Occurrence of Coenzyme Forms of Vitamin B12 in a Cultured Purple Laver (Porphyla yezoensis)

Porphyra yezoensis (Susabinori, an edible purple laver), which was cultured aseptically for 12 weeks and then lyophilized, contained 50±2 μg/g of vitamin B₁₂ per 100 g dry weight. Coenzyme forms of vitamin B₁₂ (about 60% of the total vitamin B₁₂) were found in the cultured purple laver aseptically, which may have the ability to biosynthesize the coenzymes.     

Characterization of a Corrinoid Compound in the Edible (Blue-Green) Alga,Suizenji-nori

The edible blue-green alga (cyanobacterium), Suizenji-nori, contained 143.8±22.4 μg of vitamin B₁₂ per 100 g dry weight of the alga (mean±SE, n=4). A corrinoid compound was purified from the dried Suizenji-nori, and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified corrinoid compound were not identical to those of true vitamin B₁₂, but to those of pseudovitamin B₁₂ which is inactive for humans.     

Production Potency of Folate,Vitamin B12, and Thiamine by Lactic Acid Bacteria Isolated from Japanese Pickles

Hypoallergenic wheat products in the form of pasta-like noodles and bread were fabricated. Wheat flour was added with a 0.6-fold weight of water dissolving collagenase to obtain hypoallergenic flour batter. Methods for producing hypoallergenic pasta-like noodles and bread from the batter were designed. In the noodle making, the batter was mixed with an oligosaccharide with a mild sweetness, a surfactant, and salt, and the mixture was extruded under heating. Both retorting and refrigerating processes were applied to the noodles. In the bread making a mixture of the batter, glucose, citric acid, sodium hydrogen carbonate and salt was baked at 180°C. The addition of glucose contributed to generation of faborable flavor and color.     

Plasma Trace Elements, Vitamin B12, Folate, and Homocysteine Levels in Cirrhotic Patients Compared to Healthy Controls

Increased serum homocysteine (Hcy) can induce liver diseases and can play a remarkable role in hepatic disorders. The purpose of the present study therefore was to investigate the relationship between serum vitamin B(12), folate, zinc and copper, cysteine, and Hcy level differences between cirrhotic patients and healthy subjects. We studied 32 cirrhotic patients (12 females and 20 males) aged 45 +/- 11 years and 32 control subjects (12 females and 20 males) aged 39 +/- 9 years. There was an inverse correlation between Hcy and vitamin B(12) in controls (r = -0.442, p < 0.011) but not in cirrhotic patients (r = -0.147, not significant). Also, mean plasma folate was decreased in cirrhotic patients compared to controls (p < 0.001). Copper increased whereas zinc decreased significantly in cirrhotic patients. A positive correlation was seen between the Cu/Zn ratio and Cu in controls (r = 0.690, p < 0.01), but the correlation between the Cu/Zn ratio and Cu was not significant in the cirrhotic group. Negative correlations were seen between plasma concentration of zinc and the Cu/Zn ratio in controls and cirrhotic patients (r = -0.618, p < 0.01 and r = -0.670, p < 0.01, respectively). Cirrhotic patients displayed multiple abnormalities, including changes in cysteine metabolism and in zinc and copper levels. Although hyperhomocysteinemia is known as an atherogenic and thrombogenic risk factor for cardiovascular disease, it might also be a risk factor for cirrhotic patients. Plasma Hcy, vitamin B(12), and folic acid measurement may be useful in the evaluation of cirrhotic patients.     

Application of vitamin B(12)-targeting site on Lactobacillus helveticus B-1 to vitamin B(12) assay by chemiluminescence method

Lactobacillus helveticus B-1 is assumed to have a vitamin B(12)-targeting (or B(12)-binding) site on the cells, since the binding reaction of vitamin B(12) with L. helveticus B-1 cells proceeded instantly and quantitatively. This reaction is specific to complete B(12) compounds, cobalamins, and can be used for a vitamin B(12) assay method by chemiluminescence. The calibration graph was linear from 0.1 to 10.0 ng/mL. The B(12) contents in oyster and sardine were 75.9 and 39.4 microg/100g, respectively. These values were very close to those obtained using a chemilumi-ADVIA Centaur immunoassay system with intrinsic factor and to those obtained by microbiological assays.     

The Aeration-Dependent Effect of Vitamin B12 on DNA Biosynthesis in Methylobacterium dichloromethanicum

The effect of vitamin B₁₂ (cobalamin) on DNA biosynthesis in Methylobacterium dichloromethanicum was studied. When cultivated in media with methanol or dichloromethane, the bacterium produced approximately 10 g corrinoids per gram dry biomass, compared to about 7 g/g when cultivated on ethanol or succinate. Exogenous adenosylcobalamin (AdoCbl) stimulated DNA biosynthesis in M. dichloromethanicum cells grown under poor aeration, the effect being mediated by AdoCbl-dependent ribonucleotide reductase. In vitro studies showed that M. dichloromethanicumalso has AdoCbl-independent ribonucleotide reductase. Under good aeration, exogenous AdoCbl had no effect on DNA biosynthesis, while hydroxyurea suppressed it. These data suggest that AdoCbl-independent ribonucleotide reductase, which is likely to be activated by oxygen, plays an important part in DNA biosynthesis when M. dichloromethanicum is cultured with good aeration, whereas AdoCbl-dependent ribonucleotide reductase is active under conditions of poor aeration.     

Vitamin B12, a chlorophyll-related analog to pheophytin a from marine brown algae, promotes neurite outgrowth and stimulates differentiation in PC12 cells

We previously isolated an analog to chlorophyll-related compounds, pheophytin a, from the marine brown alga Sargassum fulvellum and demonstrated that it is a neurodifferentiation compound. In the current study, we investigated the effects of the pheophytin a analog vitamin B₁₂ on PC12 cell differentiation. In the presence of a low level of nerve growth factor (10 ng ml⁻¹), vitamin B₁₂ demonstrated neurite outgrowth-promoting activity in PC12 cells. The effect was dose-dependent in the range of 6–100 μM. In the absence of nerve growth factor, vitamin B₁₂ did not promote differentiation. To investigate the mechanism for this effect, we conducted differentiation assays and western blot analysis with signal transduction inhibitors and found that vitamin B₁₂ did not promote PC12 cell differentiation in the presence of K252a or U0126 inhibitors. These results suggest that vitamin B₁₂ stimulates PC12 cell differentiation through enhancement of the mitogen-activated protein kinase signal transduction pathway, which is also induced by nerve growth factor. Thus, vitamin B₁₂ may be a good candidate for treatment of neurodegenerative diseases such as Alzheimer’s disease.     

Effects of maternal vitamin B12 deficiency from end of gestation to weaning on the growth and haematological and immunological parameters in mouse dams and offspring

Vitamin B₁₂-deficiency may induce specific symptoms as neurological alterations and unspecific symptoms such as anaemia and growth retardation. In this study, maternal vitamin B₁₂ deficiency from end of gestation to weaning was evaluated in mouse dams, which was provoked by feeding a vitamin B₁₂-deficient diet. The animals were divided into two groups (control and deficient). The control group received the vitamin B₁₂-deficient diet supplemented with commercial vitamin B₁₂. Compared to the control, the vitamin B₁₂-deficient dams and their offspring showed a significant decrease of body weight (by 20 and 39%, respectively), serum vitamin B₁₂ concentration (by 61 and 67%, respectively), haematological values as haematocrit (25 and 26%, respectively), and IgA producer cells (by 36 and 54%, respectively). In both, vitamin B₁₂-deficient mouse dams and their offspring, histological alterations of small intestine were observed, whereas growth retardation occurred in the offspring only. This experimental murine model allows assessing the incidence of maternal cobalamin deficiency in offspring and would be useful for evaluating novel adjuncts such as functional foods to prevent vitamin B₁₂ deficiency.     

Methionine,folic acid and vitamin B12 in growing-finishing pigs: Impact on growth performance and meat quality

Growth performance, metabolic variables, and meat quality were measured in 78 growing-finishing pigs using supplements of 0 (C), or 0.2% of DL-methionine (M), and three combinations of folic acid [mg/kg] and cyanocobalamin [μg/kg], respectively 0 and 0 (V0), 10 and 25 (V1), and 10 and 150 (V2) in a 2 × 3 factorial arrangement. Feed conversion was lower (p = 0.05) in M than in C pigs during the growing period (0–4 weeks). Both V1 and V2 treatments increased plasma vitamin B₁₂ (p < 0.01) and decreased plasma homocysteine (p < 0.01). Plasma 5-methyl-tetrahydrofolates were the lowest, highest and intermediate in V0, V1 and V2 pigs (p < 0.04), respectively. In V2 meat, folates were 32% higher, vitamin B₁₂, 55% higher and homocysteine, 28% lower than in V0 (p < 0.01). Oxidative stability of the fresh meat was similar among treatments during a storage period of 42 days. Therefore, methionine supplements improved growth performance during the growing period. Vitamin supplements interacted with the methionine cycle pathway, increased vitamin content of pork meat but did not improve oxidative stability of the fresh meat during storage.     

C-terminal truncation of a bovine B12 trafficking chaperone enhances the sensitivity of the glutathione-regulated thermostability

The human B₁₂ trafficking chaperone hCblC is well conserved in mammals and non-mammalian eukaryotes. However, the C-terminal ∼40 amino acids of hCblC vary significantly and are predicted to be deleted by alternative splicing of the encoding gene. In this study, we examined the thermostability of the bovine CblC truncated at the C-terminal variable region (t-bCblC) and its regulation by glutathione. t-bCblC is highly thermolabile (T_m = ∼42℃) similar to the full-length protein (f-bCblC). However, t-bCblC is stabilized to a greater extent than f-bCblC by binding of reduced glutathione (GSH) with increased sensitivity to GSH. In addition, binding of oxidized glutathione (GSSG) destabilizes t-bCblC to a greater extent and with increased sensitivity as compared to f-bCblC. These results indicate that t-bCblC is a more sensitive form to be regulated by glutathione than the full-length form of the protein. [BMB Reports 2013; 46(3): 169-174]     

Structural features of a gene encoding the vacuolar H+-ATPase c subunit from a marine red alga, Porphyra yezoensis.

We report the nucleotide sequence of a gene encoding the c ('16 kDa') subunit of the vacuolar-type H+-ATPase (V-ATPase) from a marine red alga, Porphyra yezoensis. A cDNA clone was isolated from a leafy gametophyte cDNA library and analyzed for the sequence. The genomic DNA sequence was directly determined by nested PCR. The structural gene contained four introns within a coding sequence of 483 base pairs which encodes a polypeptide of 161-amino acids with four hydrophobic transmembrane-spanning regions. Comparison of the deduced amino acid sequences showed higher similarity to the land plant Oryza sativa (69.1%) than to the Ulvophyceae Acetabularia acetabulum (64.1%). The mRNA was detected both in the leafy gametophytes and filamentous sporophytes.     

Pseudo vitamin B12 or 5-hydroxybenzimidazolyl-cobamide are the corrinoids found in methanogenic bacteria

Thirteen species of methanogenic bacteria were analyzed for corrinoids. Pseudo vitamin B₁₂ (Co-[-(7-adenyl)]-cobamide) was the predominant cobamide of methanococcales and Methanoplanus. All other methanogens contained factor III (Co-[-(5-hydroxybenzimidazolyl)]-cobamide). Vitamin B₁₂ (Co-[-(5,6-dimethylbenzimidazolyl)]-cobamide) was not detected in any of these archaebacteria. Their cobamide content was 100 to 1400 nmol per gram cell dry weight, indicating that abundant cobamides are essential for methanogens.     

Processing of glutathionylcobalamin by a bovine B12 trafficking chaperone bCblC involved in intracellular B12 metabolism

Glutathionylcobalamin (GSCbl) is a biologically relevant vitamin B₁₂ derivative and contains glutathione as the upper axial ligand thought formation of a cobalt-sulfur bond. GSCbl has been shown to be an effective precursor of enzyme cofactors, however processing of the cobalamin in intracellular B₁₂ metabolism has not been fully elucidated. In this study, we discovered that bCblC, a bovine B₁₂ trafficking chaperone, catalyzes elimination of the glutathione ligand from GSCbl by using the reduced form of glutathione (GSH). Deglutathionylation products are base-off cob(II)alamin and glutathione disulfide, which are generated stoichiometrically to GSH. Although cob(I)alamin was not detected due to its instability, deglutathionylation is likely analogous to dealkylation of alkylcobalamins, which uses the thiolate of GSH for nucleophilic displacement. The catalytic turnover number for the deglutathionylation of GSCbl is ?1.62 ± 0.13 min⁻¹, which is, at least, an order of magnitude higher than that for elimination of upper axial ligands from other cobalamins. Considering the prevalence of GSH at millimolar concentrations in cells, our results explain the previous finding that GSCbl is more effective than other cobalamins for synthesis of enzyme cofactors.     

Effects of nutrition on zinc, folic acid, and vitamin B12 levels during pregnancy

In this article, we report the results of a case control study carried out on 290 Turkish pregnant women at 5–24 wk of gestation to determine their zinc, folic acid, and vitamin B₁₂ levels in relation to their nutritional and socioconomic status. The women were divided into two groups (n=145 each), depending on the stage of gestation. Group I consisted of women in the first trimester of gestation; those in the second trimester were allocated into group II. Twenty-five age-matched, healthy nonpregnant women were selected as controls. The nutritional status of the subjects was determined by means of a survey. Based on this, the women were subdivided into three subgroups: malnourished (PN), moderately nourished (MN), and well nourished (WN). Also from the survey, the socioeconomic status was classified as good (G) or bad (B). A statistically significant decrease on zinc and folic acid was observed in group I women, as their socioeconomic status worsened. In group II, zinc decreased only in the PN and MN subgroups, p<0.001. The folic acid and vitamin B₁₂ levels did not change significantly during the second trimester of gestation in all subgroups. Regarding zinc, nutrition, or dietary habits are more relevant than socioeconomic status, but poor nutrition affects folic acid levels only during the first trimester of gestation.     

Vitamin B(12) incorporated with multiwalled carbon nanotube composite film for the determination of hydrazine

Electrochemically active composite film containing multiwalled carbon nanotubes (MWCNTs) and vitamin B₁₂ was synthesized on glassy carbon, gold, and indium tin oxide electrodes by the potentiodynamic method. The presence of MWCNTs in the composite film (MWCNT–B₁₂) modified electrode mediates vitamin B₁₂’s redox reaction, whereas vitamin B₁₂’s redox reaction does not occur at bare electrode. The electrochemical impedance spectroscopy studies reveal that MWCNTs present in MWCNT–B₁₂ film enhance electron shuttling between the reactant and electrode surface. The surface morphology of bare electrode, MWCNT film. and MWCNT–B₁₂ composite film was studied using atomic force microscopy, which reveals vitamin B₁₂ incorporated with MWCNTs. The MWCNT–B₁₂ composite film exhibits promising enhanced electrocatalysis toward hydrazine. The electrocatalysis response of hydrazine at MWCNT film and MWCNT–B₁₂ composite film was measured using cyclic voltammetry and amperometric current–time (i–t) curve techniques. The linear concentration range of hydrazine obtained at MWCNT–B₁₂ composite film using the i–t curve technique is 2.0 μM–1.95 mM. Similarly, the sensitivity of MWCNT–B₁₂ composite film for hydrazine determination using the i–t curve technique is 1.32 mA mM⁻¹ cm⁻², and the hydrazine’s limit of detection at MWCNT–B₁₂ composite film is 0.7 μM.     

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

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

Vitamin B12 transport in Escherichia coli K12 does not require the btuE gene of the btuCED operon

Summary Transport of vitamin B₁₂ across the cytoplamic membrane ofEscherichia coli requires the products ofbtuC andbtuD, two genes in thebtuCED operon. The role ofbtuE, the central gene of this operon, was examined. Deletions withinbtuE were constructed by removal of internal restriction fragments and were crossed onto the chromosome by allelic replacement. In-frame deletions that removed 20% or 82% of thebtuE coding region did not affect expression of the distalbtuD gene. These nonpolar deletions had little effect on vitamin B₁₂ binding (whole cells or periplasmic fraction) and transport. They did not affect the utilization of vitamin B₁₂ or other cobalamins for methionine biosynthesis, even in strains with decreased outer membrane transport of vitamin B₁₂. ThebtuE mutations did not impair adenosyl-cobalamin dependent catabolism of ethanolamine or repression ofbtuB expression. Thus, despite its genetic location in the transport operon, thebtuE product plays no essential role in vitamin B₁₂ transport.