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.     

Flow injection chemiluminescence determination of vitamin B12 using on-line UV-persulfate photooxidation and charge coupled device detection

A sensitive chemiluminescence method for vitamin B(12) using a charge-coupled device (CCD) photodetector combined with on-line UV-persulfate oxidation in a simple continuous flow system has been developed. The principle for the determination of vitamin B(12) is based on the enhancive effect of cobalt (II) on the chemiluminescence reaction between luminol and percarbonate in alkaline medium. In addition, percarbonate has been investigated and proposed as a powerful source of hydrogen peroxide as oxidant agent in this chemiluminescence reaction. The digestion of vitamin B(12) to release the cobalt (II) is reached by UV irradiation treatment in a persulfate medium. The CCD detector, directly connected to the flow cell, is used with the continuous flow manifold to obtain the full spectral characteristics of cobalt (II) catalyzed luminol-percarbonate reaction. The vitamin B(12) oxidation process and chemical conditions for the chemiluminescence reaction were investigated and optimized. The increment of the emission intensity was proportional to the concentration of vitamin B(12) , giving a second-order calibration graph over the cobalt (II) concentration range from 10 to 5000 μg L(-1)(r(2) = 0.9985) with a detection limit of 9.3 μg L(-1). The proposed method was applied to the determination of vitamin B(12) in different kinds of pharmaceuticals.     

Magnetic Nanoparticle-Based Solid-Phase Extraction of Vitamin B12 from Pharmaceutical Formulations

In the present study, a novel quantitative method, namely magnetic nanoparticle-based solid-phase extraction (MSPE), was applied to extract vitamin B(12) from pharmaceutical formulations. The technique involves the use of Fe(3)O(4) nanoparticles modified by sodium dodecyl sulfate (SDS) as an efficient adsorbent for solid-phase extraction of vitamin B(12). Collection of magnetic nanoparticles (MNPs) from aqueous solution was simply achieved by applying external magnetic field. The analyte was desorbed from MNPs using alkali 1-propanol. The extracted analyte was analyzed by using flow injection inductively coupled plasma-optical emission spectrometry. Factors affecting the extraction efficiency were investigated and optimized. Under the optimum conditions, enhancement factor of 184, linear dynamic range of 2.5-500 μg L(-1) with correlation of determination (R(2) > 0.999), and limit of detection of 1.0 μg L(-1) were obtained for vitamin B(12). The percent relative standard deviation based on five-replicate determination was less than 6.2%. The method was successfully applied for extraction and determination of vitamin B(12) in different types of pharmaceutical samples such as multivitamin tablet, effervescent tablet, and injection sample. The results showed that the proposed method based on SDS-Fe(3)O(4) MSPE was a simple, accurate, and highly efficient approach for analysis of vitamin B(12).     

The Status of Vitamin B12 and Folate among Chinese Women: A Population-Based Cross-Sectional Study in Northwest China

Objective

To assess the status of the vitamin B12 and folate of Chinese women living in northwest China.

Methods

A population-based cross-sectional study was conducted in 2008 among Chinese women aged 10–49 years living in Shaanxi province of northwest China. A stratified multistage random sampling method was adopted to obtain a sample of 1170 women. The women were interviewed for collection of their background information and their plasma vitamin B12 and folate were measured with the immunoassay method. The status of both vitamins was evaluated and the prevalence of deficiency was estimated.

Results

The median value of the women was 214.5 pg/mL for vitamin B12 and 4.6 ng/mL for folate. The urban women had a significantly higher vitamin B12 (254.1 vs. 195.9 pg/mL) but lower folate (4.4 vs. 4.7 ng/mL) than rural women. Total prevalence of deficiency was 45.5% (95% CI: 42.6%∼48.4%) for vitamin B12 and 14.7% (95% CI: 12.6%∼16.8%) for folate. About 36% of women presented vitamin B12 deficiency alone, 5.2% belonged to folate deficiency alone and 9.5% was combined deficiency in both vitamins. More than 25% of the women were in marginal vitamin B12 status (200–299 pg/mL) and 60% in marginal status of folate (3–6 ng/mL). About 75.2% of rural women with folate deficiency were deficient in vitamin B12 and 46% for urban women. Quantile regression model found decreasing coefficient of folate status across 73 different quantiles of vitamin B12, which indicated that the women with folate deficiency had lower vitamin B12 significantly compared with those with no deficiency.

Conclusions

The deficiency of vitamin B12 and folate is still prevalent among the Chinese women in northwest China. Vitamin B12 deficiency could be more serious and the improvement of poor vitamin B12 status should be invoked when practicing the supplementation of folate against the neural tube defects in northwest China.     

Uptake and physiological function of vitamin B12 in a photosynthetic unicellular coccolithophorid alga, Pleurochrysis carterae

The photosynthetic coccolithophoid alga, Pleurochrysis (Hymenomonas) carterae, could take up and accumulate exogenous vitamin B12, most of which was converted into the coenzyme forms of vitamin B12. Two vitamin B12-dependent enzyme activities (methylmalonyl-CoA mutase, 2.6+/-0.4 nmol/min/mg protein and methionine synthase, 85.1+/-38.9 pmol/min/mg protein) could be found in a cell homogenate of the vitamin B12-supplemented alga. Most of the methylmalonyl-CoA mutase activity and 19.2% of the vitamin B12 accumulated by the algal cells were recovered in the macromolecular fractions with Mr of 150 kDa, although the remaining vitamin B12 was found only in free vitamin B12 fractions.     

Gluconeogenesis from propionate in kidney and liver of the vitamin B12-deficient rat

1. Kidney-cortex slices and the perfused livers of vitamin B(12)-deficient rats removed propionate from the incubation and perfusion media at 33 and 17% respectively of the rates found with tissues from rats receiving either a normal or a vitamin B(12)-supplemented diet. There was a corresponding fall in the rates of glucose synthesis from propionate in both tissues. 2. The addition of hydroxocobalamin or dimethylbenzimidazolylcobamide coenzyme to kidney-cortex slices from vitamin B(12)-deficient rats in vitro failed to restore the normal capacity for propionate metabolism. 3. Although the vitamin B(12)-deficient rat excretes measurable amounts of methylmalonate, no methylmalonate production could be detected (probably because of the low sensitivity of the method) when kidney-cortex slices or livers from deficient rats were incubated or perfused with propionate. 4. The addition of methylmalonate (5mm) to kidney-cortex slices from rats fed on a normal diet inhibited gluconeogenesis from propionate by 25%. 5. Methylmalonate formation is normally only a small fraction of the flux through methylmalonyl-CoA. This fraction increases in vitamin B(12)-deficient tissues (as shown by the urinary excretion of methylmalonate) presumably because the concentration of methylmalonyl-CoA rises as a result of low activity of methylmalonyl-CoA mutase (EC 5.4.99.2). Slow removal of methylmalonyl-CoA might depress propionate uptake owing to the reversibility of the steps leading to methylmalonyl-CoA formation.     

Vitamin B12 sources and bioavailability

The usual dietary sources of vitamin B(12) are animal foods, meat, milk, egg, fish, and shellfish. As the intrinsic factor-mediated intestinal absorption system is estimated to be saturated at about 1.5-2.0 microg per meal under physiologic conditions, vitamin B(12) bioavailability significantly decreases with increasing intake of vitamin B(12) per meal. The bioavailability of vitamin B(12) in healthy humans from fish meat, sheep meat, and chicken meat averaged 42%, 56%-89%, and 61%-66%, respectively. Vitamin B(12) in eggs seems to be poorly absorbed (< 9%) relative to other animal food products. In the Dietary Reference Intakes in the United States and Japan, it is assumed that 50% of dietary vitamin B(12) is absorbed by healthy adults with normal gastro-intestinal function. Some plant foods, dried green and purple lavers (nori) contain substantial amounts of vitamin B(12), although other edible algae contained none or only traces of vitamin B(12). Most of the edible blue-green algae (cyanobacteria) used for human supplements predominantly contain pseudovitamin B(12), which is inactive in humans. The edible cyanobacteria are not suitable for use as vitamin B(12) sources, especially in vegans. Fortified breakfast cereals are a particularly valuable source of vitamin B(12) for vegans and elderly people. Production of some vitamin B(12)-enriched vegetables is also being devised.     

Human plasma R-type vitamin B12-binding proteins. II. The role of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B12-binding protein in the plasma transport of vitamin B12.

The normal human granulocyte vitamin B12-binding protein, transcobalamin I, and transcobalamin III, have been labeled with 125I-labeled N-succinimidyl 3-(4-hydroxyphenyl)propionate and utilized for plasma clearance studies performed with rabbits. Both moieties of 125I-labeled granulocyte vitamin B12-binding protein-[57Co]vitamin B12 were cleared rapidly from the plasma (is less than 90% by 5 min) by the liver. After 30 min, the bulk of the 125I reappeared in the plasma in small molecular weight (less than 1000) form and was rapidly excreted in the urine. After 60 min the bulk of the [57Co]vitamin B12 reappeared in the plasma bound to rabbit transcobalamin II and was subsequently taken up by a variety of tissues. Approximately 15% of the 125I-labeled granulocyte vitamin B12-binding protein-[57Co-a1vitamin B12 was excreted intact into the bile during the period from 10 to 80 min after injection. The hepatic uptake of the protein-vitamin B12 complex was blocked by the prior injection of desialyzed fetuin but not by native fetuin. Similar results were obtained with 125I-labeled transcobalamin III-[57Co]vitamin B12. Approximately 90% of both moieties of 125I-labeled transcobalamin I-[57Co]vitamin B12 had prolonged plasma survivals similar to that of 125I-labeled bovine serum albumin. After treatment with neuraminadase, both moieties of the 125I-labeled transcobalamin I-[57Co]vitamin B12 complex were cleared rapidly from the plasma by the liver in a manner that was indistinguishable from that observed in the case of untreated granulocyte vitamin B12-binding protein and transcobalamin III. These observations indicate that desialyzed transcobalamin I and the native forms of the granulocyte vitamin B12-binding protein and transcobalamin III are cleared from plasma by the mechanism elucidated by Ashwell and Morell (Ashwell, G., and Morell A. G. (1974) Adv. Enzymol. 41, 99-128) that is capable of clearing a wide variety of asialoglycoproteins. These observations have implications concerning the function of the human R-type vitamin B12-binding proteins, the nature of the enterohepatic circulation of vitamin B12, the biological significance of the mechanism described by Ashwell and Morell, and the etiology of the increased plasma concentration of human R-type protein that occurs frequently in chronic myelogenous leukemia and occasionally in hepatocellular carcinoma and other solid tumors.     

An effective and simplified pH-stat control strategy for the industrial fermentation of vitamin B12 by Pseudomonas denitrificans

In order to improve the productivity of vitamin B(12) by Pseudomonas denitrificans carried out in a 120-m(3) fermenter, the effect of pH on vitamin B(12) biosynthesis was investigated. Results obtained from shake flask experiments showed that the feeding of carbon source (beet molasses or glucose) and methyl-group donor (betaine or choline chloride) significantly influenced the pH and the biosynthesis of vitamin B(12). In contrast to beet molasses or choline chloride, using glucose as a feed medium and betaine as a methyl-group donor, pH could be maintained at a stable range. As a result, higher vitamin B(12) production was achieved. Accordingly, an effective and simplified pH-stat control strategy was established for the fermentation of vitamin B(12) in a 120-m(3) industrial fermenter. When the new pH control strategy was applied, pH was stably kept in the range of 7.15-7.30 during fermentation. Thus, 214.3 mug/mL of vitamin B(12) was achieved.     

Novel fermentation process strengthening strategy for production of propionic acid and vitamin B12 by Propionibacterium freudenreichii

An efficient fermentation-strengthening approach was developed to improve the anaerobic production of propionic acid and vitamin B12 by co-fermentation with Propionibacterium freudenreichii. Vitamin B12 production from glucose resulted in relatively high productivity (0.35 mg/L h) but a low propionic acid yield (0.55 g/g). By contrast, glycerol gave a high propionic acid yield (0.63 g/g) but low productivity (0.16 g/L h). Co-fermentation of glycerol and glucose with a gradual addition strategy gave high yields (propionic acid: 0.71 g/g; vitamin B12: 0.72 mg/g) and productivities (propionic acid: 0.36 g/L h; vitamin B12: 0.36 mg/L h). Finally, the integrated feedstock and fermentation system strengthening strategy was demonstrated as an efficient method for the economic production of bio-based propionic acid and vitamin B12.     

Synthesis and biological activity of ribose-5'-carbamate derivatives of vitamin B(12)

Twelve biologically active derivatives of vitamin B(12) (cyanocobalamin) have been synthesized in which spacers were attached to the ribose-5'-hydroxyl group of vitamin B(12). Their potential to act as oral delivery agents for proteins, nanospheres, or immunogens using the vitamin B(12) uptake system was evaluated by determining their affinity for intrinsic factor (IF) and non-IF. The ribose-5'-hydroxyl group of vitamin B(12) was activated through the use of 1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyldi(1,2, 4-triazole) (CDT), or di(1-benzotriazolyl) carbonate (DBTC). Subsequent addition of an aminoalkane, diaminoalkane, or alkane diacid dihydrazide gave rise to vitamin B(12) derivatives suitable for attachment to various proteins, peptides, or nanospheres to enable oral delivery utilizing the vitamin B(12) uptake system. The ribose-5'-carbamate derivatives were found to possess similar affinity for intrinsic factor as that of the e-monocarboxylic acid of vitamin B(12). The affinity for non-IF was similar to cyanocobalamin or even higher for some of the smaller derivatives. Polysciences nanoparticles derivatized with vitamin B(12) 5'-carbamate adipic dihydrazide into CaCo-2 cells showed significantly higher levels of transport of the particles, when compared to unmodified particles.     

Cyanocobalamin (vitamin B12) conjugates with enhanced solubility

Cyanocobalamin (vitamin B12) is an essential nutrient as well as a very useful carrier in drug delivery. Conjugates of vitamin B12 are investigated due to their wide range of therapeutic applications. We report the synthesis of six vitamin B12 conjugates, and the effect of conjugation on their solubilities and stabilities in various media. We reveal here that vitamin B12 can be released readily if a 2'-hydroxyl group is conjugated rather than the 5'-hydroxyl group, and the solubility (thus the equivalents of vitamin B12) could be enhanced as much as 19-fold, by simple conjugates such as glycolates. Findings disclosed here provide insights into the reactivities of vitamin B12 conjugates, the design of future prodrugs and similar conjugated moieties using vitamin B12.     

Vitamin B12 and Progression of White Matter Lesions. A 2-Year Follow-Up Study in First-Ever Lacunar Stroke Patients

In cross-sectional studies periventricular white matter lesions (WML) were related to low plasma levels of vitamin B12. Whether low vitamin B12 levels are also related to progression of WML is still unknown. We studied baseline vitamin B12 levels and its association with progression of WML over 2 years of follow-up in first-ever lacunar stroke patients. In 107 first-ever lacunar stroke patients in whom baseline brain MRI and vitamin B12 status were available, we obtained a follow-up brain MRI after 2 years. We assessed progression of periventricular WML (pWML) and deep WML (dWML) using a visual WML change scale. We studied the relationship between baseline levels of plasma vitamin B12 and progression of WML after 2 years of follow-up by binary logistic regression analyses. Vitamin B12 deficiency was more frequent in patients with progression of pWML compared to those without progression (41.9% and 19.7% respectively, p = 0.02). Corrected for sex and age, progression of pWML was associated with lower baseline levels of vitamin B12 (OR 1.42 per 50 unit decrease, 95% CI 1.00-1.92). Vitamin B12 levels were not associated with progression of dWML. In conclusion progression of pWML after 2 years of follow-up relates to low levels of vitamin B12 at baseline in first-ever lacunar stroke patients. Whether this population could benefit from vitamin B12 supplementation is unknown and requires further investigation.     

Deamination and reductive deamination of (2-amino-5, 6-dimethylbenzimidazolyl)cobamide. Preparation of (2-hydroxy-5, 6-dimethylbenzimidazolyl)cobamide and (5, 6-dimethyl-[2(-14)C]-benzimidazolyl)cobamide

(2-Amino-5, 6-dimethylbenzimidazolyl)-cobamide (III) is transformed to (2-hydroxy-5, 6-dimethylbenzimidazolyl) cobamide (IV) by nitrous acid. Exchange of the NH2-group by hydrogen with nitrous acid/hypophosphorous acid yields vitamin B12 (I). This reaction completes a cycle vitamin B12 (I)----[carboxy(2-cyanoamino-4,5-dimethylphenyl)amino]cobamide+ ++ (II)----(2-amino-5,6-dimethylbenzimidazolyl)cobamide (III)----vitamin B12 (I), which allows chemical 14C-labelling of vitamin B12. In this procedure cyanogen bromide, which is necessary for the first step, was labelled with [14C] cyanide. By the following reactions a vitamin B12 was formed in which C-2 of the 5, 6-dimethylbenzimidazole moiety is labelled.     

The effect of antimetabolites on the microbial biosynthesis of vitamin B12 and related compounds

Summary Two compounds which are both antimetabolites and precursors of vitamin B12, o-phenylendiamine, and 5,6-dimethylbenzimidazole, stimulated the production of vitamin B12 by Propionibacterium freudenreichii at concentrations which were subinhibitory for growth. The stimulatory effect of the compounds depended not only on their concentration, but also on the time of addition. During cultivation, two chromatographically distinguishable fractions with vitamin B12 activity were formed. At concentrations which stimulated production of vitamin B12, only the biosynthesis of true vitamin B12 (cyanocobalamin) took place, while the biosynthesis of the analogue with a higher molecular weight was inhibited.     

Vitamin B12 absorption: Mammalian physiology and acquired and inherited disorders

Background

Vitamin B12 (cobalamin) is a cobalt-containing compound synthesized by bacteria and an essential nutrient in mammals, which take it up from diet. The absorption and distribution of dietary vitamin B12 to the organism is a complex process involving several gene products including carrier proteins, plasma membrane receptors and transporters. Disturbed cellular entry, transit or egress of vitamin B12 may lead to low vitamin B12 status or deficiency and eventually hematological and neurological disorders.

Objective

The aim of this review is to summarize the causes leading to vitamin B12 deficiency including decreased intake, impaired absorption and increased requirements. Under physiological conditions, vitamin B12 bound to the gastric intrinsic factor is internalized in the ileum by a highly specific receptor complex composed by Cubilin (Cubn) and Amnionless (Amn). Following exit of vitamin B12 from the ileum, general cellular uptake from the circulation requires the transcobalamin receptor CD320 whereas kidney reabsorption of cobalamin depends on Megalin (Lrp2).Whereas malabsorption of vitamin B12 is most commonly seen in the elderly, selective pediatric, nondietary-induced B12 deficiency is generally due to inherited disorders including the Imerslund-Gräsbeck syndrome and the much rarer intrinsic factor deficiency. Biochemical, clinical and genetic research on these disorders considerably improved our knowledge of vitamin B12 absorption.This review describes basic and recent findings on the intestinal handling of vitamin B12 and its importance in health and disease.     

Factors affecting formiminoglutamic acid excretion in vitamin B12 deficiency

1. Formiminoglutamic acid, a product of the catabolism of histidine, is excreted in abnormally large amounts in the urines of vitamin B(12)-deficient rats and of vitamin B(12)-deficient sheep; the excretion is reduced to negligible amounts after administration of vitamin B(12). 2. After administration of certain methyl donors to vitamin B(12)-deficient rats or sheep urinary excretion of formiminoglutamic acid is temporarily decreased. 3. Irrespective of the pteroylglutamic acid status of the animals neither vitamin B(12)-deficient rats nor vitamin B(12)-deficient sheep have the ability to deal efficiently with histidine. 4. In sheep, urinary excretion of formiminoglutamic acid is increased after administration of aminopterin; treatment with pteroylglutamic acid restores the ability of the animal to deal with the catabolic products of histidine. 5. The possible functions of vitamin B(12) and methionine in relieving a virtual deficiency of pteroylglutamic acid are discussed.     

Megaloblastic anemia,associated with surgically produced gastrointestinal abnormalities

Two of the mechanisms for vitamin B(12) deficiency, leading to megaloblastic anemia, are the result of surgically produced abnormalities of the gastrointestinal tract. The basic mechanism is different for each lesion. Total gastrectomy results in complete lack of intrinsic factor which is necessary for vitamin B(12) absorption. It is believed that if patients survive long enough and are not given prophylactic vitamin B(12) therapy, all would develop megaloblastic anemia. Intestinal anastomosis leading to stasis of intestinal contents, with overgrowth of bacteria may cause vitamin B(12) deficiency through bacterial interference with the utilization of vitamin B(12). Use of radioactive vitamin B(12) (cobalt(60)-labeled B(12)) has led to a better understanding of the pathogenesis of both types of megaloblastic anemia. The radioactive vitamin provides a useful tool for study of its absorption from the gastrointestinal tract.     

Uptake of a microbially-produced vitamin (B12) by soybean roots

Vitamin B12 (Cyanocobalamin) is one of the vitamins believed to be produced exclusively by microorganisms. Although soil is a rich source of vitamin B12, systematic study as to possible uptake of this vitamin by the plant roots is lacking. This study was undertaken to investigate, under water culture conditions, the uptake of [⁵⁷Co]-cyanocobalamin by soybean (Glycine max (L.) Merr.). In the range of 10 to 3200 mol L^–1, uptake of vitamin B12 was a linear function of the vitamin concentration in the nutrient solution. Depending on the vitamin concentration, 12 to 34% of the total absorbed vitamin was transported to the plant shoots, with proportionally more vitamin B12 transported at higher vitamin concentrations. Aeration of the rooting medium with nitrogen gas significantly increased the total uptake and the percentage of vitamin transported to the shoots. Addition of respiration inhibitor dinitrophenol to the nutrient solution did not affect the total uptake or the partitioning of the vitamin. Root temperature (5–30°C) did not affect the total uptake but significantly altered the partitioning of the vitamin between the roots and the shoots. Foliar-applied vitamin B12 was not translocated to any considerable degree to other plant parts, indicating that phloem transport does not contribute to the distribution of this vitamin within the plant. It is suggested that adding manure (which is rich in this vitamin) to the soil could increase soil and thus plant content of vitamin B12. This could be of importance in raising the intake of this vitamin by people living by choice or necessity on vegetarian diets who are usually threatened by vitamin B12 deficiency.     

维生素B12的光解研究及发酵液中维生素B12检测新方法的建立

采用HPLC检测法研究5,脱氧腺苷钴胺素和甲基基钴胺素的水溶液在不同光源及不同照度下的光解情况,结果表明随着光能量的增加光解速度加快。利用脱氧腺苷钴胺素的光解性质,探索了一种检测发酵液中维生素B12含量的新方法。将含有钴胺素的细胞破碎液完全光解后,测定光解后的产物羟基钴胺素,以此来确定维生素B12产量。此方法简便快速、重复性好,可应用于发酵生产维生素B12的各个环节。