Polymorphism of 14C vitamin D3 binding protein in cattle and water buffalo serum

Cattle and water buffalo sera labelled with vitamin D₃[¹⁴C] (300 and 480 individual samples respectively) were subjected to starch gel electrophoresis followed by autoradiography in an attempt to identify a possible polymorphism of the proteins capable of binding this vitamin.
Three phenotypes controlled by two codominant autosomal alleles were identified in cattle while in water buffalo six phenotypes controlled by three codominant autosomal alleles were observed.     

Polymorphism of vitamin B12 [57Col binding proteins in rabbit serum

When rabbit serum labelled with vitamin B₁₂[⁵⁷Co] was subjected to starch gel electrophoresis and au;oradiography, three phenotypes of proteins capable of binding vitamin B₁₂ were observed. Family data revealed that these phenotypes (called TC-A, TC-AB and TC-B) are controlied by two codominant alleles (TC^A and TC^B), at an autosomic locus. Proteins capable of binding vitamin B₁₂ both in vivo and in vitro are commonly referred to as Transcobalamins and can be found in the serum of numerous animal species (for a review, see Glass, 1974; Allen, 1975; Stenman, 1975). Furthermore, Daiger et al. (1975a) have described seven different patterns of vitamin B₁₂ binding proteins which occur in human plasma and which are presumably controlled by four alleles. The present paper describes experiments in which both starch gel electrophoresis and autoradiography are used to identify three phenotypes of rabbit serum proteins responsible for binding vitamin B₁₂ in vitro. It was found that these three phenotypes are controlled by two allelic codominant genes, at an autosomic locus. Individual serum samples (30 μl), obtained from 385 White New Zealand rabbits varying in age from one month to three years, were incubated with 0.1 ng of vitamin B₁₂[⁵⁷Co] (specific activity: 180 μCi/μg; Lot 247; Radiochemical Centre, Amersham, England) at 37°C for 30 minutes. Starch gel electrophoresis and autoradiography were performed as described by Geldermann (1970) and Daiger et al. (1975b), respectively. Electrofocusing (pH range 3.5–9.5) was conducted in the 2117 Multiphor apparatus (LKB, Bromma, Sweden) according to the manufacturer's instructions. The resulting pH gradient was measured with a surface pH electrode (Ingold, Zürich, Switzerland).     

Genetically determined electrophoretic variants of the major urinary protein (Mup) complex in mouse urine

The major urinary protein (Mup-complex) excreted in mouse urine, has been studied electrophoretically both on starch gel and on cellogel.
On stargel six anodally migrating protein bands were observed. These bands are designated component 3, 2', 2, 1, and 4 (i.e. two bands) in the order of decreasing mobility toward the anode. The slower protein band of component 4 on starch gel was not observed on cellogel.
By testing mouse inbred strains, we were able to dinstinguish five male and four female Mup phenotypes. Test crosses suggested a four-allelic ( a, b, c, d, ) variation with regard to components 2', 2 and 1: 'group A' strains showed component 1, 'group B' strains components 1 and 2, 'group C' and 'group F strains none, and 'group D' strains showed components 1 and 2'. Component 3 may be encoded by another Mup locus, although no crossing-over has been observed: presence (A, B, D, and F strains), absence (C strains). Insufficiently reproducible demonstration of the variation with regard to component 4, forced us to exclude this component for strain distinction.
The Mup phenotypes described, can be useful for the detection of certain strain contaminations, especially if F₁ hybrid Mup phenotypes are distinguishable     

Holotranscobalamins in B12 and non B12 requiring prokaryotes and eukaryotes

Transcobalamins, vitamin B₁₂ binding proteins, deliver B₁₂ to cell surface receptors which then permit B₁₂ to cross cell membranes for metabolic use. There is little documentation concerning B₁₂ binding proteins in bacteria and protists. We found that prokaryotes and eukaryotes requiring B₁₂, as well as those protists synthesizing B₁₂, also produce several transcobalamins for functionally transporting B₁₂ similar to humans.     

Cobalamin (vitamin B12) binding, phylogeny, and synteny of human transcobalamin

Selected residues in a highly conserved 15-residue region, 174SVDTAAMAGLAFTC L188 of human transcobalamin (TC), a cobalamin (Cbl: vitamin B12) binding protein, were subjected to site-directed mutagenesis. The mutant constructs were expressed in TC-deficient fibroblasts or in vitro to assess the effect of these mutations on Cbl binding. Phylogenetic analyses and protein parsimony indicated that TC evolved earlier than other mammalian Cbl-binding proteins, intrinsic factor and haptocorrins, and divergence occurred between mouse/rat and human dispersing TC gene to different chromosomes. These studies show that (a) two of the three polar residues, S174, T177, or D176 and two of the three conserved alanine residues, A179 and A184 present in the 15-residue evolutionary conserved region are essential for Cbl-binding by human TC, and (b) TC gene is transferred in a syntenic manner to different chromosomes, at least before the divergence of mouse/rat and human.     

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.     

The Escherichia coli outer membrane cobalamin transporter BtuB: structural analysis of calcium and substrate binding, and identification of orthologous transporters by sequence/structure conservation

Gram-negative bacteria possess specialized active transport systems that function to transport organometallic cofactors or carriers, such as cobalamins, siderophores, and porphyrins, across their outer membranes. The primary components of each transport system are an outer membrane transporter and the energy-coupling protein TonB. In Escherichiacoli, the TonB-dependent outer membrane transporter BtuB carries out active transport of cobalamin (Cbl) substrates across its outer membrane. Cobalamins bind to BtuB with nanomolar affinity. Previous studies implicated calcium in high-affinity binding of cyanocobalamin (CN-Cbl) to BtuB. We previously solved four structures of BtuB or BtuB complexes: an apo-structure of a methionine-substitution mutant (used to obtain experimental phases by selenomethionine single-wavelength anomalous diffraction studies); an apo-structure of wild-type BtuB; a binary complex of calcium and wild-type BtuB; and a ternary complex of calcium, CN-Cbl and wild-type BtuB. We present an analysis of the binding of calcium in the binary and ternary complexes, and show that calcium coordination changes upon substrate binding. High-affinity CN-Cbl binding and calcium coordination are coupled. We also analyze the binding mode of CN-Cbl to BtuB, and compare and contrast this binding to that observed in other proteins that bind Cbl. BtuB binds CN-Cbl in a manner very different from Cbl-utilizing enzymes and the periplasmic Cbl binding protein BtuF. Homology searches of bacterial genomes, structural annotation based on the presence of conserved Cbl-binding residues identified by analysis of our BtuB structure, and detection of homologs of the periplasmic Cbl-binding binding protein BtuF enable identification of putative BtuB orthologs in enteric and non-enteric bacterial species.     

Development and application of an α-face-specific radioimmunoassay for vitamin B12

The first development of an α-face-specific radioimmunoassay for vitamin B₁₂ is described. Sheep, fed a cobalt-deficient diet, and immunized with a conjugate between Co-β carboxypropyl cobalamin and keyhole limpet hemocyanin, were used to produce antisera. The antisera crossreacted with Co-β derivatives of vitamin B₁₂, but did not crossreact with the α-face vitamin B₁₂ analog cobinamide. The antisera were used to develop a sensitive and reproducible radioimmunoassay that was free from contamination with the nonspecific vitamin B₁₂ binding protein, R-protein. Both the radioimmunoassay and measurements of plasma concentrations of methylmalonic acid were applied to the diagnosis of cobalt/vitamin B₁₂ deficiency in sheep. The assay correlated well with a commercially available radioassay and did not falsely detect normal vitamin B₁₂ concentration in plasma samples containing elevated concentrations of methylmalonic acid.     

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.     

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.     

Glutathione increases the binding affinity of a bovine B12 trafficking chaperone bCblC for vitamin B12

Intracellular B₁₂ metabolism involves a B₁₂ trafficking chaperone CblC that is well conserved in mammals including human. The protein CblC is known to bind cyanocobalamin (CNCbl, vitamin B₁₂) inducing the base-off transition and convert it into an intermediate that can be used in enzyme cofactor synthesis. The binding affinity of human CblC for CNCbl was determined to be K_d = ≈6–16 μM, which is relatively low considering sub-micromolar B₁₂ concentrations (0.03–0.7 μM) in normal cells. In the current study, we discovered that the base-off transition of CNCbl upon binding to bCblC, a bovine homolog of human CblC, is facilitated in the presence of reduced form of glutathione (GSH). In addition, GSH dramatically increases the binding affinity for CNCbl lowering the K_d from 27.1 ± 0.2–0.24 ± 0.09 μM. The effect of GSH is due to conformational change of bCblC upon binding with GSH, which was indicated by limited proteolysis and urea-induced equilibrium denaturation of the protein. The results of this study suggest that GSH positively modulates bCblC by increasing the binding affinity for CNCbl, which would enhance functional efficiency of the protein.     

Interaction of vitamin B1 with bovine serum albumin investigation using vitamin B1-selective electrode: potentiometric and molecular modeling study

Vitamin B₁ or thiamin is one of the B vitamins. All B vitamins help the body to convert food (carbohydrates) into fuel (glucose), which produces energy. The B vitamins are necessary for healthy skin, eyes, hair, and liver. It also could help the nervous system function properly, and is necessary for brain functions. Drug interactions with protein can affect the distribution of the drug and eliminate the drug in living systems. In this study, the binding of thiamine hydrochloride (vitamin B₁) to bovine serum albumin (BSA) was evaluated using a new proposed vitamin B₁ (thiamine)-selective membrane electrode under various experimental conditions, such as pH, ionic strength, and protein concentration; in addition molecular modeling was applied as well. The binding isotherms plotted based on potentiometric data and analyzed using the Wyman binding potential concept. The apparent binding constant was determined and used for the calculation of intrinsic Gibbs free energy of binding. According to the electrochemical and molecular docking results, it can be concluded that the hydrophobic interactions and hydrogen binding are major interactions between BSA and vitamin B₁.     

Decrease of trace elements in erythrocytes and plasma after removal of dental amalgam and other metal alloys

The objective of this study was to determine the concentration changes of 13 elements in erythrocytes and plasma after the removal of dental amalgam, and other metal alloys. Blood samples from 250 patients were collected, separated into erythrocytes and plasma, and analyzed by inductively coupled plasma-mass spectrometry. The 250 patients were divided into 3 groups (Negative, Zero, and Positive) depending on their estimation of quality of life in an earlier study. Magnesium in plasma, selenium and mercury in plasma, and erythrocytes showed decreased concentrations after amalgam removal in all groups (p<0.05). Titanium in plasma, copper in plasma, and erythrocytes and zinc in plasma exhibited decreased concentrations after amalgam removal in the Negative and Positive groups (p<0.05), Silver in plasma and gold in erythrocytes decreased in the Zero and Positive groups after amalgam removal (p<0.05). Copper in erythrocytes and silver and gold in plasma showed higher concentrations after amalgam removal in the Negative compared to the Positive group (p<0.05), suggesting that patients in the Negative group excrete metals slowly. Moreover, the cobalt levels in plasma were lowest in the Negative group and only this group showed a significant increase in vitamin B₁₂ levels in blood after amalgam removal.     

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.     

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.     

Effect of nitrous oxide on nickel deprivation in rats

Because nickel may have a biological function in a pathway in which vitamin B₁₂ is important, an experiment was performed to determine the effects of nitrous oxide exposure in rats deprived of nickel. Exposure to nitrous oxide (N₂O) causes inactivation of cobalamin and a subsequent decrease in the vitamin B₁₂-dependent enzymes methionine synthase and methylmalonyl CoA mutase. Rats were assigned to dietary groups of 12 in a factorially arranged experiment with dietary variables of nickel (0 or 1 μg/g) and vitamin B₁₂ (0 or 50 ng/g). After 6 wk, one-half of the rats from each dietary group were exposed to 50% N₂O/50% O₂ for 90 min/d for the last 28 d of the experiment. Vitamin B₁₂, N₂O, or their interaction had numerous effects; classical findings included N₂O-induced reduction in plasma vitamin B₁₂ and decreases in the vitamin B₁₂-dependent enzymes. Inactivation of vitamin B₁₂ by N₂O, however, did not exacerbate signs of nickel deprivation, possibly because the rats were able to metabolically compensate to N₂O exposure. Mention of a trademark or proprietary product in this article does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.