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).     

Role of Third Serum Vitamin B12 Binding Protein in Vitamin B12 Transport

A third vitamin B₁₂ binding protein present in normal serum has been shown to participate in transport of labelled vitamin B₁₂ absorbed from the gut. All three vitamin B₁₂ binding proteins in serum were labelled at the same time after oral administration of vitamin B₁₂, implying that “free” vitamin B₁₂ reached the portal blood from the gut mucosa.     

Correlation of Serum and Urine Vitamin B12

The relation between the serum vitamin B₁₂ level and the daily loss of vitamin B₁₂ in urine was examined in patients with normal serum vitamin B₁₂ levels and in patients suffering from vitamin B₁₂ deficiency. A linear correlation was found between the two measurements, suggesting that the serum vitamin B₁₂ level is a governing factor in the urinary loss of vitamin B₁₂. The contribution by this loss to the total loss of vitamin B₁₂ from the body is small under normal circumstances but becomes quantitatively more important with the depletion of body stores.     

Free and Total Vitamin B12 in Cerebrospinal Fluid

Free and total vitamin B₁₂ levels in serum and cerebrospinal fluid (CSF) were bioassayed, since there were no available data on the relationship between free and total vitamin B₁₂ in CSF or between free vitamin in serum and CSF vitamin B₁₂. The subjects were 43 neurological patients. Serum levels were normal in 40 of 43 patients. Values for free and total vitamin B₁₂ in CSF were the same in 42 of 43 patients. Mean CSF vitamin B₁₂ was 21 μμg./ml. In 17 cases CSF vitamin B₁₂ equalled free vitamin B₁₂ level in serum, in 16 cases CSF vitamin B₁₂ was lower than the free level in serum, and in 10 cases CSF vitamin B₁₂ was higher than the free vitamin level in serum. There was no apparent diagnostic correlation. The findings suggest that vitamin B₁₂ is not bound in CSF and that there is some selective control of passage of vitamin B₁₂ across the blood-CSF barrier.     

Vitamin B12 Status in Pregnancy among Immigrants to Britain

Haemoglobin, serum vitamin B₁₂, and serum and red cell folate levels have been measured in 322 pregnant immigrant women in London at their first booking and in a proportion at 34 weeks of gestation and postnatally. The Indian, East-African Indian, and Pakistani and Bangladeshi patients showed significantly lower initial mean serum vitamin B₁₂ levels than the European group, the levels being lower in Hindu and Sikh patients than in Moslems. The patients of West Indian, Indian, and East-African Indian origin showed significantly lower initial mean haemoglobin levels than the immigrants from European countries. Though there was no overall correlation between haemoglobin and serum vitamin B₁₂ level the incidence of hypersegmented polymorphs and macrocytosis in the peripheral blood was highest in the Indian and East-African Indian patients, and both these features were particularly frequent in patients with subnormal serum vitamin B₁₂ levels. Only one patient, however, had overt megaloblastic anaemia due to vitamin B₁₂ deficiency. The Indian patients whose red cell folate levels were less than 200 ng/ml also had a lower mean serum vitamin B₁₂ level than those with red cell folate levels greater than 200 ng/ml. The Indian patients had smaller babies than the Europeans but this was not related to the differences in vitamin B₁₂ status between the two groups. However, out of 39 babies of the Indian group 5 (13%) showed subnormal serum vitamin B₁₂ levels in the first 10 days of life, the lowest level being 120 pg/ml.Though there was an overall statistically significant fall in serum vitamin B₁₂ between first booking and 34 weeks of pregnancy there was no significant fall in serum vitamin B₁₂ in those who initially had subnormal levels. Thus many Indian women are vitamin B₁₂ deficient in pregnancy, and this is associated with morphological blood abnormalities in many cases, but megaloblastic anaemia due to this deficiency is relatively infrequent.     

Mouse transcobalamin II metabolism: The effects of antibiotics on the clearance of vitamin B12 from the serum transcobalamin II-vitamin B12 complex and the reappearance of the free serum transcobalamin II in the mouse

The mechanism of the clearance of vitamin B₁₂ from the serum transcobalamin II-vitamin B₁₂ (Tc-II-B₁₂) complex and the reappearance of free Tc-II in mouse have been studied. When a saturating dose of vitamin B₁₂ is given parenterally to normal mice, a portion of the Tc-II-bound vitamin B₁₂ is rapidly cleared and free Tc-II promptly reappears until it reaches a constant level in 6–8 h. The remaining vitamin B₁₂ is cleared slowly from the rest of the Tc-II-B₁₂ complex. In cycloheximide or puromycin-treated mice, free Tc-II fails to reappear and the bound Tc-IIdecreases. Treatment with actinomycin D has no effect on the reappearance of free Tc-II. The probable mechanism of this inhibition is discussed. The results suggest that mouse serum Tc-II has a stable messenger RNA template and a fast turnover. The free Tc-II which reappears in the serum after Tc-II has been saturated with vitamin B₁₂, appears to be newly synthesized.     

Transport of vitamin B12 into mouse leukemia cells.

Transport of [⁵⁷Co]cyanocobalamin (vitamin B₁₂) into L1210 murine leukemia cells, mediated by transcobalamin-II, is a biphasic process. The primary step, which occurs very rapidly (within the first minute), appears to involve binding of the B₁₂-transcobalamin complex to the external membrane of the cells; at 37 °C and pH 7.3, apparent K_m and V values are 180 pM and 5.9 pmol/min/10⁹ cells, respectively. This step is relatively insensitive to temperature, has an apparent pK_a of 6.0, and is inhibited by EDTA; Ca²⁺ or Mg²⁺ can reverse the inhibition. The slower secondary step appears to involve translocation of the vitamin into the cell; V for this step is 0.4 pmol/min/10⁹ cells. Temperature and pH optima are 30 ° and 6.5?7.0, respectively. This step is stimulated by glucose and inhibited by cyanide, arsenite, arsenate, p-chloromercuriphenylsulfonate and dinitrophenol. Labeled B₁₂ in an amount corresponding to that taken up in the initial step can be released by: (a) resuspending the cells in a B₁₂-free medium; (b) addition of unlabeled B₁₂-transcobalamin-II; and (c) addition of EDTA. The material released is chromatographically and functionally identical with B₁₂-transcobalamin-II.     

Release of unsaturated vitamin B12 binding capacity from human granulocytes by the calcium ionophore A23187

The ionophore A23187, in the presence of calcium ions, was capable of eliciting the release of granule-associated unsaturated vitamin B₁₂ binding capacity from human granulocytes. The ionophore-induced extrusion of unsaturated vitamin B₁₂ binding capacity was concentration, time and temperature-dependent. The release was blocked by 2-deoxyglucose and was unaccompanied by cytotoxicity (trypan-blue uptake and lactate dehydrogenase release). The unsaturated vitamin B₁₂ binding capacity release was accompanied by the release of lysozyme, a specific granule marker enzyme.     

A comparison between different concentrations and sources of cobalt in goat kid nutrition

There have been extensive studies in sheep and cattle considering cobalt (Co) supplementation and its effects on vitamin B₁₂ concentrations in the body. However, there are limited studies on goats. The aim of this study was to compare two different sources of Co (sulfate v. glucoheptonate) at two different concentrations (0.25 and 0.5 mg/kg dry matter) in goat kid nutrition, and to evaluate the effects of these supplements on performance, serum vitamin B₁₂, blood biochemistry and rumen volatile fatty acids. For this purpose, 30 weaned male goat kids were randomly allotted to five treatments. Serum vitamin B₁₂ increased during the trial in the Co-supplemented groups. Co supplementation increased serum glucose concentrations. On day 35, Co-supplemented groups had greater glucose concentrations compared with control. Propionic+iso-butyric acid concentrations increased only in the 0.5 mg Co glucoheptonate treatment (P<0.05). Our results suggest that, despite the two sources of Co proving mostly similar, the main advantage of Co glucoheptonate compared with Co sulfate was in the ruminal synthesis of vitamin B₁₂. However, although providing Co at National Research Council recommendation levels maintained vitamin B₁₂ above or at normal concentrations, Co supplementation of the Co sufficient basal diet increased vitamin B₁₂ and glucose concentrations.     

Vitamin B12 deficiency-induced increase of osteoclastic bone resorption caused by abnormal renal resorption of inorganic phosphorus via Napi2a

Vitamin B₁₂ deficiency is a risk factor for bone disorders via mechanisms not fully understood. In this study, an increase in serum inorganic phosphorus (Pi) concentrations was associated with a vitamin B₁₂ deficiency. Napi2a, a renal cotransporter for Pi reabsorption, accumulated on plasma membranes in a vitamin B₁₂ deficiency suggests that vitamin B₁₂ plays an important role in Pi homeostasis.     

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.     

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.     

VITAMIN B12, THIAMINE,AND BIOTIN CONTENTS OF MARINE PHYTOPLANKTON1

An extraction procedure was developed for determining vitamin B₁₂, thiamine, and biotin contents of marine phytoplankton. Phytoplankters were collected either by centrifugation or by retention on a glass fiber filter, then heated at 100 C for I hr in 100 ml of vitamin-free seawater acidified to pH 3.5 with HCl. The extract, after debris removal, was filter-sterilized and analyzed, for vitamin B₁₂, thiamine, and biotin with standard vitamin assay procedures. The vitamin contents of haeodactylum tricornutum, Skeletonema costatum, Stephanopyxis turris, and occolithus liuxleyi were determined during growth in batch cultures. P. tricornutum (non-vitamin requirer) growing in aerated cultures contained 0.29–0.96 ng B₁₂, 5–15 ng thiamine, and 0.45–1.70 ng biotin/mg C. Under similar conditions S. costatum (B₁₂-requirer) contained about 0.06 ng B₁₂, 5–36 ng thiamine, and 0.16–2.10 ng biotin/mg C. The concentrations of vitamin were generally similar during some portion of the growth curve, eg, logarithmic growth. The vitamin B₁₂, content of S. costatum growing under nonaerated conditions decreased when medium B₁₂, was reduced. The biotin content did not change when medium B₁₂ was decreased. The thiamine content per unit weight of C. huxleyi (thiamine-requirer) growing with either 10 or 120 ng/liter thiamine decreased under both medium concentrations, indicating no net synthesis of the vitamin.     

Nature of vitamin B12 binding by native serum,seromucid, and highly purified fractions from chicken serum

1.

The nature of the binding of vitamin B₁₂ by two highly purified fractions from chicken serum was compared with the binding by native chicken serum and seromucoid. The following results were obtained:     

Bile and detergent interaction with the radioassay for vitamin B12 binders using protein and dextran-covered charcoal

Bile acids and detergents such as Triton X-100, sodium dodecyl sulfate, and sodium deoxycholate interfered with the radioassay for vitamin B₁₂ binders. The effect was dependent on the concentration of the detergent. The detergents and bile acids were found, by use of [¹⁴C]glycocholic acid, to bind firmly to the covered charcoal in the assay, thereby blocking the binding of free vitamin B₁₂ to the charcoal. An increased ⁵⁷Co-labeled vitamin B₁₂ concentration was therefore observed in the final supernatant in the assay, giving rise to an apparent but false vitamin B₁₂-binding capacity.     

Assessment of Deoxyuridine Suppression Test in Diagnosis of Vitamin B12 or Folate Deficiency

Deoxyuridine (dU) suppression tests have been performed on virtually all marrow samples aspirated at this hospital over the past 12 months. Of the 110 samples studied 26 gave abnormal results, and these 26 samples came from patients deficient in either vitamin B₁₂ or folate. The dU suppression test was found to be of particular value in the diagnosis of vitamin B₁₂ or folate deficiency in non-anaemic patients with macrocytosis and equivocal changes in marrow morphology and in patients in whom the serum vitamin B₁₂ or red cell folate levels were within the normal range.     

Production of vitamin B12 by a fermentor with a hollow-fiber module

Summary To improve the microbial production of vitamin B₁₂, we applied a hollow-fiber module to cultivation of the vitamin producers. By the removal of growth inhibitors, very high concentrations of cells and vitamin B₁₂ were obtained comparing to the batch culture. We obtained 227 g dry cells/l and 52 mg vitamin B₁₂/l with Propionibacterium shermanii and 33.4 g dry cell/l and 92.5 mg vitamin B₁₂/l with Butyribacterium methylotrophicum by this cultivation.     

脱氮假单胞菌生产维生素B12过程中粪卟啉Ⅲ的测定及发酵调控

脱氮假单胞菌发酵生产维生素B₁₂过程中,副产物粪卟啉Ⅲ的积累对产物的代谢合成和分离提取有很大的影响。建立了发酵液中粪卟啉Ⅲ的HPLC快速测定方法,发酵液处理后直接进样测定,检测线性范围为12~275 μg/ml,重复性好。研究了不同供氧水平、二氧化碳浓度和pH值对发酵过程中维生素B₁₂和粪卟啉Ⅲ代谢合成的影响,并在120吨发酵罐中进行了发酵过程优化控制。结果表明:在发酵过程产物的合成期控制适当的供氧水平、控制二氧化碳浓度在8.6±0.8%、维持pH值在7.0±0.12能明显抑制卟啉Ⅲ的生物合成,同时使维生素B₁₂产量提高15%。     

Vitamin B12 Excretion in Patients with Various Skin Diseases

The excretion in the urine of ⁵⁸Co after an oral dose of ⁵⁸Co vitamin B₁₂ given together with intrinsic factor has been found to be reduced in a number of patients with psoriasis, eczema, and other less common dermatoses. There is a correlation between the abnormality and the extent of the rash. A reduced glomerular filtration rate was found in a few of the patients in whom it was measured, and this must have been responsible, at least in part, for the reduced excretion of vitamin B₁₂ in these patients, but abnormal vitamin B₁₂ excretion also occurred in the absence of impaired renal function. Our evidence is insufficient to show whether malabsorption or increased tissue utilization of vitamin B₁₂ was the explanation in other cases. Certainly a number of patients had steatorrhoea, and in these it is most likely that malabsorption was the major factor. In patients without steatorrhoea a lone malabsorption of vitamin B₁₂ cannot be excluded. A decreased serum concentration of vitamin B₁₂ was found in only one of the patients.     

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.