, Robson, Walker, and Barkhan, 1968) and the second that serum increased the binding of B12 by intrinsic factor (Rothenberg, 1961;Raven et al, 1968). By the incorporation of B12-deficient serum into the intrinsic factor control it was possible to eliminate the problem of falsely low values found previously in the assay of crystalline solutions and of some sera (Raven et al, 1968).As a result of these observations the radioisotope method has been improved and simplified and in this 180 ,uc/ug were tested and all were found to be satisfactory. Those of low specific activity (10 to 15 uc/,ug) were used in a concentration of 1,000 pg/ml, while those of high specific activity (70 to 180 uc/ug) were used in a concentration of 250 pg/ml. The increased cost of the high specific activity preparations is outweighed by their advantages in providing higher count rates in the samples and an increase in sensitivity of the method at low serum B12 levels; the high specific activity 57CoB12 is recommended for routine assays. Each batch of 57CoB12 is diluted to the required concentration according to the Amersham values and the B12 concentration of the diluted solution checked by a microbiological method. When the isotope method is working satisfactorily it may be used for checking new batches of 57CoB12 solutions and there is no need to rely on...

Vitamin B 12 binding by intrinsic factor.

“…The results shown: of a typical experiment are Raven, Robson, Walker, and Barkhan (1969) for measurement of serum vitamin B12 levels, the pH of the extract after autoclaving serum plus hydrochloric acid is 1-6. In the radioimmune assay of Ardeman and Chanarin (1963)forintrinsic factor and for type II antibody to intrinsic factor the pH remains batween 7 and 8.…”

Experience with a commercial kit for the radioisotopic assay of vitamin B12in serum: the Phadebas B12Test

“…The reproducibility of the Phadebas method both within a single assay and in different assays was similar to that of the method of Raven et al (1969).…”

“…Thirty-three of the sera were from patients with untreated pernicious anaemia, the diagnosis having been proven by peripheral blood and bone marrow examination, serum vitamin B12 and folate assays, 59 . ; J. L. Raven for 526 sera by the Phadebas method and the radioisotopic method of Raven et al (1969).…”

“…Fifty sera were assayed by the Phadebas method using the recommended three-hour incubation time Phadebas extraction process One hundred and six sera were assayed by the radioisotopic method of Raven et al (1969) using the N/4 HCl extraction process employed by that method and the assays were repeated by the same method, modified to incorporate the glutamic acid extraction process used by the Phadebas assay. There was little difference between the results obtained with the two extraction processes (fig 6).…”

Assay of vitamin B 12 .

“…have reported that the radioisotopic method of Raven, Robson, Walker, and Barkhan (1969) Into some 270 pages, Dr Bernard Knight has packed a mass of information primarily of value to the general practitioner or junior hospital practitioner but by no means confined to them. Most pathologists will find in this book a great deal of valuable information, easily obtainable through an excellent index.…”