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 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.     

Comparative studies on intrinsic factor and cobalophilin in different parts of the gastrointestinal tract of the pig

The vitamin B₁₂ binders in the pig pyloric mucosa gastric and intestinal juice from the upper gastrointestinal tract were fractionated into only two molecular forms, classified as intrinsic factor and cobalophilin. The unsaturated vitamin B₁₂-binding power due to cobalophilin was lower in the intestinal than in the gastric juice. Electrofocusing revealed that intrinsic factor and cobalophilin in the intestinal juice contained more of the `neutral'-type isoproteins, and the suggestion is made that this is due to enzyme activity. The gastric-juice intrinsic factor contained more acidic isoproteins, which supports the hypothesis that carbohydrate is added on to the polypeptide chain of this protein before it is secreted into gastric juice. The gastric- and intestinal-juice cobalophilins, studied also by electrofocusing, differed from that of pyloric mucosa and they appeared to be of salivary origin. With regard to molecular dimensions there was no significant difference between the intrinsic factors and cobalophilins from all sources studied. All cobalophilins had molecular weights by the formula of Svedberg of approx. 92500, Stokes radii of 4.62nm and sedimentation coefficients of 5.15S. The corresponding values for the intrinsic factors were 63600, 3.57nm and 4.38S. In addition, the intrinsic factors exhibited similar avidities for binding to the solubilized ileal intrinsic-factor receptor. Also the intrinsic factors and cobalophilins, irrespective of their source, bound to the analogous specific xenoantibodies with the same avidity. The present results demonstrate that intrinsic factor remains practically unaltered during its passage through the proximal intestine and render unlikely the speculations made about the presence of an endogenous binder for intrinsic factor as well as the existence of a `pancreatic intrinsic factor'. In addition, they are compatible with the theory that the interference by undegraded cobalophilin may be the reason for the abnormal vitamin B₁₂ absorption observed in patients with pancreatic insufficiency.     

Identification and characterization of intrinsic factor and cobalophilin from pig ileal and pyloric mucosa

Pig ileal mucosa was found to bind about 240 ng vitamin B₁₂/g and to contain two vitamin B₁₂-binding proteins. One was highly active in the Schilling test, behaved immunologically as intrinsic factor and was responsible for about half of the total vitamin B₁₂-binding capacity. The other binder was identified as cobalophilin (R-protein). Immunochemical purification of these proteins from pig ileum and pylorus was performed and the molecular characteristics (sedimentation and diffusion coefficients, Strokes radii, frictional ratios and molecular weights) of their vitamin B₁₂ complexes were estimated. Isoelectric focusing revealed differences between the ileal and pyloric intrinsic factors but not between the cobalophilins. The mean isoelectric points of the pyloric and ileal intrinsic factors were pH 5.79 and 5.30, respectively, whereas the corresponding figures for the cobalophilins were 4.13 and 4.10.     

One pathway can incorporate either adenine or dimethylbenzimidazole as an alpha-axial ligand of B12 cofactors in Salmonella enterica

Corrinoid (vitamin B₁₂-like) cofactors contain various α-axial ligands, including 5,6-dimethylbenzimidazole (DMB) or adenine. The bacterium Salmonella enterica produces the corrin ring only under anaerobic conditions, but it can form “complete” corrinoids aerobically by importing an “incomplete” corrinoid, such as cobinamide (Cbi), and adding appropriate α- and β-axial ligands. Under aerobic conditions, S. enterica performs the corrinoid-dependent degradation of ethanolamine if given vitamin B₁₂, but it can make B₁₂ from exogenous Cbi only if DMB is also provided. Mutants isolated for their ability to degrade ethanolamine without added DMB converted Cbi to pseudo-B₁₂ cofactors (having adenine as an α-axial ligand). The mutations cause an increase in the level of free adenine and install adenine (instead of DMB) as an α-ligand. When DMB is provided to these mutants, synthesis of pseudo-B₁₂ cofactors ceases and B₁₂ cofactors are produced, suggesting that DMB regulates production or incorporation of free adenine as an α-ligand. Wild-type cells make pseudo-B₁₂ cofactors during aerobic growth on propanediol plus Cbi and can use pseudo-vitamin B₁₂ for all of their corrinoid-dependent enzymes. Synthesis of coenzyme pseudo-B₁₂ cofactors requires the same enzymes (CobT, CobU, CobS, and CobC) that install DMB in the formation of coenzyme B₁₂. Models are described for the mechanism and control of α-axial ligand installation.     

Megaloblastic Anemia Associated with Surgically Produced Gastrointestinal Abnormalities

Two of the mechanisms for vitamin B₁₂ 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 B₁₂ absorption. It is believed that if patients survive long enough and are not given prophylactic vitamin B₁₂ therapy, all would develop megaloblastic anemia.Intestinal anastomosis leading to stasis of intestinal contents, with overgrowth of bacteria may cause vitamin B₁₂ deficiency through bacterial interference with the utilization of vitamin B₁₂.Use of radioactive vitamin B₁₂ (cobalt⁶⁰-labeled B₁₂) 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.     

Systemic lupus erythematosus

Imerslund-Gräsbeck syndrome (IGS) or selective vitamin B₁₂ (cobalamin) malabsorption with proteinuria is a rare autosomal recessive disorder characterized by vitamin B₁₂ deficiency commonly resulting in megaloblastic anemia, which is responsive to parenteral vitamin B₁₂ therapy and appears in childhood. Other manifestations include failure to thrive and grow, infections and neurological damage. Mild proteinuria (with no signs of kidney disease) is present in about half of the patients. Anatomical anomalies in the urinary tract were observed in some Norwegian patients. Vitamin B₁₂ absorption tests show low absorption, not corrected by administration of intrinsic factor. The symptoms appear from 4 months (not immediately after birth as in transcobalamin deficiency) up to several years after birth. The syndrome was first described in Finland and Norway where the prevalence is about 1:200,000. The cause is a defect in the receptor of the vitamin B₁₂-intrinsic factor complex of the ileal enterocyte. In most cases, the molecular basis of the selective malabsorption and proteinuria involves a mutation in one of two genes, cubilin (CUBN) on chromosome 10 or amnionless (AMN) on chromosome 14. Both proteins are components of the intestinal receptor for the vitamin B₁₂-intrinsic factor complex and the receptor mediating the tubular reabsorption of protein from the primary urine. Management includes life-long vitamin B₁₂ injections, and with this regimen, the patients stay healthy for decades. However, the proteinuria persists. In diagnosing this disease, it is important to be aware that cobalamin deficiency affects enterocyte function; therefore, all tests suggesting general and cobalamin malabsorption should be repeated after abolishment of the 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.     

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.     

Higher Prevalence of Metformin-Induced Vitamin B12 Deficiency in Sulfonylurea Combination Compared with Insulin Combination in Patients with Type 2 Diabetes: A Cross-Sectional Study

Long-term and high-dose treatment with metformin is known to be associated with vitamin B₁₂ deficiency in patients with type 2 diabetes. We investigated whether the prevalence of B₁₂ deficiency was different in patients treated with different combination of hypoglycemic agents with metformin during the same time period. A total of 394 patients with type 2 diabetes treated with metformin and sulfonylurea (S+M group, n = 299) or metformin and insulin (I+M group, n = 95) were consecutively recruited. The vitamin B₁₂ and folate levels were quantified using the chemiluminescent enzyme immunoassay. Vitamin B₁₂ deficiency was defined as vitamin B₁₂≤300 pg/mL without folate deficiency (folate>4 ng/mL). The mean age of and duration of diabetes in the subjects were 59.4±10.5 years and 12.2±6.7 years, respectively. The mean vitamin B₁₂ level of the total population was 638.0±279.6 pg/mL. The mean serum B₁₂ levels were significantly lower in the S+M group compared with the I+M group (600.0±266.5 vs. 757.7±287.6 pg/mL, P<0.001). The prevalence of vitamin B₁₂ deficiency in the metformin-treated patients was significantly higher in the S+M group compared with the I+M group (17.4% vs. 4.2%, P = 0.001). After adjustment for various factors, such as age, sex, diabetic duration, duration or daily dose of metformin, diabetic complications, and presence of anemia, sulfonylurea use was a significant independent risk factor for B₁₂ deficiency (OR = 4.74, 95% CI 1.41–15.99, P = 0.012). In conclusion, our study demonstrated that patients with type 2 diabetes who were treated with metformin combined with sulfonylurea require clinical attention for vitamin B₁₂ deficiency and regular monitoring of their vitamin B₁₂ levels.     

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.     

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.     

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.     

Characterization of a Vitamin B12 Compound in the Edible Purple Laver,Porphyra yezoensis

The edible purple laver, Porphyra yezoensis, contained 51.49±1.51 μg of vitamin B₁₂ compounds per 100 g dry weight of the laver (mean±SEM, n=4). A vitamin B₁₂ compound was purified from the lyophilized purple laver and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified pink-colored compound were identical to those of authentic vitamin B₁₂, but not to those of vitamin B₁₂ analogues inactive for humans.     

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.     

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.     

Biochemical Studies During the Development of the Chick Embryo

The days when the concentration of conjugated- and free- vitamin B₁₂ per wet weight g increased or decreased agreeded with that of nucleic acids in the chick embryo from 3rd through 18th day of incubation. It is intereresting that the day of increasing or decreasing of those compounds concentration corresponds with the important stages through the growth of embryo. The changes of these compounds in the egg contents free of embryo were estimated. Although a considerable large amount of free vitamin B₁₂ was contained in the embryo, the most vitamin B₁₂ in the egg content except the embryo existed in the conjugated state. It seems that the amount of total vitamin B₁₂ in the egg during the incubation is relatively constant.     

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.     

Occurrence of Coenzyme Forms of Vitamin B12 in a Cultured Purple Laver (Porphyla yezoensis)

Porphyra yezoensis (Susabinori, an edible purple laver), which was cultured aseptically for 12 weeks and then lyophilized, contained 50±2 μg/g of vitamin B₁₂ per 100 g dry weight. Coenzyme forms of vitamin B₁₂ (about 60% of the total vitamin B₁₂) were found in the cultured purple laver aseptically, which may have the ability to biosynthesize the coenzymes.     

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.