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.     

Exploring the onset of B12-based mutualisms using a recently evolved Chlamydomonas auxotroph and B12-producing bacteria

Cobalamin (vitamin B₁₂) is a cofactor for essential metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonize the algal phycosphere, forming stable communities that gain preferential access to photosynthate and in return provide compounds such as B₁₂. Extended coexistence can then drive gene loss, leading to greater algal–bacterial interdependence. In this study, we investigate how a recently evolved B₁₂-dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B₁₂ producers, its growth in co-culture was slower than the B₁₂-independent wild-type, suggesting that high bacterial B₁₂ provision may be necessary to favour B₁₂ auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B₁₂ makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B₁₂ release provided an advantage in co-cultures. We hypothesize that, given the right conditions, bacteria that release more B₁₂ may be selected for, particularly if they form close interactions with B₁₂-dependent algae.     

GROWTH OF VITAMIN B12-REQUIRING MARINE DIATOMS IN MIXED LABORATORY CULTURES WITH VITAMIN B12-PRODUCING MARINE BACTERIA12

The vitamin B₁₂ requirement of several marine diatoms can be satisfied in B₁₂^?limited laboratory cultures by heterotrophic marine bacteria isolated from the same waters and from sediments. The bacteria can utilize diatom excretory products, or the remains of dead diatom cells, in the production of the vitamin. The growth of 12 B₁₂¹? requiring diatoms (7 genera) in mixed cultures with 14 different bacteria (without added B₁₂) was compared to the growth of those same diatoms in axenic cultures with excess added B₁₂. Diatom growth was generally rapid in the first few days, followed by sustained, slower growth. The diatom yields in mixed cultures ranged from 0.8 to 84% of the yields in axenic cultures with added B₁₂. In a detailed study of one mixed culture, increases in diatom densities were paralleled by increases in cell densities of the bacterium during the first few days of exponential diatom growth. During the period of slow diatom growth, when diatom densities oscillated but steadily increased, the decreases in diatom densities were associated with increased bacterial growth. This suggests that death of a fraction of the B₁₂-limited diatom population releases sufficient organic matter to stimulate growth of the bacteria and their subsequent excretion of B₁₂; this B₁₂ in turn stimulates further growth of the diatoms. Diatom-bacteria interactions leading to the production of B₁₂ may be important in maintaining viable populations of B₁₂-requiring diatoms in nutrient-poor waters during periods between blooms, when conditions are unfavorable for rapid growth.     

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)     

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.     

Biochemical Studies on Vitamin B12 Part XI

In Part X, the authors published studies on vitamin B₁₂-like activity of cobalt-porphyrin-derivative prepared from radish-leaves, and the physiological significance of the compound as an active component in the intermediary metabolism of vitamin B₁₂ was described. , Afterward, pure chlorophyll a and b were isolated by column chromatography and the vitamin B₁₂-like activities of Co-porphyrin a and b were repeatedly confirmed.

Recently, effect of Co-porphyrin on the biosynthesis of real vitamin B₁₂ has been tested and the confirmation was also successfully completed by bioautography and paper electrophoresis.

In the present communication, it is discussed as of special importance that cobalt-porphyrin may be the active principle for the formation in vivo of vitamin B₁₂.     

Smoking in Pregnancy and Vitamin B12 Metabolism

In pregnancy the level of serum vitamin B₁₂ is lower in women who smoke than in non-smokers. This finding occurs independently of social class, parity, or level of haemoglobin. In addition, the mean serum B₁₂ level tends to be less in women who are anaemic and is less in those women who have smaller babies. These findings may be an effect of the cyanide content of tobacco smoke, since cyanide may be detoxified by a mechanism which depletes the stores of vitamin B₁₂ in the body.     

Hooked to vitamin B12 since 1955: A historical perspective

In our pioneering work in 1956, two binders of vitamin B₁₂ (B₁₂) alias cobalamin (Cbl) were identified in gastric juice, S with slow electrophoretic mobility, a 70 kD protein with intrinsic factor (IF) activity and another rapid (R), not IF active but probable digestion product. Numerous sources contained a protein immunologically identical to R (haptocorrin, Hc). Another IF-active component (I) was found. Isoelectric focusing showed that S, I and R were assemblies of “isoproteins” with different pI's due to varying glycosidation. Isolation of S, I and R in microquantities was achieved in 1962 using a series of ion exchange chromatographies and gel filtration. Ponderable products were obtained in 1965–1966. The B₁₂-IF complex was a dimer, contained 13% carbohydrate and showed a different absorption spectrum than B₁₂. Using the Schilling test, B₁₂ absorption was shown to require Ca⁺⁺, bound in vitro to the ileal receptor and IF, but most of Ca⁺⁺ could be removed with sialidase. The receptor–substrate complex contained Ca⁺⁺ and carbohydrate. The purified receptor was shown to contain two main subunits. The Imerslund–Gräsbeck syndrome was discovered 1958–1960; it is caused by mutations in either of two genes, cubilin or amnionless, which form the multiligand receptor cubam. Testicular biopsies during and after B₁₂-treated deficiency showed remarkable improvement after therapy. Studies of the turnover of radioactive B₁₂ revealed biliary and fecal excretion, enterohepatic circulation and allowed calculation of biological half-life and daily need. The B₁₂ coenzymes largely behaved like B₁₂. To study whether radiocobalt in B₁₂ was representative of the rest of the B₁₂ molecule, ³²P and ⁵⁷Co labeled hydroxocobalamins were biosynthesized and shown to behave identically when given simultaneously to rats. The complex metabolism of B₁₂ explains the pathogenesis of B₁₂ deficiencies. Some of its mechanisms are not restricted to B₁₂, e.g. the endocytosis of B₁₂-IF also applies to other macromolecules.     

Relationships between biotin and vitamin B12. Effects of biotin and vitamin B12 on folic acid metabolism

1. The effects of dietary biotin compared with vitamin B₁₂ on the total content and on the distribution of the various folate derivatives in the liver of rats given a biotin-free diet have been studied. The effect of both vitamins on the conversion in vitro of folic acid into citrovorum factor in the same experimental conditions was also examined. 2. In biotin-treated rats as well as in vitamin B₁₂-treated rats the total content of folic acid-active substances measured microbiologically by Pediococcus cerevisiae, Streptococcus faecalis and Lactobacillus casei is significantly higher than that in biotin-deficient rats. The liver distribution of various folate derivatives in the three groups of animals is also markedly modified. 3. The amount of citrovorum factor formed in systems with liver homogenate of rats receiving biotin or vitamin B₁₂ is higher than that with liver homogenates of deficient rats. 4. The results obtained demonstrate the influence of biotin in the metabolism of folic acid, and the similar actions at this level of both biotin and vitamin B₁₂. These results are discussed in relation to the participation of the two vitamins in the metabolism of C₁ units, as a biochemical interpretation of the relationships between vitamin B₁₂ and biotin.     

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.     

Methylations in vitamin B12 biosynthesis and catalysis

Vitamin B₁₂ is an essential biomolecule that assists in the catalysis of methyl transfer and radical-based reactions in cellular metabolism. The structure of B₁₂ is characterized by a tetrapyrrolic corrin ring with a central cobalt ion coordinated with an upper ligand, and a lower ligand anchored via a nucleotide loop. Multiple methyl groups decorate B₁₂, and their presence (or absence) have structural and functional consequences. In this minireview, we focus on the methyl groups that distinguish vitamin B₁₂ from other tetrapyrrolic biomolecules and from its own naturally occurring analogues called cobamides. We draw information from recent advances in the field to understand the origins of these methyl groups and the enzymes that incorporate them, and discuss their biological significance.     

Vitamin B12-dependent methionine synthesis in Rhizobium meliloti

Methionine, among the various additions to the medium, could only replace cobalt ion or vitamin B₁₂ required for the growth of $\text{Rhizobium meliloti}$. It was demonstrated that there exists a vitamin B₁₂-dependent terminal step in the methionine synthesis, that is, N⁵CH₃-tetrahydrofolate-homocysteine transmethylase, which can also catalyze the methyl transfer from CH₃B₁₂ to homocysteine, in the cell-free extracts of Rhizobium meliloti. These facts seem to indicate that the vitamin B₁₂-dependent pathway to methionine functions mainly among the B₁₂-dependent enzymatic systems in the wild-type symbionts and this is the chief nutritional significance of cobalt.     

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.     

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.     

Decreased Brain Levels of Vitamin B12 in Aging,Autism and Schizophrenia

Many studies indicate a crucial role for the vitamin B₁₂ and folate-dependent enzyme methionine synthase (MS) in brain development and function, but vitamin B₁₂ status in the brain across the lifespan has not been previously investigated. Vitamin B₁₂ (cobalamin, Cbl) exists in multiple forms, including methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), serving as cofactors for MS and methylmalonylCoA mutase, respectively. We measured levels of five Cbl species in postmortem human frontal cortex of 43 control subjects, from 19 weeks of fetal development through 80 years of age, and 12 autistic and 9 schizophrenic subjects. Total Cbl was significantly lower in older control subjects (> 60 yrs of age), primarily reflecting a >10-fold age-dependent decline in the level of MeCbl. Levels of inactive cyanocobalamin (CNCbl) were remarkably higher in fetal brain samples. In both autistic and schizophrenic subjects MeCbl and AdoCbl levels were more than 3-fold lower than age-matched controls. In autistic subjects lower MeCbl was associated with decreased MS activity and elevated levels of its substrate homocysteine (HCY). Low levels of the antioxidant glutathione (GSH) have been linked to both autism and schizophrenia, and both total Cbl and MeCbl levels were decreased in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice, which exhibit low GSH levels. Thus our findings reveal a previously unrecognized decrease in brain vitamin B₁₂ status across the lifespan that may reflect an adaptation to increasing antioxidant demand, while accelerated deficits due to GSH deficiency may contribute to neurodevelopmental and neuropsychiatric disorders.     

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.     

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.     

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.