Factor-vitamin B12 Research Articles

Recognizing, treating and understanding pernicious anaemia.

Recognizing, treating and understanding pernicious anaemia.

Intrinsic factor and vitamin B12 complex‐loaded poly[lactic‐co‐(glycolic acid)] microspheres: preparation, characterization and drug release

AbstractBACKGROUND: Vitamin B12 is an essential vitamin required by all mammals. Absorption of vitamin B12 is facilitated by binding of intrinsic factor–vitamin B12 complex to specific receptors in the ileum. In humans a deficiency of this vitamin or a lack of intrinsic factor leads to pernicious anaemia. The major objective of the present study was to prepare intrinsic factor–vitamin B12 complex‐loaded poly[lactic‐co‐(glycolic acid)] (PLGA)‐based microparticles and to investigate their release kinetics.RESULTS: PLGA copolymer was synthesized by the ring‐opening polymerization method and characterized using gel permeation chromatography, Fourier transform infrared spectroscopy and 1H NMR. The glass transition temperature measurement showed a single Tg at 40 °C. The intrinsic factor–vitamin B12 complex‐loaded PLGA microspheres were prepared by a water‐in‐oil‐in‐water double emulsion solvent extraction/evaporation technique. An environmental scanning electron microscopy investigation demonstrated that the PLGA particles had a mean particle diameter of 38 µm. Interestingly, different drug release patterns (bi‐ and triphasic ones) were observed for vitamin B12‐loaded and intrinsic factor–vitamin B12 complex‐loaded microspheres. In contrast to the rapid release of vitamin B12 by itself, in vitro release tests showed that intrinsic factor and vitamin B12 in the complex were released from PLGA microspheres in a sustained manner over 15 days.CONCLUSION: PLGA microspheres can be an effective carrier for the intrinsic factor–vitamin B12 complex. Copyright © 2007 Society of Chemical Industry

Síndromes mielodisplásticas: diagnóstico de exclusão

Myelodysplastic syndromes are common in elderly people. Laboratory presentation includes isolated macrocytosis, anemia, isolated or combined cytopenias and dysplastic bone marrow. Diagnosis depends on exclusion of non-clonal and reversible disorders. Especially in lowest grade of the disease, with no blast excess, no ringed sideroblasts, no clonal cytogenetic abnormalities the diagnosis requires an exclusion protocol. Recent exposure to toxin, cytotoxic drugs or growth factor therapy and vitamin B12 or folate deficiency are considered absolute exclusion factors precluding the definite diagnosis. Alcohol abuse, chronic inflammatory states, auto-immune disorders, metabolic dysfunctions, hormonal disorders and viral infections must all be ruled out or interpreted with caution. Some diseases of the pluripotential stem cell must also be considered especially in hypocellular MDS. Moreover, in some cases a 6-month follow-up is necessary before a diagnosis be established.

Plasma homocysteine concentration and blood pressure in healthy Iranian adults: the Tehran Homocysteine Survey (2003–2004)

Hypertension is a modifiable risk factor for cardiovascular disease, but its aetiology has not been fully elucidated. Recently, attention has been focused on the direct relations of plasma homocysteine (Hcy) to blood pressure (BP). The purpose of this study was to determine the relationship of Hcy with BP and other cardiovascular disease risk factors. This population-based study was a part of the Cardiovascular Risk Factors Survey and has been designed and conducted based on the methodology of the MONICA/WHO Project. A total of 1214 people aged 25-64 years were recruited using cluster sampling and assessed regarding standardized methods. BP was measured in seated position after a 10-min rest period. Blood samples were gathered and analysed according to standard methods. Variables were assessed in 1191 participants (416 men; 775 women). Mean age was higher in hypertensives compared to normotensives (P < 0.001). Mean Hcy was higher in hypertensives, but significant only in men (P < 0.031). Concurrent effects of Hcy, folate and vitamin B12 on hypertension indicated that Hcy acts as a risk factor and folate and vitamin B12 as protective factors; however, after adjustment, just vitamin B12 remained as a protective factor. Although we detected a simple correlation of Hcy with BP in Iranian adults, this relationship was no longer significant after applying an adjustment. In light of our observations, it is likely that the increased Hcy levels reported in hypertensive persons are concomitant rather than a precursor of hypertension.

Pathophysiology of cubilin: of rats, dogs and men

At variance from small solutes which are reabsorbed by transporters and channels, proteins present in the proximal convoluted tubule lumen are internalized by endocytosis via a vesicular system, transferred to the lysosomes and degraded. We summarize here recent data on cubilin, a 460 kDa peripheral protein which, together with megalin, serves as a multiligand receptor providing for the reabsorption of most proteins present in the tubule lumen. Interestingly, cubilin is also expressed by the yolk sac, which has endocytic and degradative properties reminiscent of the proximal tubule and serves as the receptor for intrinsic factor-vitamin B12 complexes.

Subacute combined degeneration one century later. The neurotrophic action of cobalamin (vitamin B12) revisited.

It was exactly 100 yr ago when Russell et al (1) published the paper in which they first suggested the term “subacute combined degeneration” (SCD) to define the neuropathy observed in association with pernicious anemia (PA). The reasons for this definition were the rapid course of this neurological disease, which usually affects the peripheral nerves and the different columns of the spinal cord (SC), leading to a combination of tract degeneration (1). Although many other terms for this neurological disorder have since been tentatively introduced, SCD is generally used by most authors and will be used in this review. Russell et al (1) firmly established that SCD is a clinical entity associated with especially long-standing PA. A few years earlier, Nonne argued that SCD should be considered the neurological equivalent of PA (2), rather than a secondary state dependent on the hematopoietic disease, although the lack of a correlation between the degree of anemia and the severity of SCD became clear later on (3, 4). When the anti-PA liver factor vitamin B12 (also called cobalamin [Cbl]) was first isolated and its 3-dimensional structure determined, it came to be routinely used in the therapy of both PA and SCD, thus firmly establishing the close and widely recognized relationship between the two. For further details concerning the historical aspects of the identification of SCD and the development of our understanding of the disease over time, the reader is referred to previous broader reviews and books (5–7). It is generally accepted that the most prominent histopathological changes of SCD take the form of a diffuse but uneven degeneration of SC white matter, especially affecting the posterior and lateral columns (5–7). Although imprecise in some respects, the term “SCD” is applied to the irregular demyelination of SC white matter (so-called “spongiform demyelination” …

Recent insights into the molecular genetics of the homocysteine metabolism

The homocysteine plasma level is determined by non-genetic and genetic factors. In recent years evidence has accumulated that the total homocysteine plasma level of patients under different forms of renal replacement therapy is influenced by a common mutation at nucleotide position 677 of the gene coding for 5,10-methylenetetrahydrofolate reductase (MTHFR 677C-->T). Furthermore, compound heterozygosity for the 677T allele and a novel A-->C polymorphism at nucleotide position 1298 of MTHFR is suggested to correlate with a decrease of folate plasma concentrations. Because polymorphisms of genes coding for proteins involved in the metabolism of homocysteine may contribute to elevated total homocysteine plasma concentrations, molecular genetic analyses of the homocysteine pathways experienced a drift towards screening for candidate genes with a putative relationship to total homocysteine plasma levels. One example is the cloning of the FOLR1 gene coding for the folate-binding protein (Folbp1), which has recently been inactivated in mice, thus representing an elegant model to investigate the consequence on the homocysteine metabolism. Furthermore, the recent characterization of the CUBN gene encoding the intrinsic factor-vitamin B12 receptor (cubilin) provides a basis to identify the causative mutations in patients suffering from a hereditary syndrome of hyperhomocysteinemia that presents with megaloblastic anemia and proteinuria. This review focuses on recent insights into the molecular genetics of MTHFR, FOLR1, and CUBN, and their relationships to the metabolism of the amino acid homocysteine.

Cubilin- and megalin-mediated uptake of albumin in cultured proximal tubule cells of opossum kidney

Reabsorption of albumin from the glomerular filtrate occurs via receptor-mediated endocytosis in the proximal tubule. This process is initiated by binding of albumin in apical clathrin-coated pits, followed by endocytosis and degradation in lysosomes. Although binding sites have been characterized by kinetic studies, the receptors responsible for the binding of albumin have not been fully identified. Two giant glycoproteins, cubilin and megalin, constitute important endocytic receptors localized to the kidney proximal tubule. In the present study, we examined the colocalization of cubilin and megalin in the endocytic pathway and the relationship between the uptake of albumin and the expression of cubilin and megalin in opossum kidney (OK) proximal tubule cells by immunocytochemistry and immunoblotting. OK cells expressed both cubilin and megalin. The light microscope labeling patterns for cubilin and megalin were almost identical and were mainly located at the surface area of the cells. Cubilin and megalin were also shown to colocalize on cell surface microvilli, in coated pits, and in endocytic compartments at the electron microscope level. Endocytosed bovine serum albumin (BSA) was identified exclusively in cells expressing megalin and cubilin. Uptake of BSA-FITC was saturable and inhibited by receptor-associated protein (RAP) and by intrinsic factor-vitamin B12 complex (IF-B12) at high concentrations. Significant inhibition was also observed by specific antibodies to cubilin, and megalin and cubilin antisense oligonucleotides likewise significantly reduced albumin uptake. Egg albumin did not affect the uptake of BSA. The present observations suggest that the two receptors cubilin and megalin are both involved in the endocytic uptake of albumin in renal proximal tubule cells.

Cubilin P1297L mutation associated with hereditary megaloblastic anemia 1 causes impaired recognition of intrinsic factor–vitamin B12 by cubilin

AbstractMegaloblastic anemia 1 (MGA1) is an autosomal recessive disorder caused by the selective intestinal malabsorption of intrinsic factor (IF) and vitamin B12/cobalamin (Cbl) in complex. Most Finnish patients with MGA1 carry the disease-specific P1297L mutation (FM1) in the IF-B12 receptor, cubilin. By site-directed mutagenesis, mammalian expression, and functional comparison of the purified wild-type and FM1 mutant forms of the IF–Cbl-binding cubilin region (CUB domains 5-8, amino acid 928-1386), we have investigated the functional implications of the P1297L mutation. Surface plasmon resonance analysis revealed that the P1297L substitution specifically increases the Kd for IF–Cbl binding several-fold, largely by decreasing the association rate constant. In agreement with the binding data, the wild-type protein, but not the FM1 mutant protein, potently inhibits 37°C uptake of iodine 125–IF–Cbl in cubilin-expressing epithelial cells. In conclusion, the data presented show a substantial loss in affinity of the FM1 mutant form of the IF–Cbl binding region of cubilin. This now explains the malabsorption of Cbl and Cbl-dependent anemia in MGA1 patients with the FM1 mutation.

Cubilin P1297L mutation associated with hereditary megaloblastic anemia 1 causes impaired recognition of intrinsic factor–vitamin B12 by cubilin

Megaloblastic anemia 1 (MGA1) is an autosomal recessive disorder caused by the selective intestinal malabsorption of intrinsic factor (IF) and vitamin B12/cobalamin (Cbl) in complex. Most Finnish patients with MGA1 carry the disease-specific P1297L mutation (FM1) in the IF-B12 receptor, cubilin. By site-directed mutagenesis, mammalian expression, and functional comparison of the purified wild-type and FM1 mutant forms of the IF–Cbl-binding cubilin region (CUB domains 5-8, amino acid 928-1386), we have investigated the functional implications of the P1297L mutation. Surface plasmon resonance analysis revealed that the P1297L substitution specifically increases the Kd for IF–Cbl binding several-fold, largely by decreasing the association rate constant. In agreement with the binding data, the wild-type protein, but not the FM1 mutant protein, potently inhibits 37°C uptake of iodine 125–IF–Cbl in cubilin-expressing epithelial cells. In conclusion, the data presented show a substantial loss in affinity of the FM1 mutant form of the IF–Cbl binding region of cubilin. This now explains the malabsorption of Cbl and Cbl-dependent anemia in MGA1 patients with the FM1 mutation.

Molecular Dissection of the Intrinsic Factor-Vitamin B12 Receptor, Cubilin, Discloses Regions Important for Membrane Association and Ligand Binding

Cubilin, the receptor for intrinsic factor-vitamin B12, is a novel type of high molecular weight receptor consisting of a 27 CUB (complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domain cluster preceded by 8 epidermal growth factor repeats and a short N-terminal sequence. In addition to binding the vitamin B12-carrier complex, cubilin also binds receptor-associated protein. To delineate the structures for membrane association and ligand binding we established a panel of stable transfected Chinese hamster ovary cells expressing overlapping segments of rat cubilin. Analysis of conditioned media and cell extracts of transfected cells revealed that the N-terminal cubilin region conveys membrane association. Helical plotting of this region demonstrated a conserved amphipathic helix pattern (Lys74-Glu109) as a candidate site for hydrophobic interactions. Ligand affinity chromatography and surface plasmon resonance analysis of the secreted cubilin fragments showed ligand binding in the CUB domain region. Further dissection of binding-active fragments localized the binding site for intrinsic factor-vitamin B12 to CUB domains 5-8 and a receptor-associated protein-binding site to CUB domains 13-14. In conclusion, the N-terminal cubilin region seems crucial for membrane association, whereas the CUB domain cluster harbors distinct sites for ligand binding.

The intrinsic factor-vitamin B12 receptor, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of high-density lipoprotein.

Cubilin is the intestinal receptor for the endocytosis of intrinsic factor-vitamin B12. However, several lines of evidence, including a high expression in kidney and yolk sac, indicate it may have additional functions. We isolated apolipoprotein A-I (apoA-I), the main protein of high-density lipoprotein (HDL), using cubilin affinity chromatography. Surface plasmon resonance analysis demonstrated a high-affinity binding of apoA-I and HDL to cubilin, and cubilin-expressing yolk sac cells showed efficient 125I-HDL endocytosis that could be inhibited by IgG antibodies against apoA-I and cubilin. The physiological relevance of the cubilin-apoA-I interaction was further emphasized by urinary apoA-I loss in some known cases of functional cubilin deficiency. Therefore, cubilin is a receptor in epithelial apoA-I/HDL metabolism.

The Intrinsic Factor-Vitamin B12 Receptor, Cubilin, Is Assembled into Trimers via a Coiled-coil α-Helix

A large protein was purified from bovine kidney, using selective extraction with EDTA to solubilize proteins anchored by divalent cation-dependent interactions. An antiserum raised against the purified protein labeled the apical cell surface of the epithelial cells in proximal tubules and the luminal surface of small intestine. Ten peptide sequences, derived from the protein, all matched the recently published sequences for rat (Moestrup, S. K., Kozyraki, R., Kristiansen, M., Kaysen, J. H., Holm Rasmussen, H., Brault, D., Pontillon, F., Goda, F. O., Christensen, E. I., Hammond, T. G., and Verroust, P. J. (1998) J. Biol. Chem. 273, 5235-5242) and human cubilin, a receptor for intrinsic factor-vitamin B12 complexes, identifying the protein as bovine cubilin. In electron microscopy, a three-armed structure was seen, indicating an oligomerization of three identical subunits. This model was supported by the Mr values of about 1,500,000 for the intact protein and 440,000 for its subunits obtained by analytical ultracentrifugation. In a search for a potential assembly domain, we identified a region of heptad repeats in the N-terminal part of the cubilin sequence. Computer-assisted analysis supported the presence of a coiled-coil alpha-helix between amino acids 103 and 132 of the human cubilin sequence and predicted the formation of a triple coiled-coil. We therefore conclude that cubilin forms a noncovalent trimer of identical subunits connected by an N-terminal coiled-coil alpha-helix.

The Intrinsic Factor-Vitamin B12 Receptor and Target of Teratogenic Antibodies Is a Megalin-binding Peripheral Membrane Protein with Homology to Developmental Proteins

The present report shows the molecular characterization of the rat 460-kDa epithelial glycoprotein that functions as the receptor facilitating uptake of intrinsic factor-vitamin B12 complexes in the intestine and kidney. The same receptor represents also the yolk sac target for teratogenic antibodies causing fetal malformations in rats. Determination of its primary structure by cDNA cloning identified a novel type of peripheral membrane receptor characterized by a cluster of eight epidermal growth factor type domains followed by a cluster of 27 CUB domains. In accordance with the absence of a hydrophobic segment, the receptor could be released from renal cortex membranes by nonenzymatic and nonsolubilizing procedures. The primary structure has no similarity to known endocytic receptors but displays homology to epidermal growth factor and CUB domain proteins involved in fetal development, e.g. the bone morphogenic proteins. Electron microscopic immunogold double labeling of rat yolk sac and renal proximal tubules demonstrated subcellular colocalization with the endocytic receptor megalin, which is expressed in the same epithelia as the 460-kDa receptor. Furthermore, megalin affinity chromatography and surface plasmon resonance analysis revealed a calcium-dependent high affinity binding of the 460-kDa receptor to megalin, which thereby may mediate its vesicular trafficking. Due to the high number of CUB domains, accounting for 88% of the protein mass, we propose the name cubilin for the novel receptor.

Characterization of an Epithelial ∼460-kDa Protein That Facilitates Endocytosis of Intrinsic Factor-Vitamin B12 and Binds Receptor-associated Protein

By using receptor-associated protein (RAP) as an affinity target, an intrinsic factor-vitamin B12 (IF-B12)-binding renal epithelial protein of approximately 460 kDa was copurified together with the transcobalamin-B12-binding 600-kDa receptor, megalin. IF-B12 affinity chromatography of renal cortex membrane from rabbit and man yielded the same approximately 460-kDa protein. Binding studies including surface plasmon resonance analyses of the protein demonstrated a calcium-dependent and high affinity binding of IF-B12 to a site distinct from the RAP binding site. The high affinity binding of IF-B12 was dependent on complex formation with vitamin B12. Light and electron microscope autoradiography of rat renal cortex cryosections incubated directly with IF-57Co-B12 and rat proximal tubules microinjected in vivo with the radioligand demonstrated binding of the ligand to endocytic invaginations of proximal tubule membranes followed by endocytosis and targeting of vitamin B12 to lysosomes. Polyclonal antibodies recognizing the approximately 460-kDa receptor inhibited the uptake. Immunohistochemistry of kidney and intestine showed colocalization of the IF-B12 receptor and megalin in both tissues. In conclusion, we have identified the epithelial IF-B12-binding receptor as a approximately 460-kDa RAP-binding protein facilitating endocytosis.

Imerslund‐Gräsbeck Syndrome in a Saudi Family

Imerslund-Gräsbeck syndrome, an autosomal recessive trait of defective uptake of intrinsic factor-vitamin B12 complex by terminal ileum, is described in a Saudi family (two siblings and their first cousin). This rare disease has previously been reported only in Northern Europe and North African Jewish ethnic groups.

291 Growth hormone receptors in human breast cancer: Correlation with oestrogen receptor status

Cubilin- and megalin-mediated uptake of albumin in cultured proximal tubule cells of opossum kidney.Reabsorption of albumin from the glomerular filtrate occurs via receptor-mediated endocytosis in the proximal tubule. This process is initiated by binding of albumin in apical clathrin-coated pits, followed by endocytosis and degradation in lysosomes. Although binding sites have been characterized by kinetic studies, the receptors responsible for the binding of albumin have not been fully identified. Two giant glycoproteins, cubilin and megalin, constitute important endocytic receptors localized to the kidney proximal tubule.In the present study, we examined the colocalization of cubilin and megalin in the endocytic pathway and the relationship between the uptake of albumin and the expression of cubilin and megalin in opossum kidney (OK) proximal tubule cells by immunocytochemistry and immunoblotting.OK cells expressed both cubilin and megalin. The light microscope labeling patterns for cubilin and megalin were almost identical and were mainly located at the surface area of the cells. Cubilin and megalin were also shown to colocalize on cell surface microvilli, in coated pits, and in endocytic compartments at the electron microscope level. Endocytosed bovine serum albumin (BSA) was identified exclusively in cells expressing megalin and cubilin. Uptake of BSA-FITC was saturable and inhibited by receptor-associated protein (RAP) and by intrinsic factor-vitamin B12 complex (IF-B12) at high concentrations. Significant inhibition was also observed by specific antibodies to cubilin, and megalin and cubilin antisense oligonucleotides likewise significantly reduced albumin uptake. Egg albumin did not affect the uptake of BSA.The present observations suggest that the two receptors cubilin and megalin are both involved in the endocytic uptake of albumin in renal proximal tubule cells.

Demonstration of vitamin B12 analogues in human sera not detected by microbiological assay.

Sera were absorbed with polyacrylamide beads to which purified human intrinsic factor was attached. This procedure removed the vitamin B12 analogues which are measured by microbiological assay with Lactobacillus leichmannii and Euglena gracilis and which are measured in an isotope dilution method using intrinsic factor. Such sera still contained B12 analogues that were assayed in an isotope dilution method using a non-intrinsic factor vitamin B12 binder. Such vitamin B12 analogues make up approximately half of the total vitamin B12 analogues in human serum.

Bile acid inhibition of vitamin B12 binding by intrinsic factor in vitro.

The effect of conjugated and unconjugated bile acids on the binding of vitamin B12 to intrinsic factor was investigated. The dihydroxy bile acids (deoxycholic, glycodeoxycholic, taurodeoxycholic, glycochenodeoxycholic, and taurochenodeoxycholic) inhibit the binding of intrinsic factor to vitamin B12 at physiological concentrations. On the other hand, the trihydroxy bile acids (cholic, glycocholic, and taurocholic) are not effective in this respect. The inhibition is dependent both on concentration and time, and its pattern is similar to that previously reported for duodenal juice. On column chromatography, there is a close correlation between the degree in intrinsic factor inhibition and the total acid concentration in the duodenal juice. The binding of vitamin B12 by R protein in saliva is not affected by bile acids. The results show that bile acids at concentrations found in duodenal juice inhibit intrinsic factor vitamin B12 binding. It is suggested that this observation may have physiological significance for vitamin B12 absorption.

Proceedings of the Australian Society for Medical Research.

Proceedings of the Australian Society for Medical Research.