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PubMed1993

Cobalamin metabolism in cultured human chorionic villus cells.

Cobalamin (Cbl, vitamin B12) metabolism was analyzed in cultures of human chorionic villus (CV) cells obtained at 9-10 weeks of gestation. CV cells were shown to synthesize transcobalamin II (TCII) and to possess a high affinity receptor for that molecule. The cells bound and internalized radioactive cyanocobalamin (CN[57Co]Cbl) complexed to TCII. This internalized CN[57Co]Cbl was found to be converted to both methylCbl and adenosylCbl, the two intracellular coenzyme forms of Cbl, and bound to the two known intracellular Cbl requiring enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase. Both enzyme systems were found to be functional in the intact cell by demonstrating the incorporation of the radioactive label from both [14C]CH3-tetrahydrofolate and [14C]propionate into acid insoluble products. MS activity was also detected in lysed cell material. CV cells were shown not to be auxotrophic for methionine since they were able to utilize homocysteine in place of methionine for cell division. Since CV cells are capable of performing many of the complex events associated with Cbl metabolism, it may be possible to use these cells to diagnose genetic defects of Cbl metabolism.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1998

Genetic defects of folate and cobalamin metabolism.

Deficient activity of an enzyme can result from a defect in the conversion of the vitamin to a co-enzyme as well from an abnormal apo-enzyme or disturbed binding of coenzyme to enzyme. Conversion of dietary vitamin to intracellular active co-enzyme can be complex and require many physiological and biochemical processes including stomach release of bound vitamin, intestinal uptake, carriers/transport, blood transport, cellular uptake, intracellular release and intracellular compartmentalisation. Disorders of malabsorption (food cobalamin malabsorption, intrinsic factor deficiency and abnormal enterocyte cobalamin processing) and transport proteins (transcobalamin II deficiency, R-binder deficiency) mostly lead to disturbed function of the two cobalamin requiring enzymes, methylmalonyl CoA mutase and methionine synthase. Defects of early steps of intracellular cobalamin (cblF, cbl C/D) result in marked deficiencies of both cobalamin co-enzymes and homocystinuria combined with methylmalonic aciduria. Defective synthesis of adenosyl cobalamin in the cbl A/B defects leads to methylmalonyl CoA mutase. Isolated methionine synthase deficiency is also classified as a cobalamin disorder due to its associated deficient formation of methylcobalamin. Folate disorders include methylene-tetrahydrofolate reductase deficiency and glutamate formimino-transferase deficiency. In addition a hereditary disorder of intestinal folate transport has been described. Less well established are disorders of dihydrofolate reductase, methenyl-tetrahydrofolate cyclohydrolase, and defects of cellular folate uptake.

Absorption↗
PubMed2006

Vitamin B12 and vitamin B6 supplementation is needed among adults with phenylketonuria (PKU).

Phenylketonuria (PKU) is caused by an autosomal recessive deficiency of the enzyme phelnylalanine hydroxylase leading to a failure to convert phenylalanine to tyrosine. To avoid irreversible neurological damage because of increased phenylalanine, treatment is instituted rapidly after birth. We examined 31 adult PKU patients living on a less protein-restricted diet. Theoretically, these PKU patients had an increased risk of developing vitamin B(12) and B(6) deficiency because of a limited intake of animal products. Besides laboratory tests (n = 31) we obtained clinical information (n = 30) and detailed information on food consumption (n = 28). Three-quarters of the patients had early biochemical signs of vitamin B(12) deficiency. In spite of a normal folate status, 9 (29%) had a plasma homocysteine above 12 micromol/L. In accord with these findings, the food questionnaires indicated that 11 (39%) patients received less than the recommended daily vitamin B(12), and 20 (71%) received less vitamin B(6) than recommended. A significant association was found between reduced vitamin B(12) intake and both reduced serum cobalamins (p = 0.04) and reduced serum transcobalamin saturation (p = 0.03). Eleven patients took a vitamin pill daily, and these patients had a significantly lower plasma homocysteine compared to the rest. The present study suggests that adult PKU patients were at increased risk of developing vitamin B(12) deficiency, and their intake of vitamin B(6) was below the recommended daily intake. In conclusion PKU patients need continuing dietary guidance throughout adult life, and considering the risks, costs and potential benefits, daily vitamin supplementation seems justified in these patients.

Adolescent↗
PubMed1981

Purification and characterization of rabbit haptocorrin.

Rabbit haptocorrin (R-binder) has been purified from serum by affinity chromatography on cobalamin-Sepharose and Blue Sepharose. It proved to be a protein with a relative molecular mass of 60 000 and an amino acid content very similar to that of other haptocorrins (Nexø, E. and Olesen, H. (1981) in B12 (Dolphin, D., ed.), Wiley Interscience, New York/London, in the press). The pH optimum (pH 6-9) for binding of cyanocobalamin and the affinity to dicyanocobinamide were like those of human and hog haptocorrins. In spectral studies, the extinction coefficient of cyanocobalamin at 363 nm (gamma 1-band) increased by about 16% on binding to rabbit haptocorrin. Binding of azidocobalamin gave spectra changes similar to those for binding to rabbit transcobalamin.

Amino Acids↗
PubMed1991

Synthesis and secretion of cobalamin binding proteins by opossum kidney cells.

Opossum kidney epithelial cells synthesize and secrete two Cobalamin (Cbl) binding proteins of Mr 66,000 and 43,000. When grown on culture inserts, the apical medium contained both these proteins while the basolateral medium contained only the 43 kDa Cbl binder. Colchicine, a microtubule disruptive drug, increased two fold the apical but not the basolateral secretion of the Cbl binding proteins. Although the opossum Cbl binders did not cross react with anti-serum raised to Cbl binders from other species, the identity based on Cbl binding and size suggest that the 66 kDa and 43 kDa proteins are haptocorrin and transcobalamin II.

Animals↗
PubMed1977

A radioimmunoassay for the R-type binders of cobalamin.

A radioimmunoassay of R-type binders of cobalmin was devised and tested. The assay incorporated an antibody against purified human salivary R binder as the binding reagent. The labeled ligand was cyano[57Co]cobalamin bound to the R binder of pooled human saliva. The standard source of unlabeled ligand was also from human saliva of known R binder content. The assay was responsive to R binders of several sources, to either pure R or that of crude sources and equally to R binder saturated or unsaturated with cobalamin. It was not responsive to transcobalamin II. The assay was reproducible and reliable.

Antibody Specificity↗
PubMed2005

The binding properties of the human receptor for the cellular uptake of vitamin B12.

Cellular uptake of vitamin B(12) (cobalamin, Cbl) is mediated by a receptor expressed on the plasma membrane that binds transcobalamin (TC) saturated with Cbl and internalizes the TC-Cbl by endocytosis. A few reports have described the characterization of the receptor protein. However, many discrepancies have emerged in the functional and structural properties of the receptor and therefore, the identity and primary structure of this protein remains unconfirmed. In this report, we provide evidence of a 58 kDa monomeric protein as the likely receptor for the uptake of TC-Cbl and that the functional activity is not associated with a 72/144 kDa monomer/dimer with immunoglobulin Fc structural domain that has been purported to be the receptor in a number of publications.

Biological Transport↗
PubMed1998

Transport and endogenous release of vitamin B12 in the dually perfused human placenta.

The kinetics for vitamin B12, cobalamin (Cbl), transfer across the human placenta, and the retention of the endogenous Cbl and its release into the maternal and fetal compartments were investigated in a dually perfused human term placenta in vitro. After 4 hours of perfusion following a single bolus injection (peak maternal perfusate 94 fmol/ml) of 57Co-Cbl into the maternal reservoir, the maternal [57Co-Cbl] rapidly decreased whereas the fetal [57Co-Cbl] was only 9% of the final maternal [57Co-Cbl]. Even though there was a limited transfer of 57Co-Cbl to the fetus, the placenta rapidly accumulated Cbl. At 4 hours, 18% of the initial 57Co-Cbl dose was in the placenta; only 3% of the initial dose was in the fetal perfusate. Also after 4 hours, [57Co-Cbl] was bound > 95% to transcobalamin (TC)I/III-like proteins in the maternal perfusate, whereas Cbl was bound to TCI/III- and TCII-like proteins with some free in the fetal perfusate. In the cytosol, > 95% of the [57Co-Cbl] was bound (80% to TCII-like and 19% to TCI/III-like proteins). When no exogenous Cbl was added, total endogenous [Cbl] in the maternal circulation increased with time during 8 hours of perfusion at a rate of 0.25 +/- 0.12 pmol/gm per hour. Only 2% of this Cbl was free, whereas 98% was bound to specific binding proteins. Neither plateau values nor equilibration with the fetal side were noted. In the fetal circulation, there was a release of Cbl at a rate of 0.015 +/- 0.003 pmol/gm per hour, which was 99.99% bound. Thus the human placenta rapidly concentrates Cbl in the perfused lobule with little distribution of Cbl to nonperfused areas. Therefore, the human placenta modulates the asymmetric transfer of vitamin B12 on the basis of release of specific Cbl-binding proteins (TCI/III- and TCII-like) into the maternal and fetal perfusates.

Female↗
PubMed2003

Cobalamin transport proteins and their cell-surface receptors.

The primary function of cobalamin (Cbl; vitamin B12) is the formation of red blood cells and the maintenance of a healthy nervous system. Before cells can utilise dietary Cbl, the vitamin must undergo cellular transport using two distinct receptor-mediated events. First, dietary Cbl bound to gastric intrinsic factor (IF) is taken up from the apical pole of ileal epithelial cells via a 460 kDa receptor, cubilin, and is transported across the cell bound to another Cbl-binding protein, transcobalamin II (TC II). Second, plasma TC II-Cbl is taken up by cells that need Cbl via the TC II receptor (TC II-R), a 62 kDa protein that is expressed as a functional dimer in cellular plasma membranes. Human Cbl deficiency can develop as a result of acquired or inherited dysfunction in either of these two transmembrane transport events. This review focuses on the biochemical, cellular and molecular aspects of IF and TC II and their cell-surface receptors.

Cell Membrane↗
PubMed2006

A longitudinal study of serum cobalamins and its binding proteins in lactating women.

OBJECTIVE: To examine longitudinal changes in serum cobalamins, transcobalamin (TC) and haptocorrin (HC) during lactation and to investigate the influence of vitamin B12 supplementation on these parameters. DESIGN: A 9-month follow-up study. SUBJECTS AND METHODS: Lactating mothers (N=89) including 23 supplemented with vitamin B12 (1-18 microg/daily), 41 partly supplemented and 25 not supplemented. Blood samples collected 3 weeks (baseline) and 4 and 9 months post-partum were analysed for cobalamins, TC and HC. Both the total concentration and the cobalamin-saturated form (holo) of TC and HC were analysed. RESULTS: No significant differences were observed in serum cobalamins or its binding proteins related to supplementation with vitamin B12 or the duration of lactation. Serum cobalamins remained unchanged from 3 weeks to 9 months post-partum. Total TC (holoTC) (median+/-s.e. pmol/l) decreased between 3 weeks (710+/-23 (85+/-12)) and 9 months (602+/-21 (76+/-11)) (P<0.0001 (P=0.0002)), whereas total HC (holoHC) increased from (422+/-11 (300+/-9)) at 4 months to (455+/-13 (317+/-10)) to 9 months post-partum (P<0.0001 (P<0.0001)). CONCLUSION: We report a decrease in TC and an increase in HC during a 9-month period post-partum. No differences were observed between the vitamin B12-supplemented and the unsupplemented groups. Thus, supplementation with vitamin B12 has no impact on the circulating level of serum cobalamins or its binding proteins in a Danish population of lactating mothers.

Adult↗
PubMed1975

Analytical subcellular fractionation of human granulocytes with reference to the localization of vitamin B12-binding proteins.

1. A granulocyte-rich fraction was isolated from blood of a patient with chronic granulocytic leukaemia and from blood of a normal subject and the cells were disrupted in isotonic sucrose. The nuclei were removed by low-speed centrifugation and the post-nuclear supernatant was fractionated by centrifugation on sucrose density gradients. 2. The subcellular organelles in the gradients were detected with marker enzymes by the use of highly sensitive assay techniques. Similar results were obtained with granulocytes from both subjects. 3. Unsaturated vitamin B12-binding proteins were almost exclusively localized to the specific granules. Chromatographic analysis of these proteins showed them to have approximately equal proportions of transcobalamins I and III. Vitamin B12 assayed microbiologically could not be detected in the specific granule fractions.

Blood Proteins↗
PubMed1977

Intracellular binding of radioactive hydroxocobalamin to cobalamin-dependent apoenzymes in rat liver.

We identified previously an intracellular cobalamin (Cbl) binding protein(s) in cultured human fibroblasts, distinct from known Cbl "R" binders and absent from mutant cells deficient in the synthesis of the two Cbl coenzymes. In order to further characterize this binding activity, we have investigated its homologue in rat liver. After being transported to the liver by the serum protein transcobalamin II, [57Co]Cbl was bound by at least two distinct proteins, one cytosolic, the other mitochondrial. Labeled Cbl bound to cytosolic protein faster than or prior to the mitochondrial protein. With time there was a decline in radioactivity associated with the cytosolic binder and a coordinate increase in that associated with the mitochondrial binder. Although both proteins cochromatographed on Sephadex G-150 and had apparent molecular weights of 120,000, they were separated into two discrete components by polyacrylamide gel electrophoresis and by DEAE-cellulose chromatography. The cytosolic binder cochromatographed with N5-methyltetrahydrofolate:homocysteine methyltransferase activity (5-methyltetrahydropteroyl-L-glutamate:L-homocysteine S-methyltransferase, EC 2.1.1.13); the mitochondrial one with methylmalonyl CoA mutase activity (methylmalonyl-CoA CoA-carbonylmutase, EC 5.4.99.2). These proteins were distinguished further by the chemical forms of [57Co]Cbl found with them, hydroxocobalamin and methylcobalamin with the cytosolic protein and adenosylcobalamin with the mitochondrial one. These results suggest that intracellular Cbl binding activity in rat liver can be accounted for by attachment of Cbl to the two known Cbl-dependent apoenzymes, methylmalonyl CoA mutase and methyltetrahydrofolate methyltransferase. The mechanism and significance of the observered binding protein deficiency in mutant human fibroblasts must, therefore, be re-evaluated.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1975

Vitamin B12-binding proteins in human gastric mucosa. General pattern and demonstration of intrinsic factor isoproteins typical of mucosa.

Extracts of human stomach mucosa were shown to contain immunoreactive intrinsic factor and small amounts of R protein and transcobalamin II. In gel filtration the mucosal vitamin B12-intrinsic factor complex behaved as though its molecular weight were smaller than the corresponding complex in gastric juice. In isoelectric focusing both the gastric juice and mucosal vitamin B12-intrinsic factor complexes resolved into numerous isoproteins. These findings were verified by DEAE-cellulose chromatography. The mucosal intrinsic factor isoprotein pattern contained, in addition to the isoproteins found in gastric juice, components with higher isoelectric points -- that is, pH 5.73-6.64.

Blood Proteins↗
PubMed1975

Characterization of a cobalamin-binding plasma protein from a patient with hepatoma.

A cobalamin-binding protein has been purified by affinity chromatography from plasma of a patient with hepatoma and a 10,000-fold increase in the concentration of the plasma cobalamin-binding capacity. The protein behaved as normal transcobalamin I in gel filtration, agar gel electrophoresis, immunoelectrophoresis, precipitation by ammonium sulphate, and cobalamin-binding studies. The protein contained 38 per cent carbohydrate, and the relative molecular mass based on amino acid and carbohydrate analyses was 69,000. The molar absorption coefficient of cyanocobalamin bound to the protein was determined to be 36,000 at 362 nm. On amino acid sequencing, one amino terminal was found, and the first 13 residues were determined as Glu-Ile-Ser/Cys-Glu-Val-Ser/Cys-Glu-Glu-Asx-Tyr-Ile-Arg-Leu/Ile.

Amino Acid Sequence↗
PubMed1998

Cobalamin (vitamin B12) and holotranscobalamin changes in plasma and liver tissue in alcoholics with liver disease.

OBJECTIVE: We wanted to know if alterations in plasma cobalamin (B12) concentration and B12 carriers, e.g., holotranscobalamins (holo TC), occur in blood and liver tissue from patients with severe alcoholic liver disease. Our purpose was to test the hypothesis that liver disease may disrupt B12 distribution. METHOD: Total B12, as well as B12 bound to transcobalamin I, II, III (holo TC), were measured to determine their concentration in plasma and in liver tissue; Poteriochromonas malhamensis--a protozoan reagent served to measure only metabolically active (true) B12. Total B12 as distributed in holo TC in plasma and liver tissue of healthy subjects (controls) were compared to patients with severe alcoholic liver disease. RESULTS: Severe liver disease initiates highly elevated B12 levels in plasma and a lowered liver tissue total B12 concentration. The percent of B12 distributed to holo TC II is significantly depleted during liver disease. In contrast, holo TC I and III are elevated in plasma during liver disease and contain more B12 than controls. Total B12 and B12 distributed to TC are lower in diseased liver tissue. CONCLUSION: Severe alcoholic liver disease involves leakage of total B12 from liver tissue into the plasma. Holo TC I and III concentration increases in plasma; this preserves the high plasma B12 from being excreted. However, plasma holo TC II B12 distribution is decreased, indicating that there is a depression of exogenous B12 entering the plasma and tissues. In severe liver disease, liver tissue B12 binding and storage by TC is disrupted and causes B12 to leak out of the liver into the circulation. Eventually liver disease could produce enough severe tissue B12 deficits to cause metabolic dysfunction despite elevated plasma total B12. Elevation of plasma B12, accompanied by a lowering of holo TC II distribution, seemed to be a useful index of liver disease severity suggesting preventive treatment.

Humans↗
PubMed2006

A daily intake of approximately 6 microg vitamin B-12 appears to saturate all the vitamin B-12-related variables in Danish postmenopausal women.

BACKGROUND: Recommended daily intakes of vitamin B-12 vary between 2 and 6 microg. OBJECTIVE: The objective was to examine the associations between vitamin B-12 intake and markers of vitamin B-12 deficiency. DESIGN: We studied 98 Danish postmenopausal women aged 41-75 y. Serum cobalamin, transcobalamin (TC) saturated with vitamin B-12 (holo-TC), TC saturation (holo-TC/total TC), methylmalonic acid (MMA), and total homocysteine (tHcy) were measured to assess vitamin B-12 status. Dietary intakes of vitamin B-12 were determined from 7-d weighed food records. Gastric pH measurement and the alkali-challenge test were performed with the use of Heidelberg radiotelemetric capsules. RESULTS: The total intake of vitamin B-12 ranged from 1.2 to 23.9 microg/d. All vitamin B-12-related variables, except gastric pH, correlated significantly with total vitamin B-12 intake. Those taking supplements (54%) had higher circulating concentrations of cobalamin and TC saturation and lower concentrations of MMA and tHcy than did those not taking supplements. All subjects were divided into quintiles according to their total vitamin B-12 intake. For all the variables analyzed, the curves appeared to level off at a daily vitamin B-12 intake of approximately 6 microg; the median (and 25th-75th percentiles) for cobalamin was 380 (270-480) pmol/L, for holo-TC was 119 (92-162) pmol/L, for MMA was 0.12 (0.14-0.17) micromol/L, and for tHcy was 9.75 (8.3-11.40) micromol/L (n = 58). CONCLUSION: A daily vitamin B-12 intake of 6 microg appeared to be sufficient to correct all the vitamin B-12-related variables measured in the postmenopausal Danish women in this study.

Administration, Oral↗
PubMed1978

Vitamin B12-binding proteins in serum and plasma in various disorders. Effect of anticoagulants.

Because of recent developments in the study of vitamin B12-binding proteins, the levels of the three serum binders were compared in serum and plasma samples from subjects with various disorders. The results allow the following conclusions: (1) As previously reported, transcobalamin (TC) III and to a lesser extent TC I are artifactually elevated in serum. The appear to be released in vitro during the clotting process, presumably from granulocytes. (2) Blood cells of patients with polycythemia vera release exceedingly large amounts of TC I and TC III in vitro. (3) The above findings support, but do not prove, at least a partial granulocytic source of TC I. Nevertheless, factors other than granulocytes influence TC I levels, as disorders characterized by increased TC I (most prominently chronic myelogenous leukemia but also several cases of cancer) manifest relatively little cellular release of TC I in vitro. (4) Despite the serum artifact, the serum abnormalities described in various conditions were seen in plasma also, even though the actual values of themselves were lower in plasma. The chief exception was TC III, which was elevated in plasma only in polycythemia vera (and in a few cases of leukocytosis). (5) EDTA-NaF anticoagulant is not suitable, as it causes plasma dilution, thus explaining previous reports of TC II level differences between serum and plasma. EDTA is therefore a preferable anticoagulant for vitamin B12-binding protein studies, although it too may not be ideal.

Anticoagulants↗
PubMed1983

An unusual case of autoimmune agranulocytosis with total absence of myeloid precursors: demonstration of diverse sources of R binder for cobalamin in plasma and secretions.

An unusual autoimmune agranulocytosis featured intense plasmacytosis, hyperglobulinemia, and total absence of myeloid precursors. Remission was induced with prednisone and cyclophosphamide on two separate occasions, producing reversal of all the above abnormalities. Anti-granulocyte antibodies were demonstrated in relapse by staphylococcal protein A assay. During agranulocytosis, levels of the R binder for cobalamin in the blood were greatly diminished (20-60 pg/mL unsaturated binding capacity). Moreover, the acquired R binder deficiency was accompanied by the previously undescribed phenomenon of a shift of endogenous serum cobalamin to transcobalamin II; only 10-19% of endogenous cobalamin was attached to R binder. In remission, unsaturated R binder rose to normal levels and R binder also carried 78-91% of endogenous cobalamin. This confirms that granulocytes are responsible for most of the circulating R binder in humans. In contrast, salivary R binder concentration was normal throughout the patient's course, indicating that secretory R binder is nongranulocytic in origin. These data establish that R binder has more than one source in humans.

Adult↗