Uptake Of Cobalamin Research Articles

An update on cobalamin deficiency in adults

Cobalamin (vitamin B12) deficiency is particularly common in the elderly (>65 years of age), but is often unrecognized because of its subtle clinical manifestations; although they can be potentially serious, particularly from a neuropsychiatric and hematological perspective. In the general population, the main causes of cobalamin deficiency are pernicious anemia and food-cobalamin malabsorption. Food-cobalamin malabsorption syndrome, which has only recently been identified, is a disorder characterized by the inability to release cobalamin from food or its binding proteins. This syndrome is usually caused by atrophic gastritis, related or unrelated to Helicobacter pylori infection, and long-term ingestion of antacids and biguanides. Besides these syndromes, mutations in genes encoding endocytic receptors involved in the ileal absorption and cellular uptake of cobalamin have been recently uncovered and explain, at least in part, the hereditary component of megaloblastic anemia. Management of cobalamin deficiency with cobalamin injections is currently well codified, but new routes of cobalamin administration (oral and nasal) are being studied, especially oral cobalamin therapy for food-cobalamin malabsorption.

New insights into carrier binding and epithelial uptake of the erythropoietic nutrients cobalamin and folate

In addition to malnutrition several genetic and acquired conditions may affect the homeostasis of cobalamin (vitamin B12) and folate, leading to megaloblastic anemia and other diseases. The present review describes new insight into protein handling of cobalamin and folate. The recent solution of the three-dimensional structure of the cobalamin binder transcobalamin shows two separate domains enclosing the vitamin. This structure apparently also applies for the other homologous cobalamin binders, intrinsic factor and haptocorrin. Genetic studies of inherited cobalamin malabsorption and biochemical studies have now revealed that the functional receptor for uptake of intrinsic factor-vitamin cobalamin complexes also is a complex itself consisting of two different gene products, cubilin and amnionless. A role in folate uptake of megalin, an endocytic receptor for epithelial uptake of various proteins including transcobalamin, is now also indicated by the observation that megalin can mediate uptake of soluble folate receptor. New data show the structure of cobalamin carriers and reveal novel proteins involved in the epithelial uptake of cobalamin and folate. Genetic abnormalities in three different genes encoding proteins in the epithelial uptake of cobalamin are now known to cause malabsorption of cobalamin and megaloblastic anemia.

Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type

Methylmalonic aciduria and homocystinuria, cblC type (OMIM 277400), is the most common inborn error of vitamin B(12) (cobalamin) metabolism, with about 250 known cases. Affected individuals have developmental, hematological, neurological, metabolic, ophthalmologic and dermatologic clinical findings. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood. The cblC locus was mapped to chromosome region 1p by linkage analysis. We refined the chromosomal interval using homozygosity mapping and haplotype analyses and identified the MMACHC gene. In 204 individuals, 42 different mutations were identified, many consistent with a loss of function of the protein product. One mutation, 271dupA, accounted for 40% of all disease alleles. Transduction of wild-type MMACHC into immortalized cblC fibroblast cell lines corrected the cellular phenotype. Molecular modeling predicts that the C-terminal region of the gene product folds similarly to TonB, a bacterial protein involved in energy transduction for cobalamin uptake.

Effects of interferon beta on transcobalamin II-receptor expression and antitumor activity of nitrosylcobalamin.

The ubiquitous plasma membrane transcobalamin II receptor (TC II-R) mediates uptake of cobalamin (Cbl; vitamin B12), an essential micronutrient. Tumors often require more Cbl than normal tissue, and increased Cbl uptake may result from increased TC II-R expression. To examine whether Cbl could therefore be used as a carrier molecule to target a chemotherapy drug, we tested an analogue of Cbl with nitric oxide as a ligand, nitrosylcobalamin (NO-Cbl). Because interferon beta (IFN-beta) has antitumor effects and increases expression of some membrane receptors, we examined whether it may enhance the effects of NO-Cbl. Antiproliferative effects of NO-Cbl were assessed in 24 normal and cancer cell lines. Xenograft tumors of human ovarian cancer NIH-OVCAR-3 cells were established in athymic nude mice, and tumor growth was monitored after treatment with NO-Cbl and IFN-beta, both individually and concomitantly. TC II-R expression and apoptosis was monitored in vitro and in vivo. RNA protection assays and mitochondrial membrane potential assays were used to distinguish the extrinsic and intrinsic apoptotic pathways, respectively. Cancer cell lines were more sensitive to NO-Cbl (with ID(50)s [the dose that inhibits growth by 50%] as low as 2 microM) than normal cell lines (with ID(50)s of 85-135 microM). Single-agent NO-Cbl and IFN-beta treatment of NIH-OVCAR-3 xenografts induced tumor regression, whereas combination treatment induced tumor eradication. IFN-beta treatment increased TC II-R expression in vitro and uptake of [(57)Co]cobalamin in vivo. Compared with NIH-OVCAR-3 cells treated with NO-Cbl, cells treated with NO-Cbl and IFN-beta were more apoptotic and expressed higher mRNA levels of various apoptosis-associated genes. No changes in mitochondrial membrane potential were observed in cells treated with NO-Cbl. NO-Cbl inhibited tumor growth in vivo by activating the extrinsic apoptotic pathway. The increased expression of TC II-R induced by IFN-beta resulted in enhanced antitumor effects with NO-Cbl both in vitro and in vivo.

Vitamin B12 metabolism after urinary diversion with a Kock ileal reservoir.

To elucidate the influence of construction of a Kock reservoir for urinary diversion using 70 cm of the distal ileum on vitamin B12 metabolism. Blood samples for determination of cobalamin concentrations were drawn up to 18 years after construction of a Kock reservoir in 97 patients. Preoperative values were obtained in 20 of these patients. Pre- and postoperative Schilling tests and analyses of methylmalonic acid and homocysteine concentrations were performed in subgroups. The operation did not induce a significant decrease in cobalamin uptake as reflected in the pre- and postoperatively performed Schilling tests. No significant decline in cobalamin concentration postoperatively was noted. The value of analysis of methylmalonic acid and homocysteine concentrations in finding patients with cobalamin deficiency is limited by the fact that kidney function influences the results. Ten per cent of the patients did develop true cobalamin deficiency. A postoperative vitamin B12 value below 200 pmol/l indicates a 50% risk of later cobalamin deficiency. The construction of a Kock reservoir does not per se cause cobalamin deficiency. Substitution should be instituted at a postoperative concentration below 200 pmol/l.

Folate and cobalamin uptake by human placenta in complicated pregnancies: Prematurity, preeclampsia and fetal neural tube defects

The purpose of the present study was to evaluate whether folate and cobalamin uptakes by microvillous membrane vesicles (MMV) from human placenta are affected in pregnancy complications such as prematurity, preeclampsia, and fetal neural tube defects. MMVs were obtained from pooled placental samples from adult women with uncomplicated full-term gestations (n=4), premature delivery (n=3), preeclampsia (n=3), and fetal neural tube defects (n=2). Uptake of cobalamin bound to transcobalamin (TC) was several times higher than the uptake of free cobalamin or cobalamin bound to haptocorrin. TC-cobalamin and folate uptakes by placental MMV were not impaired in preeclampsia, but TC-cobalamin uptake was higher (p<0.001) in prematurity than in term gestations (58±7 vs 33±5 pmol cyano-[ 57Co]cobalamin/g MMV protein), whereas folate uptake was unaffected (11.0±1.0 vs 10±0.8 pmol [ 3H]folic acid/ mg MMV protein). In pregnancies affected by fetal neural tube defects, placental folate uptake was similar to that of uncomplicated pregnancies, whereas TC cobalamin uptake was lower (p<0.001; 17±3 pmol cyano-[ 57Co]cobalamin/g MMV protein). Whether the results obtained for cobalamin uptake in mature placenta is a mere consequence of placental changes in NTD-affected pregnancies or whether it reflects a situation that occurs during the closure of the neural tube in early pregnancy is still unclear. These results suggest that a defect in folate uptake is not involved in NTD etiology, but that the supply of cobalamin to embryonic cells may be impaired, which could affect folate metabolism.

Oral absorption of peptides through the cobalamin (vitamin B12) pathway in the rat intestine.

This study was aimed at examining the extent and mechanism of uptake of cobalamin (Cbl)-conjugated peptides in vitro and in vivo. To enable acquisition of quantitative absorption data of Cbl-peptides, metabolically stable octapeptides (DP3), with (Cbl-Hex-DP3) or without a hexyl spacer (Cbl-DP3), were coupled to Cbl and radiolabeled. For comparison, LHRH coupled to Cbl was used as metabolically susceptible peptide. Biological recognition of Cbl-peptides was studied in the physiological order: binding by Intrinsic Factor (IF), recognition and transport of the IF-complexes by IF-Cbl receptors (IFCR) on Caco-2 monolayers and oral absorption of the Cbl-conjugates in the rat. All Cbl-peptides bound to IF and the IF-complexes were recognized by IFCR receptors on Caco-2 monolayers. Binding was saturable and could be inhibited by a 20-fold excess of IF-Cbl, but not of Non-intrinsic Factor (NIF)-Cbl. Oral administration of these ligands to rats resulted in absorption of 53%, 45%, 42%, and 23% of the applied radioactivity for Cbl, Cbl-LHRH, Cbl-Hex-DP3, and Cbl-DP3, respectively. Simultaneous administration of a >10(5)-fold excess of unlabeled Cbl reduced uptake of all compounds to <4%. Tissue distribution and elimination of the metabolically stable Cbl-conjugates were comparable to Cbl. The endogenous Cbl uptake pathway can be exploited for oral peptide delivery as indicated by the specific and high (40-45%) uptake of metabolically stable Cbl-coupled octapeptides.

Transcobalamin II and in vitro Proliferation of Leukemic Cells

We have recently shown that antibodies to transcobalamin II (TCII) inhibit the in vitro growth of human and murine leukemic cells. This antiproliferative strategy targets the uptake of cobalamin (Cb1), an essential cofactor for two biochemical reactions in humans. To date there has been no appropriate cell culture model available to study antagonism of Cbl as a potential antiproliferative strategy. We have established cell culture conditions which allow reproducible measurements of cell proliferation that is dependent on Cbl and its carrier protein, TCII. This bioassay has allowed us to demonstrate that several monoclonal antibodies, raised against TCII, are potent inhibitors of cell proliferation and that excess Cbl abrogates this inhibitory effect. Thus, supporting our hypothesis that interference with Cbl uptake or metabolism will result in inhibition of cell proliferation. Furthermore, Cbl metabolism appears to provide a useful target for antiproliferative strategies which now involve the use of inactive Cbl analogs. In this review, we update our work on the role of targeting TCII and Cbl as an antiproliferative strategy for leukemic cells. We suggest that this strategy may provide a novel direction for anti cancer reagents.

Expression of functional intrinsic factor using recombinant baculovirus

Publisher Summary Intrinsic factor (IF) is produced in foregut tissues of mammals and mediates the uptake of cobalamin (Cbl, vitamin B I2 ). It is a protein with unusual properties, being soluble and functional at very acidic pH. This chapter discusses the expression of functional IF using recombinant baculovirus. The majority of recombinant IF produced in the baculovirus system is nonglycosylated. Insect cells do not often add the larger complex oligosaccharide side chains seen in glycoproteins of higher eukaryotes, but they do provide trimmed high-mannose units. Glycosylation of IF only during the first 24 hours could be due either to efficient glycosylation only when expression of recombinant protein is at a low level or to direct inhibition of host cell glycosylation as infection proceeds. The composition of the carbohydrate side chains of baculovirus-derived recombinant IF is not known, but it is probably different from that of native gastric IF. Despite this difference, the functional characteristics of the purified recombinant IF are identical to those of native IF.

Characterization of Monoclonal Antibodies to Epitopes of Human Transcobalamin II

Cellular uptake of cobalamin (Cbl) is mediated by transcobalamin II (TCII), a Cbl binding protein in the plasma. The TCII-Cbl complex binds to a cell surface receptor and is internalized by endocytosis. We have generated monoclonal antibodies (mAbs) to human TCII that can be distinguished into three functional types on the basis of interaction with three different regions of the protein. Type 1: Receptor blocking. This mAb binds holo-TCII and inhibits the cellular uptake of Cbl. Type 2: Cbl blocking. This mAb binds apo-TCII at or near the Cbl binding domain and inhibits the formation of holo-TCII. Type 3: Precipitating. This mAb binds both holo-TCII and apo-TCII but does not interfere with Cbl binding. Whereas type 1 and type 2 mAb, following incubation with TCII-[57Co]Cbl or apo-TCII, respectively, inhibit the uptake of radio-labeled Cbl by K562 cells, type 3 mAb has no such activity with either form of TCII. These properties of type 1 and type 2 mAb that inhibit the cellular uptake of Cbl, may serve to induce rapid Cbl deficiency and provide a model to study the effect of selective Cbl depletion on cell division and differentiation as well as on the pathways dependent on the two Cbl cofactors, methyl-Cbl and 5′-deoxyadenosyl-Cbl.

The dynamics of cobalamin utilization in L-1210 mouse leukemia cells: a model of cellular cobalamin metabolism

The uptake and metabolism of cobalamin (Cbl) has been studied in L-1210 murine leukemia cells propagating in vitro. Extracellular Cbl (protein bound and free) and intracellular Cbl (protein bound and free) were determined after culturing L-1210 cells in the presence of [ 57Co]cyanocobalamin (CN-Cbl) bound to transcobalamin II (transcobalamin, TC). The intracellular pool of free [ 57Co]Cbl increased during the first 24 h of culture and a substantial fraction of this free pool was effluxed from the cell to the medium. Upon depletion of extracellular TC-[ 57Co]CN-Cbl, the intracellular concentration of free Cbl decreased as did the efflux of Cbl to the medium. Internalized [ 57Co]CN-Cbl was converted to hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl) and 5′-deoxyadenosylcobalamin. These Cbl forms were found in both soluble (cytoplasmic) and insoluble (membrane) fractions. Intracellular protein-bound [ 57Co]Cbl fractionated with methionine synthase (MS) and methylmalonyl-CoA mutase (MU) activity. The major form of Cbl associated with the two enzymes was OH-Cbl. Cells propagated in medium containing N 5-methyltetrahydrofolate and homocysteine showed a substantial increase in MS activity which paralleled the increase in the intracellular concentration of Me-Cbl and the Cbl bound to the enzyme.

Serum ferritin and cobalamin in growth retarded fetuses

To examine fetal and maternal serum cobalamin and ferritin concentrations in pregnancies complicated by fetal growth retardation. Harris Birthright Research Centre for Fetal Medicine, King's College Hospital Medical School, London. Cross sectional study. Fetal blood samples obtained by cordocentesis from 20 growth retarded fetuses at 26 to 36 weeks of gestation. Maternal venous blood was also collected and serum ferritin and cobalamin concentrations were measured by radio-immunoassay in the fetal and maternal samples. In the growth retarded group, the mean fetal serum concentration of cobalamin was higher than the normal mean for gestation (t = 3.27, P < 0.01), and this increase was significantly associated with fetal acidaemia (r = -0.686, P < 0.001) and erythroblastosis (r = 0.731, P < 0.001). In contrast, the fetal to maternal ferritin ratio was significantly reduced; there was a nonsignificant decrease in fetal serum and an increase in maternal serum ferritin concentration. There was an association between fetal serum ferritin concentration and erythrocyte count (r = -0.612, P < 0.01). In placental insufficiency, as in postnatal starvation and Kwashiorkor syndrome, uptake and storage of cobalamin by the fetal liver may be impaired. The decrease in fetal to maternal ratio of ferritin could be the consequence of impaired placental perfusion.

Uptake of Cobalamin by Euglena Mitochondria1

Cobalamin uptake by Euglena mitochondria is a biphasic process, consisting of energy-independent cobalamin-binding to mitochondrial membranes and energy-dependent active transport. The energy-dependent phase of cobalamin uptake is not dependent on mitochondrial respiration, but on the presence of ATP within the mitochondrial matrix. The dissociation constant of the energy-independent cobalamin-binding reaction is estimated to be 0.45 nM. Inhibition of the mitochondrial cobalamin uptake by a variety of cobalamin analogues indicates that Euglena mitochondria have an absolute requirement for the complete cobalamin molecule with an alpha-axial ligand (the cobalt-coordinated nucleotide) and an intact b-propionamide side-chain. Thus, the Euglena mitochondrial cobalamin uptake system is highly specific for the cobalamin structure. The cobalamin taken up by the Euglena mitochondria cannot be exchanged with exogenous cobalamin. All of the mitochondrial cobalamin is associated with three proteins with molecular masses of > 700,000 (16.3%), 160,000 (7.4%), and 35,000 (76.3%). They occur in the soluble fraction of mitochondria, suggesting that these cobalamin-binding proteins or cobalamin-dependent enzymes play an important role in cobalamin accumulation and metabolism within the mitochondria.

The proton motive force drives the outer membrane transport of cobalamin in Escherichia coli.

Cells of Escherichia coli pump cobalamin (vitamin B12) across their outer membranes into the periplasmic space, and it was concluded previously that this process is potentiated by the proton motive force of the inner membrane. The novelty of such an energy coupling mechanism and its relevance to other outer membrane transport processes have required confirmation of this conclusion by studies with cells in which cobalamin transport is limited to the outer membrane. Accordingly, I have examined the effects of cyanide and of 2,4-dinitrophenol on cobalamin uptake in btuC and atp mutants, which lack inner membrane cobalamin transport and the membrane-bound ATP synthase, respectively. Dinitrophenol eliminated cobalamin transport in all strains, but cyanide inhibited this process only in atp and btuC atp mutant cells, providing conclusive evidence that cobalamin transport across the outer membrane requires specifically the proton motive force of the inner membrane. The coupling of metabolic energy to outer membrane cobalamin transport requires the TonB protein and is stimulated by the ExbB protein. I show here that the tolQ gene product can partly replace the function of the ExbB protein. Cells with mutations in both exbB and tolQ had no measurable cobalamin transport and thus had a phenotype that was essentially the same as TonB-. I conclude that the ExbB protein is a normal component of the energy coupling system for the transport of cobalamin across the outer membrane.

Increased uptake and accumulation of cobalamin by multiple myeloma bone marrow cells as a possible cause of low serum cobalamin

Increased uptake and accumulation of cobalamin by multiple myeloma bone marrow cells as a possible cause of low serum cobalamin

Different corrinoid specificities for cell growth and the cobalamin uptake system in Euglena gracilis z

A variety of cobalamin analogues synthesized chemically or microbiologically were used to study the corrinoid specificities of cell growth and the cobalamin uptake system in Euglena gracilis z. Although Euglena could not utilize most of the analogues for cell growth, benzimidazolyl cobamide, cyanocobalamin-O 5-phosphate, and cyanocobalamin-b-, -d-and -e-monocarboxylates had effects similar to that of cyanocobalamin on cell growth. It is suggested that Euglena cells have the ability to synthesize ‘complete cobalamin’ from the acid derivatives (amidation reaction) and/or the phosphate derivative (dephosphorylation reaction). Inhibition of uptake of radiolabelled cyanocobalamin caused by addition of various analogues indicates that both the α-lower axial ligand (the cobalt-coordinated nucleotide) and the (b)-propionamide side-chain of the cobalamin molecule are essential for the cobalamin uptake system in Euglena. These results indicate that there are different corrinoid specificities for cell growth and the cobalamin uptake system in E. gracilis z.

Immunogold localization of intrinsic factor in rat and human stomach

There are several proteins that mediate uptake and transport of dietary cobalamin (vitamin B12). The gastric intrinsic factor (IF) is one of these which binds cobalamin in the gut lumen and allows for efficient intestinal absorption of the ingested vitamin. The IF has a rather novel and complicated secretory pathway and precise knowledge of the expression and cellular distribution will help us to understand the recognized defects in vitamin B12 absorption and metabolism. Earlier studies using autoradiographic and immunocytochemical methods have identified different IF production sites for various animals: gastric chief cells in rodents; antropyloric cells in the hog; parietal cells in humans and most other species. In the present work, we have used immunogold electron microscopy for cellular and subcellular localization of IF.

Glial cells as a model for the role of cobalamin in the nervous system: Impaired synthesis of cobalamin coenzymes in cultured human astrocytes following short-term cobalamin-deprivation

The conversion of cyanocobalamin to adenosyl- and methylcobalamin is impaired in cobalamin-deficient cultured human glial cells. In contrast cultured human skin fibroblasts retained their ability to synthesize coenzyme forms when grown in cobalamin-deficient medium. Cells were pre-conditioned by growing in cobalamin-deficient media for six weeks and then subcultured in medium containing either free or transcobalamin II-bound 57Co-cyanocobalamin. Although both coenzyme levels were low in cobalamin-deficient glial cells, the decrease in methylcobalamin was more marked than that of adenosylcobalamin. Methionine synthase and Cbl reductase activities were markedly decreased in cobalamin-deficient glial cells but were unchanged in fibroblasts cultured in cobalamin-deficient medium. Our data suggest that in glial cells, cobalamin coenzyme synthesis and function is exquisitely sensitive to short-term cobalamin deprivation. Glial cells apparently synthesize and secrete transcobalamin II since antibodies directed against the transport protein inhibit the uptake of free cobalamin.

The cDNA sequence and the deduced amino acid sequence of human transcobalamin II show homology with rat intrinsic factor and human transcobalamin I.

The cellular uptake of cobalamin (Cbl, vitamin B12) is mediated by transcobalamin II (TCII), a plasma protein that binds Cbl and is secreted by human umbilical vein endothelial (HUVE) cells. These cells synthesize and secrete TCII and, therefore, served as the source of the complementary DNA (cDNA) library from which the TCII cDNA was isolated. This full-length cDNA consists of 1866 nucleotides that code for a leader peptide of 18 amino acids, a secreted protein of 409 amino acids, a 5'-untranslated segment of 37 nucleotides, and a 3'-untranslated region of 548 nucleotides. A single 1.9-kilobase species of mRNA corresponding to the size of the cDNA was identified by Northern blot analysis of the RNA isolated from HUVE cells. TCII has 20% amino acid homology and greater than 50% nucleotide homology with human transcobalamin I (TCI) and with rat intrinsic factor (R-IF). TCII has no homology with the amino-terminal region of R-IF that has been reported to have significant primary as well as secondary structural homology with the nucleotide-binding domain of NAD-dependent oxidoreductases. The regions of homology that are common to all three proteins are located in seven domains of the amino acid sequence. One or more of these conserved domains is likely to be involved in Cbl binding, a function that is common to all three proteins. However, the difference in the affinity of TCII, TCI, and R-IF for Cbl and Cbl analogues indicates, a priori, that structural differences in the ligand-binding site of these proteins exist and these probably resulted from divergence of a common ancestral gene.

Intrinsic Factor Secretion and Cobalamin Absorption: Physiology and Pathophysiology in the Gastrointestinal Tract

Intrinsic factor is produced by the gastric parietal cell. Its secretion is stimulated via all pathways known to stimulate gastric acid secretion: histamine, gastrin, and acetylcholine. There is, however, a different mode of secretion for both substances: atropine, vagotomy, and H2 receptor antagonists inhibit both intrinsic factor and acid secretion, but secretin and the hydrogen-potassium ATPase antagonist omeprazole have no effect on intrinsic factor while substantially reducing acid secretion. Cobalamin in food is bound to animal protein. Cobalamin deficiency due to inadequate dietary intake is rarely seen in extreme vegetarians (vegans). In the stomach cobalamin is liberated from its protein binding by peptic digestion and bound to R-proteins. Hypochlorhydria or achlorhydria, whether medically induced or not, may impair cobalamin uptake. The cobalamin-R-protein complex is split by pancreatic enzymes in the duodenum, where cobalamin is bound to intrinsic factor. Pancreatic insufficiency may lead to cobalamin deficiency. Lack of intrinsic factor is the commonest cause of cobalamin deficiency; very rarely, aberrant forms of intrinsic factor are produced, but the clinical syndrome is similar. Gram-negative anaerobe bacteria bind the cobalamin-intrinsic factor complex, and bacterial overgrowth of the small intestine diminishes cobalamin resorption. Parasitic infections with fish tape-worm and Giardia lamblia are also associated with cobalamin malabsorption. The cobalamin-intrinsic factor complex binds to the ileal receptors in the terminal ileum. Cobalamin absorption may be impaired after resection or by diseases affecting more than 50 cm of the terminal ileum, such as Crohn's disease, coeliac disease, tuberculosis, lymphoma or radiation. There is clearly a wide diversity in the aetiology of cobalamin deficiency, which requires a versatile diagnostic approach.