Vitamin B12 Coenzyme Research Articles

Pharmacokinetic profiles of methylcobalamin in rats after multiple administration routes by a simple LC-MS/MS assay with a small volume of plasma

Methylcobalamin (MBL) is a vitamin B12 coenzyme and is effective for treating peripheral neuropathies. Little is known about pharmacokinetics (PK) of MBL in animals, we have developed a simple assay for MBL by using only 0.01 mL of plasma for PK of MBL in rats. Under minimal light exposure (<5 lx), MBL was extracted by a simple protein precipitation using methanol and detected by liquid chromatography with tandem mass spectrometry. MBL in rat plasma at 20–10,000 ng/mL was quantified using only 0.01 mL of plasma. Relative error and relative standard deviation met the acceptance criteria in reproducibility assessments, indicating the robustness of the assay. PK of MBL was evaluated after intravenous, intramuscular, and subcutaneous administration. PK of MBL was dose proportional at 5–20 mg/kg in both intramuscular and subcutaneous administrations. Bioavailability after the two dosing routes was complete (ca. 100 %). The incurred sample reanalysis also supported that the assay is robust. The established assay was successfully applied to PK studies in rats to find that MBL showed high bioavailability after intramuscular and subcutaneous administrations.

Characterization of the core gut microbiota of Nile tilapia (Oreochromis niloticus): indication of a putative novel Cetobacterium species and analysis of its potential function on nutrition.

The genus Cetobacterium has been considered a dominant group of gut bacteria in many freshwater fish, and members of this genus contribute to anaerobic metabolism. Because of its significant place in the gut of freshwater fish, many studies on Cetobacterium were performed. Those studies mostly focused on the temporal and spatial changes of its abundance in fish intestine, which were affected byfood or other environmentalconditions. However, only a few studies isolated strains from genus Cetobacterium and reported their characteristics. In the present study, we performed 16S rRNA sequencing of the intestinal mucosa of Nile tilapia (Oreochromis niloticus) and found that Cetobacterium sp. existed widely in the foregut, midgut and hindgut mucosa, and a strain of Cetobacterium was successfully isolated from the gut of tilapia. We sequenced its whole genome and predicted it to be a novel candidate species of Cetobacterium sp. and named it NK01. The size of its genome was 3,095,946bp, with a guanine + cytosine content of 28.8%. Among the identified genes, 2855 were predicted to be coding DNA sequences, 84 were tRNA and 34 were rRNA. We found that NK01 produced amino acids, including leucine, isoleucine, valine, glycine, alanine, phenylalanine and proline. Strain NK01 could use starch, sucrose, maltose, glucose, and mannose and synthesize and utilize glycogen. INV, GPI, malQ, malZ, sacA, scrK, glgC, glgA and glk, which were related to carbohydrate metabolism, were detected. yiaY and adhE, which oxidize ethanol to acetaldehyde and participate in a variety of metabolic pathways, were also present in the genome. No coding genes directly involved in acetate or butyrate production were detected. NK01 could also catabolize a variety of vitamins, and all genes involved in folate synthesis were detected, including folP, folC, folA and eutT, which converted vitamin B12s into vitamin B12 coenzyme. Here, we investigated the draft genome and in vitro function of Cetobacterium isolated from the intestinal tract of Nile tilapia. The results provided a preliminary understanding of the core microbiota of fish gut.

A sensitive bioanalytical assay for methylcobalamin, an endogenous and light-labile substance, in human plasma by liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study

Methylcobalamin (mecobalamin) is a vitamin B12 coenzyme and is effective for the treatment of peripheral neuropathies. As mecobalamin is an endogenous substance in human plasma at low levels and is light-labile, a sensitive assay method has been developed by using liquid chromatography with tandem mass spectrometry with particular care taken for light exposure. Stability tests performed under light showed that mecobalamin was stable in plasma in amber tubes only when exposed to <10 lx light, and thus, all the assays used for the method validation and the in-study sample assays should be performed under red light with minimal light exposure. Mecobalamin and its stable isotope, which was used as an internal standard, were extracted from 0.1 mL plasma by protein precipitation with methanol and were quantifiable in a range from 0.05 to 20 ng/mL. The reproducibility assessment showed acceptable accuracy and precision (≤15 %). Mecobalamin was stable in plasma at room temperature for 21 h when exposed to <10 lx light in amber tubes and for 205 days when stored frozen. The validated method was successfully applied to an in-study sample assay by performing a clinical pharmacokinetic study of mecobalamin, in which a successful incurred sample reanalysis was performed.

The Birth and Demise of Hypotheses on Evolution of S-Adenosyl-Lmethionine and Adenosylcobalamin

Relationships between adenosylcobalamin and S-adenosyl-Lmethionine (SAM)-dependent enzymatic radical reactions are explored with a view toward determining their evolutionary relationships. Adenosylcobalamin is a Vitamin B12-coenzyme, and the vitamin deficiency causes pernicious anemia in humans. Methionine, the precursor of SAM, is a nutritionally essential amino acid. Evidence implicates both SAM and adenosylcobalamin in the generation of the 5’-deoxyadenosyl radical as the initiator of carbon-centered radical chemistry. However, expectations of the evolutionary superiority of the structurally and chemically complex adenosylcobalamin as an initiator of radical biochemistry are contradicted by available information. It is pointed out that adenosylcobalamin functions equally well aerobically and anaerobically, whereas SAM requires strong reducing conditions and electron transfer mediated by a [4Fe–4S]1+ cluster to initiate carboncentered radical chemistry.

A Rare Combination of Ribonucleotide Reductases in the Social Amoeba Dictyostelium discoideum

Ribonucleotide reductases (RNRs) catalyze the only pathway for de novo synthesis of deoxyribonucleotides needed for DNA replication and repair. The vast majority of eukaryotes encodes only a class I RNR, but interestingly some eukaryotes, including the social amoeba Dictyostelium discoideum, encode both a class I and a class II RNR. The amino acid sequence of the D. discoideum class I RNR is similar to other eukaryotic RNRs, whereas that of its class II RNR is most similar to the monomeric class II RNRs found in Lactobacillus spp. and a few other bacteria. Here we report the first study of RNRs in a eukaryotic organism that encodes class I and class II RNRs. Both classes of RNR genes were expressed in D. discoideum cells, although the class I transcripts were more abundant and strongly enriched during mid-development compared with the class II transcript. The quaternary structure, allosteric regulation, and properties of the diiron-oxo/radical cofactor of D. discoideum class I RNR are similar to those of the mammalian RNRs. Inhibition of D. discoideum class I RNR by hydroxyurea resulted in a 90% reduction in spore formation and decreased the germination viability of the surviving spores by 75%. Class II RNR could not compensate for class I inhibition during development, and an excess of vitamin B12 coenzyme, which is essential for class II activity, did not improve spore formation. We suggest that class I is the principal RNR during D. discoideum development and growth and is important for spore formation, possibly by providing dNTPs for mitochondrial replication.

The Isolation of the Vitamin B12 Coenzyme and the Role of the Vitamin in Methionine Synthesis

The invitation to contribute a “Reflections” article has brought back many memories as I recall the events early in my career that were most significant in determining the path I have taken for more than 50 years. I could reflect on several periods in my career, such as the early work on serotonin biosynthesis and metabolism or later studies on the role of GTP and elongation factors in protein synthesis, but the period when I worked on vitamin B12 in the late 1950s and early 1960s especially stands out in my mind, and I will focus mainly on these studies.

Simultaneous determination of vitamin B12 and its derivatives using some of multivariate calibration 1 (MVC1) techniques

Resolution of binary mixtures of vitamin B12, methylcobalamin and B12 coenzyme with minimum sample pre-treatment and without analyte separation has been successfully achieved by methods of partial least squares algorithm with one dependent variable (PLS1), orthogonal signal correction/partial least squares (OSC/PLS), principal component regression (PCR) and hybrid linear analysis (HLA). Data of analysis were obtained from UV–vis spectra. The UV–vis spectra of the vitamin B12, methylcobalamin and B12 coenzyme were recorded in the same spectral conditions. The method of central composite design was used in the ranges of 10–80 mg L −1 for vitamin B12 and methylcobalamin and 20–130 mg L −1 for B12 coenzyme. The models refinement procedure and validation were performed by cross-validation. The minimum root mean square error of prediction (RMSEP) was 2.26 mg L −1 for vitamin B12 with PLS1, 1.33 mg L −1 for methylcobalamin with OSC/PLS and 3.24 mg L −1 for B12 coenzyme with HLA techniques. Figures of merit such as selectivity, sensitivity, analytical sensitivity and LOD were determined for three compounds. The procedure was successfully applied to simultaneous determination of three compounds in synthetic mixtures and in a pharmaceutical formulation.

436. Recombinant Adeno-Associated Virus Gene Therapy for Cob(I)Alamin Adenosyltransferase Deficiency

Metabolism of inactive cobalamins, both in the recycling and assimilation from dietary sources, into the B12 coenzymes is vital for various metabolic pathways. Disruption of vitamin B12 coenzyme synthesis has been linked to chronic liver disease, cancer, elevated serum homocysteine (possible risk factor for heart disease), increased rate of DNA damage and impaired cognitive function. Vitamin B12 (cyano-cobalamin, CNCbl) is a precursor of two B12 coenzymes, adenosyl-cobalamin (AdoCbl) and methyl-cobalamin (CH3Cbl), which are instable due to a labile upper ligand to the cobalt atom. The assimilation and recycling pathway consists of a multi-enzyme pathway, the last step being the conversion of cob(I)alamin to AdoCbl by the ATP dependent cob(I)alamin adenosyltransferase (ATR). In humans, methylmalonyl-CoA mutase (MCM), responsible for the conversion of propionyl-CoA to the TCA cycle intermediate succinyl-CoA, is dependent on AdoCbl. Propionyl-CoA is produced by the catabolism of the amino acids valine, isoleucine, methionine, and threonine, as well as thymine, cholesterol and odd-chain fatty acids. The impaired activity of MCM, resulting from the lack of coenzyme B12 (AdoCbl), leads to methylmalonic aciduria (MMA), a disease that is often fatal in the first years of life with a frequency of 1/50,000 births. MMA is diagnosed primarily by a buildup of methylmalonyl-CoA in the serum and in the urine. We have obtained human fibroblast cell lines from patients with known ATR deficiencies. Identification of mutations, generally seen to be a single nucleotide or missense mutation, have been performed on most. The human ATR cDNA is 753 bp, the appropriate length for use in rAAV gene therapy vector. It has been cloned into our rAAV vector driving expression of hATR from the hybrid CMV enhancer chicken-beta actin promoter and pseudotyped in AAV 2 and 8 capsids. Next, cell lines were stably transfected with an ATR-neo plasmid and selected for using G418. Levels of methylmalonic acid in the tissue culture medium is measured using tandem mass spectroscopy and an enzyme assay for the conversion of cob(I)alamin to AdoCbl with the ATR enzyme. RESULTS: Overexpression of hATR message from the pCB-hATR vector in patient and control cell lines was confirmed by semi-quantitative RT-PCR. Transgene expression in neomycin resistant clones was 3 to 7 times greater than endogenous expression. Correction of deficient patient cell lines will be assayed at varying doses of rAAV virus. Further experiments will include testing vector expressed ATR mRNA and increased enzyme acitivity in the muscle and liver tissues of C57Bl/6 mice.

Vitamin B12 Coenzyme Models: Perspectives on Recent Developments in the Chemistry of the Cobaloximes and Related Models

Discoveries on B12 models made since 1989 are assessed in the light of the advances in structural and spectroscopic methodologies. Further studies emanating in part from these advances have confirmed, often definitively, previously identified principles describing the properties of the classic simple models (cobaloximes and iminocobaloximes) and have established some new principles defining the properties of the axial Co-C and Co-N bonds; the latter are clearly relevant to enzymatic processes. Some new simple models have been proposed and studied in relation to the classic ones and to the more complicated natural cobalamins. In several cases, the influence of the steric and electronic factors have been established, sometimes in semi-quantitative terms, also with the help of studies on the rhodium analogues of cobaloximes. New spectroscopic techniques have been introduced, which have been found useful in the study of the natural cobalamins. Recent structural analyses of the binding site in some B12-based enzymes have shown that the B12 cofactors bind in the base-off form, with displacement of the benzimidazole residue from cobalt and coordination of a histidine residue of the protein chain. Such observations have stimulated new experiments aimed at defining the mechanism of the Co-C homolytic cleavage in isomerases and mutases or at testing the recently proposed mechanism for the Co-C heterolytic cleavage in methionine synthase. As a consequence, the information now available on models and on cobalamins (e.g., on the nature of the Co-S bond) is much broader in scope, and these advances have prompted this new analysis of models. The experimental aspects have advanced much more quickly than our theoretical understanding, and hence further calculations, possibly based on more sophisticated approaches, are clearly required.

A Molecular Mechanics Force Field for Alkylcobaloximes, a Model of Vitamin B12 Coenzyme – Implications of Steric and Electronic Factors in the Co–C Bond Cleavage

Molecular mechanics force field constants have been derived for alkylcobaloximes, (alkyl)Co(DH)2L (DH = monoanion of dimethylglyoxime and L = planar N-donor ligand), implementing the AMBER force field. Atomic charges have been calculated by the semiempirical ZINDO/1 method. Stretching and bending constants have been calculated by the Badger and Halgren equations, with the introduction of simple functions for the description of the electronic influence of the axial ligands on the coordination geometry. 26 parameters have been optimized, by the Simplex method, to fit 4523 bond lengths and angles of 52 alkylcobaloxime accurate crystal structures. In spite of the oversimplification of the adopted method for the description of the electronic effects, this approach provides a good description of the metal coordination geometries. The root-mean-square deviations of the calculated bond lengths and angles from the experimental values average to 0.023 Å and 1.4°, respectively. The molecular mechanics results are discussed in terms of the steric properties of the axial ligands and correlated with their calculated cone angles. The force field has been used to analyse some conformational features of these compounds, such as the influence of the rotation of the axial ligands on their coordination geometry. The calculated energy for the Co–N rotational barrier agrees well with the experimetal ones derived from dynamic and saturation 1H-NMR spectroscopy in (alkyl)Co(DH)2(2-NH2-py). In order to have more experimental data for this analysis, the crystal structures of two new cobaloximes, (CH2Cl)Co(DH)2L with L = py, 1, and 1-Me-Im, 2) have been determined. The results of a conformational analysis on ribosyl-imidazole derivatives, taken as a suitable vitamin B12 coenzyme model, suggest that the 1-Me-imidazole-like ligands have a significantly greater rotation freedom with respect to the benzimidazole-like ones, but cause similar stretchings of the Co–C bond, and a significantly less stretching of the Co–N one. Implications for the recent findings on the binding mode of the coenzyme B12 in the enzyme active site are discussed.

A Molecular Mechanics Force Field for Alkylcobaloximes, a Model of Vitamin B12 Coenzyme – Implications of Steric and Electronic Factors in the Co–C Bond Cleavage

A Molecular Mechanics Force Field for Alkylcobaloximes, a Model of Vitamin B12 Coenzyme – Implications of Steric and Electronic Factors in the Co–C Bond Cleavage

Alteration of mitochondrial DNA and RNA level in human fibroblasts with impaired vitamin B12 coenzyme synthesis

Alterations of mitochondrial (mt) nucleic acid metabolism in methylmalonic aciduria (MMA) were studied in two cell lines from skin fibroblasts of patients with mitochondrial (GM00595) or cytosolic (GM10011) defects in the biosynthesis pathways of cobalamin coenzymes. The mtDNA level increased two-fold in GM00595 cells, which carry a mt defect in the adenosylcobalamin synthesis, whereas no appreciable change was found in GM10011 cells. The content of the two rRNAs 16S and 12S mtRNAs, normalized for the mtDNA copy number, decreased by 70% and 50% in GM00595 and GM10011, respectively. The normalized content of ND1, ND2 and CO I mRNAs decreased in GM00595, but was unchanged in GM10011. Respiratory chain complex activities measured in these two cell lines were not different from control activities. These data suggest that the maintenance of the mt function is due to doubling of mtDNA and that this compensatory response takes place only in those cells in which the greater reduction of the level of rRNA might have brought the content of these transcripts below the threshold value for optimal expression of the mt genome.

Structural and computational analysis of published neutron diffraction data show that crystalline vitamin B12 coenzyme contains a strong intramolecular N-H...Ph hydrogen bond

The neutron diffraction crystal structure of vitamin B12 coenzyme [Bouquiere et al. (1993). Acta Cryst. B49, 79–89] contains a yet unnoticed intramolecular aromatic hydrogen bond donated by a propionamide side chain and accepted by the benzimidazole group. The distance from the H-atom to the aromatic midpoint is only 2.58 Å and the bond energy is calculated to be 16.7 kJ mol−1.

AXIAL-BASE EQUILIBRIUM AND THERMODYNAMIC STUDIES OF RIBOSYLCOBALAMINS

Thermodynamic and equilibria properties of two vitamin B12 coenzyme analogues, i.e. 1-methyl-5-deoxy-β-d-(−)ribofuranosylcobalamin (RibCbl) and 1-methyl-5-deoxy-2,3-isopropylidene-β-d-(−)ribofuranosylcobal- amin (Rib*Cbl) were studied by u.v.-vis. spectroscopy. Their temperature-dependent equilibria between 6-coordinate base-on species and 5-coordinate unprotonated base-off species, and pH-dependent equilibria for the protonation and displacement of 5,6-dimethylbenzimidazole at 25°C have been examined. Values of Δ H and Δ S in neutral solution and at pH 4.00, and the axial base pKa were determined. Comparing the results with those for coenzyme B12 and other cobalamins, it has been found that Δ H and Δ S values for two ribosylcobalamins fall in the range of organocobalamins, but for RibCbl*, a ca. 1 unit difference in pKa values exists. The effect of the ribofuranose structure on the Co-N(dbzm) strength is considered.

Effects of cobalt on plants

Effects of cobalt on plants

Electronic Structure of the Coenzyme Vitamin B12 and Related Systems. 1. Co(DH)2(L)(R) Compounds (DH = Dimethylglyoxime; L = NH3, py, 2-NH2py, 5,6-Dimethylbenzimidazole; R = CH3, i-C3H7, 5'-Deoxyadenosyl) as Model Systems for the Vitamin B12 Coenzyme

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTElectronic Structure of the Coenzyme Vitamin B12 and Related Systems. 1. Co(DH)2(L)(R) Compounds (DH = Dimethylglyoxime; L = NH3, py, 2-NH2py, 5,6-Dimethylbenzimidazole; R = CH3, i-C3H7, 5'-Deoxyadenosyl) as Model Systems for the Vitamin B12 CoenzymeLillian M. Hansen, P. N. V. Pavan Kumar, and Dennis S. MarynickCite this: Inorg. Chem. 1994, 33, 4, 728–735Publication Date (Print):February 1, 1994Publication History Published online1 May 2002Published inissue 1 February 1994https://doi.org/10.1021/ic00082a018RIGHTS & PERMISSIONSArticle Views202Altmetric-Citations31LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (963 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

Syntheses and Characterization of Coenzyme B12 Analogues Containing 2′-Deoxynucleosede

Abstract A new vitamin B12 coenzyme analogue 2′, 5′-dideoxyuridinyl-cobalamin and two other 2′, 5′-dideoxyribonucleosideccbalamins of adenosyl and thymidyl were synthesized by an improved method. It consists in direct tosylation of the nucleoside, followed by reaction with reduced hydroxocobalamin (vitamin B12a). All the three derivatives were characterized by UV -Vis and 1H NMR spectroscopy.

A new model compound for vitamin B12 coenzyme: synthesis and crystal structure of a tetraazamacrocyclic alkylcobalt(III) compound with a pendant coordinating alkyl arm

A novel reaction between norbornene and the cobalt(II) complex of the tetraazamacrocyclic ligand 5,14-dihydro-6,8,15,17-tetramethyldibenzo[b,i][1,4,8,11]tetraazacyclotetradecine(H2L), in the presence of oxygen, gives the 5,16-dihydro-16-(exo-norbornane-2,3-diyl-κC2)-6,8,15,17-tetramethyldibenzo[b,i][1,4,8,11]tetraazadecinato-κ4N5,9,14,18cobalt(III) cation (A); this was isolated as [mono(acetone)-{5,16-dihydro-16-(exo-norbornane-2,3-diyl-κC2)6,8,15,17-tetramethyldibenzo[b,i[1,4,8,11]tetraazadecinato-κ4N5,9,14,18}cobalt(III)] tetraphenylborate, and its X-ray crystal structure establishes the presence of a pendant alkyl–cobalt bond.

How the apoprotein elicits the function of vitamin B12 coenzyme.

How the apoprotein elicits the function of vitamin B12 coenzyme.

Normal and abnormal electron spin resonance spectra of low-spin cobalt(II)[N4]-macrocyclic complexes. A means of breaking the Co—C bond in B12 Co-enzyme

Co(cyen)† normally has an e.s.r. spectrum characteristic of a low-spin cobalt-(II) complex in a 2B2 ground state. However, its BF3 adduct, Co(cyen)· BF3 and its bis(nitro) derivative, Co(5-O2N-cyen) have 2A1 ground states. It is proposed that the ground state of this sort of system can be changed from A1 to B2 in principle by increasing the ligand field. This idea is applied to the mechanism of Co—C homolytic fission in vitamin B12 coenzyme, and it is suggested that the approach of an amine group of the 5′-deoxyadenosyl group to the corrin ligand may stabilize an intermediate of B2 symmetry. Fission of the Co—C bond may occur by a ‘staircase lock’ mechanism.