J. Van Kapel

Uptake of transcobalamin II-bound cobalamin by HL-60 cells: Effects of differentiation induction

Binding and uptake of transcobalamin II-bound cobalamin by HL-60 promyelocytic leukemia cells proceed through receptor-mediated endocytosis. The affinity constant of the receptor for transcobalamin II-cobalamin was found to be 6.1 liter/nmol and the maximal rate of uptake 12 pmol/10(9) cells/h. This uptake is mediated by about 3000 receptor sites per cell. Evidence is presented that the receptor recirculates from the cell surface to the lysosomes and vice versa. Upon differentiation induction of the cells by either DMSO in granulocytic direction or by 1,25-dihydroxy-vitamin D3 in monocytic direction a rapid decline in cellular uptake and cell surface binding of the protein-bound vitamin ensues. In particular the internalization of the complex decreases faster than all other observed signs of the ongoing differentiation process, such as reduction in the OKT9-reactive transferrin receptor, increase in lineage-specific surface markers, and decrease in [3H]thymidine incorporation and actual cell proliferation. The transcobalamin II receptor on the cell surface appears to be a proliferation-associated membrane component in human leukemic cells.

Application of a simple immunoadsorption assay for the measurement of saturated and unsaturated transcobalamin II and R-binders

In this paper an immunoadsorption method for the selective measurement of the concentrations of saturated and unsaturated transcobalamin II and R-binders in human plasma is presented. With this assay the concentrations of saturated transcobalamin II found in the plasma of 70 normal individuals ranged from 20-220 pmol per litre, with a mean of 70 pmol/l. The concentration of R-binders, carrying cobalamin, ranged from 87-491 pmol/l (mean 195 pmol/l). The 33-203 pmol/l (mean 111 pmol/l) of cobalamin analogues were found to be almost exclusively bound to the R-binder fraction. The level of saturated binding proteins is not, or only marginally, influenced by the absorption of ingested cobalamin, even with an oral dose of 15 micrograms. The concentration of transcobalamin II-bound cobalamin is apparently not determined by the availability of unsaturated binding protein. On the contrary, a positive correlation was found between the R-binder-cobalamin concentration and total R-binder level. These observations suggest that the concentration of transcobalamin II-bound cobalamin is primarily determined by the concentration of cobalamin in the tissues.

Uptake of transcobalamin II-bound cobalamin by isolated rat kidney tubule cells

The uptake and intracellular processing of transcobalamin II-bound cobalamin by isolated rat kidney tubule cells were studied. The cells absorbed the complex in a temperature-and calcium-dependent process, which could be inhibited by monensin, an inhibitor of endocytosis. Cells, loaded with a mixture of 125I- and 57Co-labelled transcobalamin II-vitamin B12, released 125I-labelled protein-degradation products, while keeping the 57Co-labelled vitamin. Protein degradation was inhibited by chloroquine and monensin, which is further evidence for a process of endocytosis, followed by intralysosomal hydrolysis of the transport protein. Transcobalamin II-vitamin B12 uptake was not fully saturable and other proteins, for example, haemoglobin, inhibited the uptake in a concentration-dependent way. Apparently the uptake proceeds through relatively unspecific protein-binding sites, probably involved in the reabsorption of filtrated proteins, although the affinity for transcobalamin II seems relatively high. Consequently, elevated urinary excretion of cobalamin is expected in patients with overflow proteinuria, and was indeed found in a patient with paroxysmal nocturnal haemoglobinuria.

Improved distribution analysis of cobalamins and cobalamin analogues in human plasma in which the use of thiol-blocking agents is a prerequisite

In this paper the quantitative analysis of cobalamins and cobalamin analogues in human plasma by means of high performance liquid chromatography (HPLC) and radioisotope dilution assay (RIDA) is described. Current methods for the extraction of cobalamins from plasma proved impracticable due to the selective loss of hydroxo- and sulphitocobalamin, caused by concomitant reduction to the Co2+ form and tight binding to thiol groups of denatured plasma proteins. Although this process can be prevented by exchange of the hydroxyl group with sulphite, azide or nitrite ions, further separation of the respective cobalamin forms from the other endogenous cobalamins by HPLC proved to be impossible. Efficient extraction of hydroxo- and sulphitocobalamin has been obtained in the presence of the thiol-blocking agent N-ethylmaleimide, which does not interfere with the subsequent chromatographic separation of any of the plasma cobalamin forms. The use of R-binder free Intrinsic Factor and salivary R-binder separately as binding substances in RIDA made it possible to analyse the distribution of biologically active cobalamins as well as cobalamin analogues over the various HPLC fractions. Analyses are reported of 15 normal human plasma samples, in which the major components were found to be methylcobalamin (46.9 +/- 4.5%, mean +/- SD) and hydroxocobalamin (40.4 +/- 7.1%, mean +/- SD). Analyses of plasma extracts in the absence of N-ethylmaleimide showed a gross relative overestimation of the amount of methylcobalamin, due to loss of hydroxocobalamin during the extraction procedure. Cobalamin analogues appeared to be evenly distributed over the cobalamin fractions in a pattern almost similar to that of the biologically active forms.

Application of heparin-conjugated Sepharose for the measurement of cobalamin-saturated and unsaturated transcobalamin II

The cobalamin-binding plasma protein transcobalamin II has a high affinity for the anticoagulant heparin. This phenomenon has been exploited in a new method for the quantification of cobalamin-saturated (holo-) and unsaturated (apo-) transcobalamin II in human plasma. Transcobalamin II is adsorbed from human plasma to heparin-conjugated Sepharose under suitable conditions and either cobalamin from adsorbed holo-transcobalamin II is measured by a radioisotope dilution assay or apo-transcobalamin II is determined by measuring the adsorbed unsaturated cobalamin-binding capacity with radioactive cobalamin. The assay results for apo- and holo-transcobalamin II are similar (r = 0.99 and 1.0, respectively) to those obtained with the established radioimmunosorbent assay using specific rabbit anti-human transcobalamin II-conjugated Sepharose. The assay cannot be carried out in heparin-anticoagulated plasma, because the free heparin competes with the immobilized heparin for the binding of transcobalamin II. The amount of heparin in plasma from patients being treated with subcutaneous or intravenous heparin is too low to interfere significantly with the measurement of transcobalamin II. Also the presence of circulating anti-transcobalamin II antibodies, as occur in some rare patients after frequent intramuscular injections of cobalamin, does not influence the assay.

The uptake of R-type cobalamin-binding protein by isolated rat liver cells

The uptake of R-type cobalamin-binding protein from human granulocytes and plasma by isolated parenchymal rat liver cells has been studied. When [57Co] cyanocobalamin-saturated granulocyte-binding protein or transcobalamin III was incubated with the liver cells in a concentration of 500 pM, more than 80% of the vitamin was taken up in 1 h. Vitamin B-12 bound to plasma transcobalamin I, however, was not taken up unless the protein was desialylated by neuraminidase from Vibrio cholerae. The uptake of iodinated pure granulocyte-binding protein, saturated with cobalamin, reached 100% and was accompanied by increasing intracellular proteolytic degradation of the binding protein. EGTA and asialo-orosomucoid completely inhibited this process of uptake and degradation, whereas partial inhibition was caused by chloroquine and colchicine. These observations provide evidence that these (asialo)-R-type cobalamin-binding proteins are taken up by the cell through the plasma membrane receptor for asialoglycoproteins by means of endocytosis followed by proteolysis of the binding protein in the lysosomes.

Purification of human transcobalamin II-cyanocobalamin by affinity chromatography using thermolabile immobilization of cyanocobalamin

Transcobalamin II-cyanocobalamin was isolated from Cohn fraction III of pooled human plasma by affinity chromatography on cyanocobalamin-Sepharose and some conventional separation methods. The affinity ligand cyanocobalamin was coupled to AH-Sepharose by a thermolabile linkage. The unsaturated binding protein was absorbed at 4 degrees C and eluted from the column at 37 degrees C as transcobalamin II-cyanocobalamin complex. The final preparation had a specific cyanocobalamin-binding capacity of 0.98 mol cyanocobalamin/mol transcobalamin II, the yield was 55% and the purification index amounted to 1.1 . 10(6). In dodecyl sulphate polyacrylamide gel electrophoresis one major protein band was observed at a molecular weight of 37 000 and a faint band at a molecular weight of 29 000. In polyacrylamide gel isolectric focusing the pure preparation turned out to be heterogeneous with isoelectric points ranging from pH 6.2 to 6.8, possibly by the occurrence of isoproteins.

Preparation of vitamin B-12-free serum for the use in vitamin B-12 radioassays.

In radioassays for serum vitamin B-12, the separation of free and bound vitamin is usually made with charcoal absorption. The specificity of this separation depends on the amount of charcoal and the protein content and constitution of the medium. The large difference in protein concentration between the samples for the dilution curve and the serum samples introduces an uncontrolled variable in the test. In order to equalize the experimental circumstances, the standard dilutions were made in serum freed from vitamin B-12 after boiling the serum for 20 min in a 4-fold dilution with glutamic acid buffer at pH 3.3 and subsequent passage over a CH-Sepharose 4-B column complexed with hog intrinsic factor (IF). The vitamin B-12-binding capacity of such an affinity column prepared from 1 g CH-Sepharose and 20 mg IF, suffices for the absorption of vitamin B-12 in 3000 ml serum from which 300 series of vitamin B-12 standard solutions can be made. Our first results with this method confirm that the charcoal absorption radioassay has become more accurate by the use of vitamin B-12-free serum in the standard dilutions.

Evaluation of a radioassay for serum folate and the effects of ascorbate and methotrexate

In this study a competitive binding assay for serum folate is evaluated with special attention to storage conditions of the serum samples and the effect of methotrexate on the assay results. Average serum folate levels were 31 nmol/1 +/- 8 (S.D.) in 59 nromal sera and 19 nmol/1 +/- 12 (S.D.) in 354 sera from hospitalized patients of which 43 values were lower than 9 nmol/1. Serum folate appeared to be stable at - 20 degrees C for at least 5 weeks. The addition of ascorbate, which is said to stabilize serum folate, caused a non-specific increase of the measured folate. Methotrexate acted competitively with the tracer folate in the assay sytem when added in vitro and elevated the folate readings. The same occurred in the serum of a patient after intravenous methotrexate.

Separation of transcobalamin II isoproteins by means of chromatofocusing

Transcobalamin II, the principal cobalamin-binding protein in human plasma, expresses a genetic polymorphism. Four more or less common alleles, denoted by X, S, M and F, have been defined earlier by means of gel electrophoretic techniques followed by autoradiography. This technique is less suitable for the analysis of individual samples and requires long exposure times. This paper describes the analysis of transcobalamin II phenotypes by means of fast protein liquid chromatofocusing. This technique has the advantage that the results of the analysis of several samples can be obtained within a day, and it also seems applicable to the preparative separation of transcobalamin II isoproteins. The sequence of elution of the isoproteins was in complete accordance with the banding pattern obtained by electrophoretic separation. The characteristic doublet bands found with polyacrylamide gel electrophoresis were less obvious in the chromatofocusing elution pattern.