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...
A novel deletion of approximately 27 kb with the 5′ breakpoint 1.5 to 2.2 kb upstream of the beta-globin gene, and the 3′ breakpoint approximately 24 kb downstream of the beta-globin gene, has been found in five members of two families from Southeast Asia (Vietnam and Cambodia). Six members of another family from China, previously reported from our laboratory, have also been shown to carry this deletion. The patients presented with mild hypochromia and microcytosis, a hemoglobin (Hb) A2 level of approximately 4.0%, and a markedly increased, heterocellularly distributed, Hb F level (14.0 to 26.0%). In vitro globin-chain synthesis showed a mild imbalance with appreciable gamma-chain compensation (alpha/beta + gamma ratio of 1.46). The 3′ end of this deletion includes the 3′HS-1, and we hypothesize that removal of this region results in the loss of its gamma-globin gene-silencing effect, which causes a markedly elevated Hb F level with a modest increase in Hb A2 levels, unlike the situation in other deletional beta zero-thalassemias. The possible influence of particular sequence variations in the locus control region 5′HS-2 and the G gamma promoter, present on the chromosome with this deletion, on the overall gamma-globin gene should also be considered.
The partial molecular characterization of a large deletion present in two members of an Indonesian-Malay family with beta-thalassemia trait is described. Polymerase chain reaction and sequencing analyses of the breakpoint identified a sequence which has previously been described in patients with the 45 kb Filipino beta 0-thalassemia deletion, i.e. a 5' breakpoint at position -4279 nucleotides 5' from the Cap site of the beta-globin gene. The 3' breakpoint is located in an L1 family of repetitive sequences at an unknown distance from the beta-globin gene. The hematological and hemoglobin data of the patients with this beta 0-thalassemia deletion further supports the concept that the unusually high Hb A2 levels are unique to deletions removing the 5' beta-globin gene region, and points to the importance of the 3' junction sequences for the regulation of Hb F levels in patients with deletional defects of the beta-globin gene cluster.
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