Myelin lipids in vitamin B12 deficiency in chicks

Lipid profile of the spinal cord myelin was studied in normal and vitamin B₁₂ deficient chicks. The significant findings were a reduction in the total galactolipids and an increase in the total phospholipids of myelin in vitamin B₁₂ deficiency. The altered molar ratios of these lipids suggest a relative immaturity of the myelin in this condition. These changes may initiate the degenerative changes in the central nervous system in vitamin B₁₂ deficiency.Vitamin B₁₂ is essential for normal functioning of both the hemopoietic and the nervous system. Sub-acute combined degeneration of the spinal cord is seen both in association with pernicious anemia and in megaloblastic anemia of dietary origin (1,2). Though many biochemical postulates (3, 4) have been advanced to explain the neurological changes, the role of vitamin B₁₂ in maintaining the integrity of myelin is still obscure. So far only in two animal species the monkey (5) and the chick (6) has myelin degeneration been reported in vitamin B₁₂ deficiency. However, in neither of these reports, have changes in the composition of myelin been described. The results of a study in chicks wherein the effects of vitamin B₁₂ on the lipid profile of myelin were investigated are reported here.     

Utilization of Hydrocarbons and Vitamin B12 Production by Bacillus badius

The accumulation of vitamin B₁₂ by Bacillus badins grown on hydrocarbon was investigated. The bacterium could assimilate n-alkanes of C₁₁–C₁₈, ethanol, fumarate, α-ketoglutarate and malate. n-Alkanes of C₁₆–C₁₈, were the best for vitamin B₁₂ production. The bacterium utilized well all of the nitrogen sources tested. Above all, ammonium dihydrogen phosphate was the best for the bacteria] growth and vitamin B₁₂ production. Addition of organic nutrients such as malt extract and meat extract, and addition of metal ions such as ferrous and cobalt promoted the growth and vitamin B₁₂ production. Interestingly, vitamin B₁₂ was produced mostly in the supernatant. The cyanoform of the corrinoid predominantly formed in the supernatant would confirm the identity with cobalamin.     

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.     

The effect of vitamin B12 and biotin on the metabolism of vitamin B12 in biotin-deficient rats

1. The effect of the administration of vitamin B₁₂ and biotin on the metabolic pattern of vitamin B₁₂ in biotin-deficient rats was studied. 2. No significant changes in the absorption and excretion of orally administered [⁵⁸Co]vitamin B₁₂ were noted either in vitamin B₁₂-treated and or in biotin-fed rats. A significant decrease of the uptake of orally given [⁵⁸Co]vitamin B₁₂ was observed in the liver and kidneys of biotin-treated rats, whereas an increase of uptake in the kidneys of vitamin B₁₂-treated rats was noted as compared with biotin-deficient animals. 3. No significant difference in the excretion of radioactivity was noted between biotin deficient and biotin-fed rats when [⁵⁸Co]vitamin B₁₂ was administered by injection. A small decrease was observed in vitamin B₁₂-treated rats. The retention of injected [⁵⁸Co]vitamin B₁₂ by major organs of biotin-treated rats was lower than that of biotin-deficient rats. A lower content of [⁵⁸Co]vitamin B₁₂ was also detected in the organs, with the exception of the kidneys, of vitamin B₁₂-treated rats. 4. These results are discussed in terms of an interrelationship between biotin and vitamin B₁₂.     

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.     

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.     

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.     

Characterization of vitamin B12 compounds in the fruiting bodies of shiitake mushroom (Lentinula edodes) and bed logs after fruiting of the mushroom

This study determined the vitamin B₁₂ content in commercially available dried fruiting bodies of shiitake mushroom, Lentinula edodes. The vitamin B₁₂ contents in dried donko-type fruiting bodies with closed caps (5.61 ± 3.90 μg/100 g dry weight), did not significantly differ from those of dried koushin-type fruiting bodies with open caps (4.23 ± 2.42 μg/100 g dry weight). The bed logs after fruiting of the mushroom also contained the vitamin B₁₂ levels similar to that in the dried shiitake fruiting bodies. To determine whether the dried shiitake fruiting bodies and their bed logs contained vitamin B₁₂ or other corrinoid compounds that are inactive in humans, we purified corrinoid compounds using an immunoaffinity column and identified vitamin B₁₂ using vitamin B₁₂-dependent Escherichia coli 215 bioautograms and liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) chromatograms. Dried shiitake fruiting bodies rarely contained an unnatural corrinoid vitamin B₁₂[c-lactone] that is inactive in humans. Given that shiitake mushroom lacks the ability to synthesize vitamin B₁₂ de novo, the vitamin B₁₂ found in dried shiitake fruiting bodies must have been derived from the bed logs.     

Reactions of vitamin B12r with polyhalogenated hydrocarbon pesticides

The reaction of vitamin B_12r, generated by photolysis of methylcobalamin under a nitrogen atmosphere, with 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), results in extensive dechlorination and formation of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) as the major products. Minor quantities of 1,1-bis(p-chlorophenyl)-2-chloroethane (DDMS), 1,1-bis(p-chlorophenyl)-2-chloroethylene (DDMU), 1,1-bis(p-chlorophenyl)ethane (DDO), and 1,1-bis(p-chlorophenyl)ethylene (DDNU) were also formed. Reaction of vitamin B_12r with DDD results in the production of DDMU and DDMS, the latter of which can react to produce DDNU and DDO. DDE and DDMU do not react with vitamin B_12r. The results obtained are suggestive of a vitamin B_12r-mediated dechlorination pathway for polyhalogenated hydrocarbon pesticides.     

Effects of Vitamin B12 Analogues with Alternations in the Side Chains of the Corrin Ring on Urinary Methylmalonate Excretion in Vitamin B12-Deficient Rats

To study how much the side chains of the corrin ring of vitamin B₁₂ are involved in the physiological roles of the vitamin, five vitamin B₁₂ analogues (cyanocobalamin-b-monocarboxylate, cyanocobalamin-d-monocarboxylate, cyanocobalamin-e-monocarboxylate, cyano-13-epicobalamin, and cyanocobalamin(c-lactam)) with alternations in the side chains were synthesized chemically and then administered orally and intravenously to vitamin B₁₂-deficient rats. Male rats fed a vitamin B₁₂-deficient diet for 11 wk developed a severe vitamin B₁₂ deficiency with a high urinary methylmalonate excretion (223.8 ± 136.2 μmol/d) and ~97% (1.2±0.7ng/g tissue) lower hepatic vitamin B₁₂ content. Oral and intravenous administration of cyanocobalamin-b-,-d-, and -e-monocarboxylates and cyano-13-epicobalamin could not improve the severe vitamin B₁₂-deficient status of the rats, indicating that the b-, d-, and e-propionamide side chains of the corrin ring of vitamin B₁₂ are important in the absorption, transport, and function of the vitamin in rats. Urinary methylmalonate excretion of the rats that were intravenously administered cyanocobalamin(c-lactam) increased twice as much as those of the other analogue-supplemented rats, suggesting that cyanocobalamin(c-lactam) act as a powerful Cbl-antagonist. The results also indicate that mammalian cells do not contain a system for synthesizing complete vitamin B₁₂ from these analogues.     

Vitamin B12 Content of Circulating Leukocytes as an Aid in the Differentiation of the Acute Leukemias

From 25 patients with acute leukemia 116 specimens of leukocytes were assayed microbiologically for total vitamin B₁₂ to determine if variation in vitamin B₁₂ content would help in differentiating the acute leukemias. The mean cell vitamin B₁₂ levels (μμg./10⁸ cells) in the different types of leukemia were: lymphoblastic 464, myeloblastic 1058 and monocytic 200. Cell vitamin B₁₂ levels above the normal range (100-800 μμg./10⁸ cells) are suggestive of myeloblastic leukemia. The only elevated cell vitamin B₁₂ levels comparable to those found in myeloblastic leukemia were in reticulum cell leukemia, and this type of leukemia was not difficult to diagnose morphologically. Blast cells contained more vitamin B₁₂ than mature cells of the same series; there was a significant positive correlation between the percentage of blast cells and cell levels of total vitamin B₁₂ in both lymphoblastic and myeloblastic leukemia.     

Enhanced anaerobic biotransformation of carbon tetrachloride with precursors of vitamin B12 biosynthesis

Relatively low concentrations of Vitamin B₁₂ are known to accelerate the anaerobic biotransformation of carbon tetrachloride (CT) and chloroform (CF). However, the addition of vitamin B₁₂ for field-scale bioremediation is expected to be costly. The present study considered a strategy to generate vitamin B₁₂ by addition of biosynthetic precursors. One of the precursors, porphobilinogen (PB) involved in the formation of the corrin ring, significantly increased the CT biotransformation rates by 2.7−, 8.8- and 10.9-fold when supplemented at 160, 500 and 900 μM, respectively. A positive control with 10 μM of vitamin B₁₂ resulted in a 5.9-fold increase in the CT-bioconversion rate. PB additions provided high molar yields of inorganic chloride (57% of CT organochlorine), comparable to that obtained with vitamin B₁₂ supplemented cultures. The primary substrate, methanol, known to induce vitamin B₁₂ production in methanogens and acetogens, was required for PB to have a significant impact on CT conversion. The observation suggests that PB’s role was due to stimulating vitamin B₁₂ biosynthesis. The present study therefore provides insights on how to achieve vitamin B₁₂ enhanced rates of CT bioremediation through the use of less complex compounds that are precursors of vitamin B₁₂. Although PB is a costly chemical, its large impact points to corrin ring formation as the rate-limiting step.     

Impact of diet management and composition on vitamin B12 concentration in milk of Holstein cows

As vitamin B₁₂ is only synthesized by bacteria, ruminant products, especially dairy products, are excellent sources of this vitamin. This study aims to identify if diet and cow characteristics could affect vitamin B₁₂ concentration in milk of dairy cows. Information on 1484 first, 1093 second and 1763 third and greater parity Holstein cows in 100 herds was collected during three consecutive milkings. During the first morning milking, all dietary ingredients given to cows were sampled and quantities offered were recorded throughout the day. Nutrient composition of ingredients was obtained by wet chemistry to reconstitute nutrient composition of the ration. Milk samples were taken with in-line milk meters during the evening milking of the 1^st day and the morning milking of the 2nd day and were analyzed for vitamin B₁₂ concentration. Milk yields were recorded and milk components were separately analyzed for each milking. Daily vitamin B₁₂ concentration in milk was obtained using morning and evening vitamin B₁₂ concentrations weighted with respective milk yield, then divided by daily yield. To decrease the number of interdependent variables to include in the multivariable model, a principal component analysis was carried out. Daily milk concentration of vitamin B₁₂ averaged 3809±80 pg/ml, 4178±79 pg/ml and 4399±77 pg/ml for first, second and third, and greater lactation cows. Out of 11 principal components, six were significantly related to daily milk concentration of vitamin B₁₂ when entered in the multivariable model. Results suggested that vitamin B₁₂ concentration in milk was positively related to percentage of fiber and negatively related to starch as well as energy of the diet. Negative relationships were noted between vitamin B₁₂ concentration in milk and milk yield as well as milk lactose concentration and positive relationships were observed between vitamin B₁₂ concentration in milk and milk fat as well as protein concentrations. The percentages of chopped mixed silage and commercial energy supplement in the diet as well as cow BW were positively related to vitamin B₁₂ in milk and percentages of baled mixed silage, corn and commercial protein supplement in the ration were negatively related to vitamin B₁₂ concentration in milk. The pseudo-R² of the model was low (52%) suggesting that diet and cow characteristics have moderate impact on vitamin B₁₂ concentration in milk. Moreover, when entering solely the principal component related to milk production in the model, the pseudo-R² was 46%. In conclusion, it suggests that studied diet characteristics have a marginal impact on vitamin B₁₂ concentration in milk variation.     

Vitamin B12-induced alterations in activities of α-glycerophosphate dehydrogenase and malic enzyme in brain of singi fish, Heteropneustes fossilis (Bloch)

Different doses of vitamin B₁₂ (0.25, 0.5, 1, 2 and 4 μg/g, injected intraperitoneally for three consecutive days) altered the activities of mitochondrial-α-glycerophosphate dehydrogenase (α-GPD) and NADP-dependent cytosolic malic enzyme (ME) in the brain of singi fish. The α-GPD activity increased at doses of 0.5, 1, 2 and 4 μg/g vitamin B₁₂. A dose of 0.5 μg/g vitamin B₁₂ induced less activity than higher doses. ME activity increased with 1, 2 and 4 μg/g of vitamin B₁₂/g. The mitochondrial and cytosolic protein content remained unchanged after vitamin B₁₂ administration. Cycloheximide treatment inhibited the vitamin B₁₂-induced increase in α-GPD and ME activity. Thus, vitamin B₁₂ is involved in the induction of some enzymes in fish brain.     

Nutrition-Related Vitamin B12 Deficiency in Patients in Pakistan with Type 2 Diabetes Mellitus not Taking Metformin

ObjectiveTo estimate the frequency of undiagnosed vitamin B₁₂ deficiency among patients with type 2 diabetes mellitus who had not taken metformin during at least the prior 5 years and to ascertain whether vitamin B₁₂ deficiency among the patients with type 2 diabetes was due to nutritional deficiency or malabsorption.MethodsSerum vitamin B₁₂ levels were measured in 44 subjects with diabetes and a mean age of 51 years (range, 40 to 70), 21 (48%) of whom had low levels (< 200 pg/mL). Of those 21 patients, 10 agreed to enroll in an intervention phase consisting of oral supplementation with mecobalamin, 1,500 μg daily for 3 months. Those patients in whom vitamin B₁₂ levels failed to normalize after oral supplementation alone would be presumed to have vitamin B₁₂ deficiency attributable to malabsorption.ResultsAlmost half of the subjects with type 2 diabetes not taking metformin had biochemically proven vitamin B₁₂ deficiency. All 10 subjects who enrolled in the intervention phase had normalization of their vitamin B₁₂ levels after 3 months of oral supplementation with mecobalamin.ConclusionWe conclude that vitamin B₁₂ deficiency is common among patients with type 2 diabetes and was related to nutrition in our study group. In addition to intensive glycemic control, vitamin B₁₂ supplementation should be considered for treatment of diabetic neuropathy. In almost 50% of patients with low vitamin B₁₂ levels, the deficiency was corrected with oral supplementation only. This, indeed, is an important finding, inasmuch as oralvitamin B₁₂ supplementation is easy, convenient, and readily accepted by patients. This finding highlights the need for aggressive and early diagnosis and treatment to avoid complications of vitamin B₁₂ deficiency. (Endocr Pract. 2010;16:205-208)     

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.     

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.     

Oxidation of compounds metabolized through folate coenzyme pathways in vitamin B12-deficient rats

The effects of vitamin B₁₂ deficiency in rats and dietary supplementation with vitamin B₁₂ and/or l-methionine plus folate on the oxidation of compounds metabolized through folate coenzyme pathways were investigated. Rats fed a vitamin B₁₂-deficient diet oxidized significantly lower amounts in 60 min of l-histidine, glycine, sarcosine, formate, and l-serine to CO₂ than vitamin B₁₂-supplemented controls. Supplementation of the deficient diet with l-methionine plus folate restored the ability to oxidize the ring-2-carbon of l-histidine, the methyl group of sarcosine, and formate to the same level as that observed in animals receiving vitamin B₁₂. In contrast, oxidation of the 1-carbon of glycine and the 3-carbon of l-serine was not restored to control levels by addition of methionine plus folate to the vitamin B₁₂-deficient diet. Inhibition of the metabolism of the 2-carbon of glycine to CO₂ was partially overcome by additional dietary methionine and folate. Glycine synthase activity in homogenates paralleled the in vivo pattern of oxidation of the 1-carbon of glycine to CO₂, whereas sarcosine dehydrogenase activity appeared to increase 2-fold in vitamin B₁₂ deficiency.     

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.     

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.