Measurement of vitamin B12-binding proteins of plasma. I. Technique

The unsaturated binding capacities (UBBC) of individual vitamin B12- binding proteins in plasma were measured by a two-step procedure. Transcobalamin II (TC II) was separated by precipitation with ammonium sulfate; the "R"-type binders remaining soluble were then divided into two components by bath separation with anion exchange on DEAE- cellulose. The two R components were designated alpha1-R (TC 1) and alpha2-R (third binder, fetal binder, PV binder, TC III). Ten normal sera were studied by this technique giving a separation into TC III and total plasma R identical to that obtained simultaneously by gel filtration. The mean UBBC of TC II was 969 plus or minus 204 pg of 57 Co B12 per ml of serum. The mean contamination of the precipitated TC III with plasma R was 3%. The UBBCs of alpha 2-R and alpha 1-R were 127 plus or minus 42 and 40 plus or minus 12 pg/ml, respectively. The mean contamination of the R fraction by TC II was 14% as evaluated by gel filtration. By isoelectric focusing it was found that the alpha1-R contained principally those components isoelectric at pH isoelectric at pH of 2.9-3.2, while alpha2-R was made up of those components isoelectric at pH of 3.6 or greater.     

Affinity chromatographic identification of vitamin B12-binding proteins in egg white

To identify vitamin B12-binding proteins in egg white, an affinity chromatographic isolation procedure was applied. A fraction tightly absorbed on vitamin B12 immobilized in agarose was eluted with 1 mM cyanocobalamin, and then separated by isoelectric focusing in sucrose density gradient. Two isolated proteins (or perhaps two forms of the same protein) of isoelectric points 6.2 and 7.2 and the same Mr 90,000 were found capable of detectable vitamin B12 binding. They are probably glycoproteins, each composed of a single polypeptide chain.     

The effect of proteolytic enzymes on the vitamin B12-binding proteins of human gastric juice and saliva.

Pepsin had no effect on the vitamin B12 binder in human saliva (R-binder), while trypsin was found to reduce the apparent molecular weight of the R-binder and to release vitamin B12 from the R-B12complex of human saliva and human gastric juice (HGJ). Trypsin had no effect on the molecular weight and biological activity of intrinsic factor (IF) in HGJ, as demonstrated by gel filtration on Sephadex G-150 and the uptake of IF-B12 by guinea pig intestinal brush borders. An extract of purified guinea pig intestinal lysosomes was also without effect on the molecular weight and the biological activity of IF but was found to release vitamin B12 from the R-B12 complex. The results support the observation that the external pancreatic secretion corrects malabsorption of vitamin B12 by an effect on the non-IF protein in the intestinal juice. Moreover, the results indicate that lysosomal enzymes are not involved in the intestinal absorption of vitamin B12.     

Vitamin B12 and vitamin B12 binding proteins in liver diseases

Serum vitamin B12 and vitamin B12 binding proteins (transcobalamins, TCS) were determined in patients with malaria, amoebic liver abscess, carcinoma of the liver, infectious hepatitis, cirrhosis and chronic myelocytic leukemia (CML) as well as in 60 blood donor subjects. Serum vitamin B12 in patients with infectious hepatitis, cirrhosis and CML were higher than that of the normal subjects. The values of unsaturated vitamin B12 binding capacity (UBBC) in patients with carcinoma of the liver, infectious hepatitis, cirrhosis were lower while that of patients with CML were higher than that of the normal subjects. A markedly increased TCI and decreased TCII was observed in patients with CML while these changes was much less in patients with other liver diseases. The difference was possibly due to a flooding of vitamin B12 from damaged liver cells into the circulation and the decreased synthesis of transcobalamins in patients with liver diseases while the increased granulocytes, the source of TCI, was much increased in patients with CML.     

Folic acid, vitamin B12 and vitamin B12 binding proteins in patients with neuroblastoma

Serum vitamin B12, serum and red cell folate and serum vitamin B12 binding proteins were determined in 18 patients with neuroblastoma, with ages ranging from 8 months to 14 years. A mean value of serum vitamin B12 level was slightly but not significantly lower than that of the normal subjects but all of them had serum vitamin B12 levels over 150 pg/ml. There was no relationship between serum vitamin B12 levels and hemoglobin, hematocrit or white cells. Transcobalamin I (TCI) was significantly increased resulting in slightly elevated UBBC and normal TBBC levels in these patients. This could be a compensatory mechanism for the low serum vitamin B12 by increasing the unsaturated vitamin B12 binding capacity of TCI. All these findings indicated that the status of vitamin B12 in patients with neuroblastoma was within the normal limits. Treatment of neuroblastoma by giving a high dose of vitamin B12 would therefore not give any direct therapeutic effect. Both serum and red cell folate concentrations were significantly lower in the group of patients. As only 2 out of 18 patients had low serum folate and none of them had red cell folate lower than the lower limit of normal subjects; therefore these patients were only in the state of negative folate balance.     

Eosinophilic granulocytes as a possible source of vitamin B12-binding protein

Leucocyte B12 and B12-binding capacity were measured by Simultrac radioassay in eosinophilic granulocytes, neutrophilic granulocytes and leucocytes obtained from patients with chronic granulocytic and lymphocytic leukaemia. It is shown that (a) eosinophils are a possible source of B12-binding protein similar to neutrophils and (b) granulocytes in myeloproliferative disorders and normal neutrophils have similar B12 and B12-binding capacity indicating that increased B12 and B12-binding capacity in myeloproliferative disorders arise from an increase in myeloid cell turnover.     

Serum vitamin-B12-binding proteins in kwashiorkor

Summary . In contrast with plasma albumin and transport proteins such as transferrin and ceruloplasmin, vitamin B₁₂-binding proteins are not decreased in Cape Coloured children with kwashiorkor. Compared with normal coloured adults the mean serum vitamin B₁₂ was increased while the unsaturated vitamin B₁₂ binding capacity (UBBC) was markedly increased mainly due to elevation of the alpha-1-globulin vitamin B₁₂ binder (alpha-binder). In normal coloured children the UBBC was also increased with mean alpha-binder intermediate between values found in kwashiorkor and normal coloured adults. The mean beta-globulin-vitamin B₁₂-binder (beta-binder) was decreased in kwashiorkor. The total serum alpha-1 and beta-globulins, showed poor quantitative correlation with alpha-and beta-vitamin B₁₂-binders in these patients. Urinary vitamin B₁₂-levels were similar in kwashiorkor, normal children and adults. In the small series of adult Cape Coloured subjects studied, serum vitamin B₁₂ and UBBC values were higher than in white adults. The alpha- and beta-binders of kwashiorkor and normal children adequately transferred ⁵⁷ Co-vitamin B₁₂ to rat liver homogenate while the alpha-binder of chronic myeloid leukaemia transferred less efficiently.     

Crohn's disease and vitamin B12 metabolism

The concentrations of vitamin B₁₂, its analogs, and the haptocorrin and transcobalamin carriers in 21 patients suffering from Crohn's disease and a group of controls (20 adults) were measured. There were no significant differences in the mean values for vitamin B₁₂, total corrinoids (vitamin B₁₂ + analogs), or vitamin B₁₂ or total corrinoids bound to haptocorrin or transcobalamin of the Crohn's and control patients. There was a significant increase in the binding capacity of transcobalamin in the Crohn's patients compared to the controls (P<0.001), but there was no difference in the binding capacities of haptocorrin. The serum concentrations of the markers of vitamin B₁₂ status, homocysteine and methylmalonic acid, showed an increase (P<0.01) in homocysteine in the Crohn's disease patients, but no change in methylmalonic acid. As the hyperhomocysteinemia was associated with normal folate concentrations, there may have been a defect in the activation of the enzyme due to altered intracellular vitamin B₁₂ status.     

The binding of vitamin B12 by serum proteins in normal and B12-deficient subjects

Endogenous B₁₂ in normal serum has been shown to be associated with α globulins (Pitney, Beard and Van Loon, 1954; Heinrich and Erdmann-Oehlecker, 1956; Mendelsohn, Watkin, Horbett and Fahey, 1958). When B₁₂ has been added to normal serum, however, most or all of the added B₁₂ has been associated with the β- and α₂-globulins (Miller, 1958). In the present investigation with radioactive ₅₇cobalt cyanocobalamin (B₁₂*) added to serum, it was possible to define at least two B₁₂-binding proteins (BP), a β-globulin, and an α₁ globulin. B₁₂* added to the serum of normal subjects migrated mainly with the β-globulins on electrophoresis, but when added to the serum of B₁₂-deficient subjects, a small fraction of the B₁₂* was also associated with the α₂-globulins.