B12 Complex Research Articles

Effect of folate supplementation on clinical chemistry and hematologic changes related to bidisomide administration in the rat.

In a chronic toxicity study in the rat, bidisomide administered as a dietary admixture produced a dose-related lowering of reticulocytes and leucocytes. Plasma alanine aminotransferase activity was increased at 300 mg/kg and decreased at 900 mg/kg. The potential mechanisms of these effects were investigated by comparing the responses in groups of male Sprague-Dawley rats receiving a control diet, or 300 or 1200 mg/kg/day bidisomide. Subsets of these groups were co-treated subcutaneously with folinic acid or with a vitamin B1, B6, B12 complex. Subsets of control and 300 mg/kg groups were maintained on a 20-25% feed restriction regimen for 3 months, to mimic the depression in body weight gain observed in animals receiving 1200 mg/kg. Body weight gains were significantly reduced at 1200 mg/kg and in all feed-restricted animals. Plasma and liver alanine aminotransferase (ALT) and plasma aspartate aminotransferase (AST) levels were also reduced at this dose level. At 300 mg/kg, plasma transaminases, glutamate dehydrogenase (GLDH) and sorbitol dehydrogenase (SDH) activities were increased. These changes were prevented in animals receiving folinic acid supplementation. Plasma glucose, triglycerides, and unsaturated and total iron binding capacities were decreased, while plasma iron levels tended to increase, mainly at the high dose. Vitamin supplementation prevented a decrease in reticulocyte counts at 300 mg/kg. Bidisomide increased urinary formimino-glutamic acid (FIGLU) excretion but did not affect methylmalonic acid (MMA) or taurine excretion. The effect on FIGLU at 1200 mg/kg was prevented by folinic acid co-treatment. Absolute liver weight was lowered at both dose levels and in feed-restricted animals. However, the relative liver weights were unaffected. Thymidine kinase and thymidylate synthase activity of the bone marrow cells were not altered by the bidisomide treatment. Except for the increase in plasma transaminase, GLDH and SDH levels at 300 mg/kg, changes in clinical chemistry parameters are considered to result mainly from nutritional restrictions. Changes in hematologic parameters appear to be related to the combination of decreased feed consumption (leukocytes) and decreased availability or utilization of folates (reticulocytes). This alteration, however, did not affect DNA synthesis in bone marrow. The prevention by folinic acid, but not by feed restriction, of the elevation of liver enzymes at 300 mg/kg is an intriguing, yet unexplained finding. There was no evidence that bidisomide affected B6 and B12 availability.

Formation of a square-planar Co(I) B12 intermediate. Implications for enzyme catalysis

X-ray edge and extended x-ray absorption fine structure (EXAFS) techniques provide powerful tools for analysis of local molecular structure of complexes in solution. We present EXAFS results for Co(I) B12 that demonstrate a four-coordinate (distorted) square-planar configuration. Comparison of EXAFS solutions for Co(I) and Co(II) B12 (collected previously; Sagi et al. 1990. J. Am. Chem. Soc. 112:8639-8644) suggest that modulation of the Co-N bond to the axial 5,6-dimethylbenzimidazole (DMB), in the absence of changes in Co-N (equatorial) bond distances, may be a key mechanism in promoting homolytic versus heterolytic cleavage. As Co-C bond homolysis occurs, the Co-N (DMB) bond becomes stronger. However, for heterolytic cleavage to occur, earlier electrochemical studies (D. Lexa and J. M. Saveant. 1976. J. Am. Chem. Soc. 98:2652-2658) and recent studies of methylcobalamin-dependent Clostridium thermoaceticum (Ragsdale et al. 1987. J. Biol. Chem. 262:14289-14297) suggest that removal of the DMB ligand (before Co-C bond cleavage) favors formation of the four-coordinate square-planar Co(I) species while inhibiting formation of the five-coordinate Co(II) B12 complex. This paper presents the first direct evidence that formation of the Co(I) B12 intermediate must involve breaking of the Co-N (DMB) bond.

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.

UV-visible spectra and equilibria of vitamin B12 complexes with phosphite ligands

UV-visible spectra and equilibria of vitamin B12 complexes with phosphite ligands

Vitamin B12 transport in Escherichia coli: energy coupling between membranes

Cells of Escherichia coli possess high-affinity active transport systems of vitamin B12 and iron-siderophore complexes. Specific outer-membrane proteins carry out the energy-dependent transport across the outer membrane, in conjunction with the TonB coupling protein. Mutagenesis experiments have identified a conserved region near the amino-terminus of the outer-membrane transporters that is necessary for energy-coupled transport. The ability of extragenic suppressor mutations in tonB to correct the transport defect indicates that TonB couples the proton-motive force to the outer-membrane proteins by direct contact.

Vitamin B12-mediated electrochemical reactions in the synthesis of natural products

Abstract

Outer-membrane permeability of bacteria.

Gram-negative bacteria evolved to survive under the conditions in which a number of hazardous compounds are abundant. The outer membrane which protects the cell interior acts as a barrier against such hazardous agents, yet the cells must incorporate the chemicals that are essential for the cellular activity. The devices that Gram-negative bacteria developed to incorporate such essence are the transmembrane pores. These pores could be subdivided into three categories: (1) pore made of porins has a weak solute selectivity; (2) pore made of lamB protein and tsx proteins hold intermediate solute specificity. and (3) pores for the diffusion of vitamin B12 and ferric ion-chelator complexes have a tight solute specificity. Porins are identified from a number of Gram-negatives and from the outer membrane of mitochondria of various sources. Studies on the diffusion properties of these outer-membrane proteins provided essential information to understand membrane transports.

Determination of the R-protein and the R-protein-vitamin B12-complex in saliva and gastrointestinal juice by FPLC Mono S cationic chromatography.

The binding of vitamin B12 to the two vitamin B12 binding proteins--intrinsic factor and R-protein--in gastrointestinal juice is pH dependent. It is therefore of importance that binding studies are carried out at pH values near the physiological pH of the gastric juice. In the present study the vitamin B12 complexes of the two vitamin B12 binders were separated at a pH of 1.8 using the cationic exchange chromatograph Mono S attached to the fast protein liquid chromatography (FPLC) system. The R-protein concentration was measured in saliva and gastric juice with high accuracy and with highly significant correlation compared to the serum-coated charcoal method of Gottlieb. The method showed a decreased recovery in duodenal juice and in samples with high bile content.

Formation of (C-C) bonds catalyzed by vitamin B12

Abstract

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.

9] Solubilization of the receptor for intrinsic factor—B12 complex from guinea pig intestinal mucosa

Publisher Summary This chapter discusses an overview of solubilization of the receptor for intrinsic factor-B 12 complex from guinea pig intestinal mucosa. The absorption of dietary vitamin B 12 from the intestine in many animal species requires the presence of intrinsic factor (IF), a glycoprotein secreted by the gastric mucosa. Normally, this protein is produced in large excess over actual requirements and is rarely a limiting factor in absorption. In vitro studies employing everted intestinal sacs, mucosal homogenates, and microvillous membranes prepared from the distal, but not the proximal, small bowel show uptake of IF-B 12 complex in the presence of a physiologic pH and calcium ions. The implication of this is that a receptor for IF-B 12 complex exists at the surface of the ileal cell. This chapter describes solubilization of guinea pig ileal mucosal homogenates employing the detergent Triton X-100. A particle-free solution was obtained that contained all the IF-B 12 receptors estimated to be present on the starting intestinal membranes. It is expected that better quantitation of IF- B12 receptor will help investigators to elucidate the mechanisms of B 12 absorption and malabsorption. The method employed for assaying and quantitating this solubilized receptor is also described in the chapter.

Dissociation of the Intrinsic Factor-Vitamin B12Complex by Bile: Contributing Factor to B12Malabsorption in Pancreatic Insufficiency

Human bile incubated with vitamin B12 bound to intrinsic factor in human gastric juice will effectively dissociate this complex, and the vitamin will transfer to non-intrinsic factor unsaturated binding protein(s) contained in bile. Preincubation of the bile with pancreatic enzymes, particularly trypsin, and pepsin, decreases this effect of bile on the intrinsic factor--vitamin B12 complex by digesting the unsaturated binder(s) in the bile. These studies help explain why there is malabsorption of tracer amounts of vitamin B12 in some patients with pancreatic insufficiency, and why this abnormality is correctable by the administration of pancreatic extract.

Perturbed angular correlation studies of Hf binding to cyanocobalamin (vitamin B12)

Gamma ray perturbed angular correlation (PAC) experiments have been carried out with 181Hf labeled cyanocobalamin. Evidence is presented which strongly indicates that Hf binds to vitamin B12 at the phosphate group linking the sugar residue to a side chain of the corrin ring system. Analysis of the time-differential PAC spectra for the crystalline Hf–B12 complex indicates a static electric quadrupole interaction at the Hf nucleus, corresponding to the electric field gradient generated by the chemical bonding. The magnitudes of the derived interaction parameters are similar to those found in Hf phosphate compounds. In aqueous solution, the Hf–B12 complex exhibits PAC spectra which appear to originate from two sources. Approximately 3/4 of the Hf nuclei experience a static electric quadrupole interaction with the same characteristic interaction frequency as in the solid, but with an increased asymmetry parameter. Approximately 1/4 of the Hf signal strength is attributable to a time-dependent quadrupole interaction with a relaxation constant indicative of an effective molecular entity comparable in size to the B12 molecule. This effect may be related to molecular motion in the solution. These results demonstrate the utility of the PAC experimental method for the study of macromolecular species in both the solid and solution forms, and opens possibilities for obtaining new information concerning the structure, orientation, and behavior of macromolecules.

A rapid polyethylene glycol assay for gastric intrinsic factor.

The use of polyethylene glycol (mol. wt 4000) in a rapid radioassay for intrinsic factor is reported. The assay is based on the observation that polyethylene glycol precipitates the intrinsic factor-vitamin B12 complex in the presence of serum from patients with pernicious anaemia having auto-antibodies against intrinsic factor. The assay was found highly reproducible (CV 1.8%) and well correlated to the classical method using coated charcoal in the separation step.

ChemInform Abstract: INTERACTION OF VITAMIN B12A WITH 8‐AZAGUANINE AND 6‐MERCAPTOPURINE: KINETIC AND THERMODYNAMIC CHARACTERIZATIONS

AbstractDie Geschwindigkeitskonstanten für die Bildung und Zersetzung der 6‐Mercaptopurin‐ und 8‐Azaguanin‐Addukte von Vitamin B123 sowie ihre Stabilitätskonstanten werden in wäßriger gepufferter Lösung als Funktion vom pH‐Wert bei 25°C bestimmt.

Effect of exocrine pancreatic insufficiency on small intestine in the mouse

A genetically conditioned mouse model of exocrine pancreatic insufficiency (epi) has been used to study the effect of the absence of lumenal proteases on small intestinal mucosal proteins. The small bowel was divided into eight equal segments. Enzyme activity was increased only in the first three segments in the case of maltase, sucrase, and lactase (all mol wt above 200,000). Alkaline phosphatase (mol wt 145,000), trehalase (mol wt 95,000), and peptidase (mol wt 175,000) activities were unaffected in proximal segments from epi mice. Proximal brush border proteins were identified and measured quantitatively by sodium dodecyl sulfate acrylamide gel electrophoresis. Those enzymes with increased activity were associated with increased amounts of protein in epi mice. Double labeled studies of protein turnover revealed a longer half-life for large brush border proteins (mol wt above 175,000) in epi mice than in normal mice. Enterokinase activity (a marker for duodenal mucosa) was nearly absent from the duodenum of epi mice. Receptors for the intrinsic factor-vitamin B12 complex (markers for ileal mucosa) were present in the ileum equally in normal and in epi mice. Enterokinase activity can be induced in epi mice by feeding its substrate trypsinogen, but not by trypsin or chymotrypsinogen. Epi mice thus retain the ability to synthesize enterokinase. Pancreatic proteases play an important role in the turnover of certain large mucosal proteins and in the induction of enterokinase.

Interaction of vitamin B12a with 8-azaguanine and 6-mercaptopurine: kinetic and thermodynamic characterizations

Rate constants for the formation, k1app., and decomposition, k–1app. of 6-mercaptopurine and 8-azaguanine adducts of vitamin B12a, B12–mpur and B12–agua, and hence their stability constants Kapp.=k1app./k–1app., have been determined in aqueous buffered solutions as a function of pH at 25.0 °C. The pH–rate profiles for the formation of B12–mpur and B12–agua are bell shaped with maxima at pH 7.5 and 7.0, respectively. Rate constants for the decomposition of B12–mpur decrease curvilinearly with increasing pH, having a short plateau in the pH 7–9 region. k–1app. Values for the decomposition of B12–agua do not change between pH 6 and 8, but they increase exponentially with increasing hydrogen-ion concentration at pH <6. Kinetic treatment of the data in terms of dissociation constants for vitamin B12a, the ligand, and the vitamin B12 complexes, and in terms of the reactivities of these species, affords pH-independent rate constants for the formation, k1, and for the decomposition, k–1, of these vitamin B12 complexes. k1 and k–1 values for B12–mpur are 800 dm3 mol–1 s–1, and those for B12–agua are 220 dm3 mol–1 s–1 and 2.0 × 10–2 s–1, respectively. The mechanism of these reactions and their pharmaceutical potential are discussed.

A radioisotope dilution assay for unlabelled vitamin B12-intrinsic factor complex employing the binding intrinsic factor antibody: probable evidence for two types of binding antibody.

A new radioisotope dilution assay for vitamin B12-intrinsic factor complex is described. The method is based on the use of the binding type intrinsic factor antibody (the binding reagent), which when combined with the intrinsic factor-vitamin B12 complex (labelled ligand), is quantitatively adsorbed onto zirconium phosphate gel at pH 6.25. The new assay has been shown to provide a measure of intrinsic factor comparable with other intrinsic factor assays, but it has the important advantage of being able to measure the unlabelled vitamin B12-intrinsic factor complex (unlabelled ligand), and will, therefore, be valuable in the study of physiological events in the gastrointestinal tract. During the study, it was found that there is some evidence for a least two types of binding intrinsic factor antibody: One which combines preferentially with the intrinsic factor-vitamin B12 complex and one which combines equally well with this complex or with free intrinsic factor.

A saturable high affinity binding site for transcobalamin II-vitamin B12 complexes in human placental membrane preparations.

Studies were designed to evaluate the binding of binding of vitamin B12 to cell membrane preparations from human placenta. The transcobalamin II-vitamin B12 complex (TCII-B12), which has a much greater affinity for the membranes than vitamin B12 alone, binds to a single saturable binding site with an approximate Ka = 7.2 mM-1. The binding requires a divalent cation and is temperature-dependent. Free TCII can compete with TCII-B12 for the binding site but has somewhat less affinity than does TCII-B12. Rat TCII-B12 has an affinity constant that is less than one-fifth that of human TCII-B12; human TCI-B12, bovine TCII-B12, hog intrinsic factor-B12 (IF-B12), and human IF-B12 do not bind to the membranes. Pretreating the membranes with trypsin causes a marked decrease in subsequent binding; this suggests the binding site includes a relatively exposed membrane protein. These data suggest that a specific cell surface receptor for the TCII-B12 complex exists in placenta. This TCII-B12 receptor can be solubilized with Triton X-100.

Effects of divalent cations on vitamin B12 adsorption to brush borders of rat intestine.

A brush border preparation from rat intestine was incubated with rat intrinsic factor-vitamin B12 complex in 0.01 M Tris-HCl buffer, pH 7.4. The 57Co-B12 uptake to brush borders was proportional to the amount of protein or to alkaline phosphatase activity in the preparations. The uptake increased with time of incubation. At 37degreesC, the uptake after incubation for 15 min was 80-85% of that for one hr. The uptake at 4degreesC was approximately 70% of that at 37degreesC. Ther was no difference as a result of adding glucose to the incubation medium. The uptake was observed in the alkaline environment above pH 6.3. Maximum uptake occurred at pH 8.0. Brush borders washed with Krebs-Ringer bicarbonate buffer (pH 7.4) exhibited no difference in B12 uptake, whether in the presence or absence of calcium ion. But brush borders washed with ethylenediaminetetraacetate exhibited no uptake when incubated in calcium-free medium. The uptake reached a maximum by addition of calcium ion at a concentration of 0.3 mM, and was not alter up to 10 mM. Addition of magnesium ion exhibited no uptake. Calcium-dependent B12 uptake was markedly inhibited by manganese ion. Magnesium ion seemed to slightly inhibit the calcium-dependent uptake.