Vitamin B12 Analogues Research Articles

A study of the acid-base transformations of oxodihydrothiochrome

The acid-base properties of oxodihydrothiochrome — the product of the redox disproportionation of the vitamin B1 catabolite thiochrome (pH=10.7, 4.5, and 0.5) — have been studied in the pH interval of 0–12 by PMR spectroscopy. It has been established that in acid medium (pH 0–1) the formation of a doubly charged ion is accompanied by a further structural transformation of the molecule of (I) with the formation of a vitamin B1 analogue — oxothiamine (II). A possible mechanism of the action of (I) as an inhibitor of thiamine-dependent enzymes is discussed in the light of the results obtained. Details of the PMR spectra of (I) and (II) are given.

Substances with biological activity of vitamin B12 formed during cultivation of Propionibacterium freudenreichii in the presence of precursors (short communications).

The papers of Kolhouse et al. and Cooper et al. described the occurrence of vitamin B12 analogues of unknown origin in blood serum. Some of these analogues may be derived from slaughter cattle raised on feed supplemented with vitamins and minerals, as was observed by Allen. Herbert et al. found vitamin B12 analogues in multivitamin preparations produced in U.S.A., and Kanazawa et al. in human liver, red cells and brain. It is not clear so far, if and how do vitamin B12 analogues interfere with vitamin B12 metabolism. When P. freudenreichii was cultivated in the presence of o-phenylenediamine and 5,6-dimethylbenzimidazole, which may be considered precursors as well as antimetabolites of vitamin B12, the stimulation of biosynthesis of substances with biological activity of vitamin B12 took place. Various signs show that these substances are probably vitamin B12 analogues. During stimulated and nonstimulated production of vitamin B12 by P. freudenreichii, two substances with vitamin B12 biological activity have always been obtained. Their relation was not stable and differed according to the conditions of cultivation. Every attempt to stimulate the biosynthesis of vitamin B12 resulted in the suppression of production of the substance with higher molecular weight, even if the biosynthesis of cobalamin (lower molecular weight) was increased. In our note we want to pay attention to the character of substances arising in the stimulated biosynthesis.

Functionalized Dithia(2,5)pyridinophanes as Vitamin B6 Analogues. Synthesis, Properties, and Catalytic Activity for Racemization Reaction

Abstract Exploration of synthetic routes to achieve designed vitamin B6 models on the structural basis of functionalized dithia(2,5)pyridinophanes from pyridoxine hydrochloride was undertaken and the catalyst potency for a racemization reaction was examined in comparison with that of pyridoxal. By the reaction of 3,4′-O-isopropylidenepyridoxine 2′,5′-dichloride with α,ω-dithiols, dithia(2,5)pyridinophanes were synthesized in good yields. For dithia[3]paracyclo[3](2,5)pyridinophanes, juncture sulfur atoms were extruded photochemically, whereas 2,(n+3)-dithia[m](2,5)pyridinophanes incompletely extruded sulfur atoms under various conditions. Because of instability and low preparative yield of 15-hydroxy-16-formyl[2]paracyclo[2](2,5)pyridinophane, dithia-containing phanes, (n+9)-hydroxy-(n+10)-formyl-2,(n+3)-dithia[m](2,5)pyridinophanes [26a (n=4), 26b (n=6), 26c (n=8); m=n+4], 17-hydroxy-18-formyl-2,11-dithia[3]paracyclo[3](2,5)pyridinophane (27), and an acyclic congener for 26a, 5′-deoxy-2′,5′-bis(ethylthio)pyridoxal (28), were eventually prepared in high yields by use of manganese dioxide in the presence of a primary amine. It was found on the basis of 1H-NMR spectral analyses that the rotations of pyridine rings in some molecules, 26a, 26b, and 27 with ring sizes equal to or less than fourteen were restricted. The racemization potency of 26a–c, 27, and 28 for sodium hydrogen l-glutamate demonstrated that these are 1.4- to 1.7-fold more potent than pyridoxal itself under certain conditions (pH 10.0, 25±0.5 °C) until ca. 80% completion of the racemization reaction. 26a was found to be quite stable (no decomposition), but the others, 26b, 26c, and 28, indicated 14, 8, and 12% decomposition, respectively. 27 was so unstable that a 63% decomposition was marked at the time of 56% completion of the racemization reacion.

Vitamin B12 absorption in cystic fibrosis.

Vitamin B12 absorption was measured in 30 patients with cystic fibrosis by means of the urinary excretion method and found to be impaired, i.e. less than 10%, in 25. The mean urinary excretion amounted to 4.7 +/- 0.8%. In all patients vitamin B12 absorption improved by the addition of trypsin (18.9 +/- 2.1%). Addition of the vitamin B12 analogue cobinamide, which prevents vitamin B12-binding by R-binders, raised the vitamin B12 absorption to 15.0 +/- 2.2%. A further improvement was obtained by the simultaneous addition of cobinamide and trypsin, 18.2 +/- 2.6%, the same value as with trypsin alone. Assuming that cobinamide addition was effective in suppressing all R-binder activity, the additional effect of trypsin suggests a second, stimulatory function of trypsin on vitamin B12 absorption, separate from R-binder-inactivation. In 5 patients only marginal improvement of vitamin B12 absorption was gained by the addition of either trypsin or cobinamide. The deficient serum vitamin B12 (110 pmol/l) in one of them indicates that the normal pancreas-substitution therapy not always implies sufficient restoration of vitamin B12 absorption.

Noncobalamin vitamin B12 analogues in human red cells, liver, and brain

Analogues of vitamin BJ2 which appear to be noncobalamin corrinoids appear to be present in human red cells, liver, and brain. Their sources, nature, and effects require study, particularly with reference to their positive and/or negative effects on vitamin B12 metabolism. In normal persons, they are concentrated in liver, with only small quantities in red cells and still smaller quantities in brain. Their concentrations in disease states will be of interest, particularly in persons with varying degrees of neurological damage associated with deficiencies in vitamin B12 or analogue metabolism.

Synthesis and characterization of substituted diaza(2,5)pyridinophanes as precursors for vitamin B6 analogs

Synthesis and characterization of substituted diaza(2,5)pyridinophanes as precursors for vitamin B6 analogs

6.15 The Effects of Soil Cobalt on Rumen Vitamin B12 Synthesis

The high level of cobalt (Co) in soil relative to herbage means that soil accidentally ingested with herbage may constitute the larger source of the element for vitamin B12 synthesis by rumen microbes.A continuous culture simulation of rumen fermentation (Rusitec: Czerkawski and Breckenridge. 1977) was used to determine the extent to which soil Co was used for microbial vitamin B12 synthesis. A low Co hay (0.044 mg/kg DM), 7.25 g DM/day, was “fed” to each of four vessels for a period of 8 days to provide resting values for vitamin production. A chalk, a sand, a clay or a loam, with Co concentrations ranging from 0.70 to 64.5 mg/kg DM (see Table 6.15.1), were then added at 0.70 g/day, to the hay in separate vessels each morning for 14 days. For a further period of 10 days, the hay in two vessels was supplemented with either chalk or loam at 1.4 g/day. Daily vitamin B12 production was measured by analysis of each vessel's effluent for cobalamin (cbl.), using a modification of the method of Green (1980). Vitamin B12 analogue production was assessed from the Lactobacillus leichmannii microbial assay of the effluent. All vessels produced a “roughage” fermentation (acetate c. 50% and propionate c. 20% of the total volatile fatty acids) with the soil supplements.

Biosynthesis of vitamin B12

Radioactivity from [1'-14C]riboflavin was incorporated into the 5,6-dimethylbenzimidazole moiety of Vitamin B12 in the aerobes Bacillus megaterium, Nocardia rugosa and Streptomyces sp. as well as in the aerotolerant anaerobe Propionibacterium freudenreichii, but not in the anaerobe Eubacterium limosum. As recently published for E. limosum, also in the anaerobe Clostridium barkeri radioactivity from [1-14C]glycine and [2-14C]glycine was found in the 5,6-dimethylbenzimidazole moiety, but not in the corrin moiety. The addition of L-[methyl-14C]methionine to C. barkeri led to the labeling of the corrin moiety and the 5,6-dimethylbenzimidazole moiety, showing that the seven "extra" methyl groups in the corrin ring as well as the two methyl groups of the base part originate from this precursor. In Clostridium thermoaceticum, forming the vitamin B12 analog 5-methoxybenzimidazolylcobamide, [1-14C]glycine and [2-14C]glycine were also incorporated into the 5-methoxybenzimidazole moiety, but not into the corrin ring. In E. limosum L-[U-14C]glutamate led to the labeling of the corrin ring of vitamin B12, but not of its base moiety. These results together with data from the literature indicate that a common biosynthetic pathway might exist for the corrinoid biosynthesis in aerobic microorganisms, and in those aerotolerant anaerobes like the Propionibacteria, which form the 5,6-dimethylbenzimidazole moiety of vitamin B12 only under aerobic conditions. They also show that this pathway differs from the pathway found in anaerobic bacteria.

Demonstration of vitamin B12 analogues in human sera not detected by microbiological assay.

Sera were absorbed with polyacrylamide beads to which purified human intrinsic factor was attached. This procedure removed the vitamin B12 analogues which are measured by microbiological assay with Lactobacillus leichmannii and Euglena gracilis and which are measured in an isotope dilution method using intrinsic factor. Such sera still contained B12 analogues that were assayed in an isotope dilution method using a non-intrinsic factor vitamin B12 binder. Such vitamin B12 analogues make up approximately half of the total vitamin B12 analogues in human serum.

Vitamin B6 requirements of nutritionally variant Streptococcus mitior.

The growth rate of three vitamin B6-dependent Streptococcus mitior (B6DS) and two non-B6DS strains in Todd-Hewitt broth, with and without vitamin B6 supplementation, was examined. Even in optimally supplemented culture media, the growth rate of the three B6DS strains was much slower than that of comparable non-B6DS strains. Uptake studies with [3H] pyridoxine suggest that these B6DS strains cannot assimilate pyridoxine. Although not transported intracellularly, pyridoxine inhibited the growth of B6DS strains in media supplemented with other vitamin B6 analogs, probably by binding to the vitamin B6 transport system and inhibiting the uptake of the other vitamin B6 analogs.

The Effect of Folate Analogues and Vitamin B12 on Provision of Thymine Nucleotides for DNA Synthesis in Megaloblastic Anemia

AbstractThe role of vitamin B12 in the folate dependent biosynthesis of thymine nucleotides is controversial. In an attempt to clarify this, three methods have been used to assess the relative efficacy of vitamin B12 (hydroxocobalamin) and various folate analogues in titrated concentrations at correcting ‘de novo’ thymidylate synthesis by megaloblastic human marrow cells: (1) The deoxyuridine (dU) suppression test which analyses the reduction in (3H)–thymidine labeling of DNA by unlabeled dU. Marrow cells were also labeled with (6–3H)-dU with assessment of (2) its incorporation into DNA and (3) the accumulation of (6–3H)-deoxyuridine monophosphate (3H-dUMP). The three methods gave similar results. In both, NB-formyl tetrahydrofolate (formyl-FH4) was the most effective agent at correcting thymidylate synthesis in megaloblastic anemia due to vitamin B12 or folate deficiency. Vitamin B12 corrected the lesion in vitamin B12 deficiency but not in folate deficiency. Tetrahydrofolate (FH4) and folic acid were effective in deficiency of vitamin B12 or folate, although in both deficiencies they were less effective than formyl-FH4. Methyl–FH4 was effective in folate deficiency but not in vitamin B12 deficiency. These results confirm the failure of methyl-FH4 utilisation in vitamin B12 deficiency. They suggest that if vitamin B12 is needed in the formylation of FH4, this is a minor role in provision of the correct coenzyme for thymidylate synthesis compared with its major role of provision of FH4 from methyl–FH4.

The effect of folate analogues and vitamin B12 on provision of thymine nucleotides for DNA synthesis in megaloblastic anemia

The role of vitamin B12 in the folate dependent biosynthesis of thymidine nucleotides is controversial. In an attempt to clarify this, three methods have been used to assess the relative efficacy of vitamin B12 (hydroxocobalamin) and various folate analogues in titrated concentrations at correcting ‘de novo’ thymidylate synthesis by megaloblastic human marrow cells: (1) The deoxyuridine (dU) suppression test which analyses the reduction in (3H)-thymidine labeling of DNA by unlabeled dU. Marrow cells were also labeled with (6–3H)-dU with assessment of (2) its incorporation into DNA and (3) the accumulation of (6–3H)-deoxyuridine monophosphate (3H-dUMP). The three methods gave similar results. In both, N6-formyl tetrahydrofolate (formyl-FH4) was the most effective agent at correcting thymidylate synthesis in megaloblastic anemia due to vitamin B12 or folate deficiency. Vitamin B12 corrected the lesion in vitamin B12 deficiency but not in folate deficiency. Tetrahydrofolate (FH4) and folic acid were effective in deficiency of vitamin B12 or folate, although in both deficiencies they were less effective than formyl-FH4. Methyl-FH4 was effective in folate deficiency but not in vitamin B12 deficiency. These results confirm the failure of methyl-FH4 utilisation in vitamin B12 deficiency. They suggest that if vitamin B12 is needed in the formylation of FH4, this is a minor role in provision of the correct coenzyme for thymidylate synthesis compared with its major role of provision of FH4 from methyl- FH4.

Chemistry of Chiral Vitamin B6 Analogs. IV. Syntheses of Chiral Pyridoxal and Pyridoxamine Analogs Having a Branched “Ansa Chain” between 2′- and 5′-Positions

Abstract Racemic 15-formyl-14-hydroxy-5,5-dimethyl-2,8-dithia[9](2,5)pyridinophane (4), an analog of pyridoxal having a branched “ansa chain” between 2′- and 5′-positions, was synthesized from 2,5-bis(chloromethyl)-3-hydroxy-O,O′-isopropylidene-4-pyridinemethanol. Optical resolution of 4 was achieved via the formation of Schiff base with 3-amino-3-deoxy-1,2:5,6-di-O-isopropylidene-d-glucofranose, giving the levorotary enantiomer of 4 (4a). Racemic 15-aminomethyl-14-hydroxy-5,5-dimethyl-2,8-dithia[9](2,5)pyridinophane (5), the same type of pyridoxamine analog as 4, and its levorotary enantiomer were synthesized from the corresponding 4 and 4a. Both levorotary and dextrorotary enantiomers of 5 were also obtained by the optical resolution of the racemate via the formation of chiral dibenzoyltartaric acid salts of 5.

Forms of vitamin B12 in radioisotope dilution assays.

Since the presence of analogues of vitamin B12 (B12, cobalamin, Cbl) has been postulated as the basis for the high values obtained by some radioisotope dilution assays (RIDA) of serum Cbl we examined serum for analogues. None could be demonstrated in the extracts of serum prepared for RIDA as sought by both direct and indirect techniques. The natural forms of serum Cbl were converted to cyanocobalamin (CN Cbl) by this process of extraction which included cyanide (CN). The correctly performed RIDA for Cbl based on R binder gave higher values than a RIDA based on intrinsic factor or than by bioassay. By exclusion, the difference appeared to be due to unidentified factors rather than the presence of analogues.

The biological activity of vitamin B6 analogs in the rat.

The effects of three vitamin B6 analogs were studied in young adult and weanling rats. The 2-ethyl analog was the most active. It produced higher growth rates, an average feed efficiency equal to the control and elevated liver glycogen levels. Xanthurenic acid excretion remained low with the 2-ethyl analog and pyridoxine in the diet and plasma amino acid concentration was low. Response to the 2-n-propyl analog was similar to that of a B6-deficient diet, with a large increase in xanthurenic acid excretion. Rats receiving the 2-isopropyl analog were intermediate between the ethyl group and B6-deficient animals in all parameters measured. These in vivo results parallel those reported in the in vitro yeast pyridoxine dehydrogenase enzyme system. The ethyl pyridoxine analog was the most active of all the analogs in the yeast dehydrogenase system, in supporting growth and in maintaining normal xanthurenic acid excretion in the rat.

Use of normal serum as a satisfactory binding protein in radioassay of Vitamin B12

Recent investigations of radioassay procedures for serum Vitamin B12 have shown a disturbing discrepancy between the clinical state of Vitamin B12 deficiency and the normal result produced for serum Vitamin B12 in a number of patients. It was estimated that this discrepancy occurred in more than 10% of Vitamin B12 deficient patients, a frequency which is unacceptably high. Investigation of the problem by Kolhouse <i>et al</i>. revealed that serum contained Vitamin B12 analogues which are not biologically active in man but are measured as Vitamin B12 in many commercial assays because of the contamination of the intrinsic factor binder with ‘R' protein. The ‘R' protein can bind the analogues whereas the intrinsic factor is specific for Vitamin B12. Kolhouse recommended that clinical laboratories and commercial manufacturers change their assays and use only purified intrinsic factor as binder, and this applied to laboratories which used human serum as the source of Vitamin B12 binding protein. The purpose of this report is to present evidence that normal human serum is a satisfactory source of Vitamin B12 binding protein and that assays using serum did not suffer the same problems that occur with commercial kits. As in other laboratories, the awareness of the authors of the problem started with a clear discrepancy between serum Vitamin B12 results using a commercial kit (Quanta-Count B12 Radioassay, Bio Rad Laboratories) and the clinical diagnosis of 5 patients. However, instead of repeating the Vitamin B12 analyses by a microbiological method, the analyses were repeated by a radioassay using normal human serum as binder. Results consistent with the patient's diagnoses were obtained in all cases. Subsequently 49 specimens with mainly low Vitamin B12 levels were assayed by the commercial radioassay kit, radioassay using serum binder and microbiological assay. Six samples gave normal levels by commercial kit but low levels by the serum binder radioassay and microbiological assay. The agreement between the serum binder radioassay and microbiological assay was good overall. Over a 12 mth period all serum radioassay results for Vitamin B12 below 350 pg/ml were checked by microbiological assay. A total of 264 samples were assayed in this way. The results showed good agreement between the 2 methods, with no major difference in classification evident. Thus it is concluded that normal human serum is a satisfactory binder for radioassay of Vitamin B12.

The effect of bacterially produced vitamin B12 analogues (cobamides) on the in vitro absorption of cyanocobalamin

An incubation system containing rat jejunoileum was used to determine whether analogues of cyanocobalamin (CNCbl) inhibited the in vitro absorption of CNCbl. The naturally occurring analogues “pseudo B12,” “factor A,” and the nonnaturally occurring analogue desde-methyl B12, were produced by guided biosynthesis with Propionibacterium arabinosum. Factor B was produced by acid cleavage of cyanocobalamin. Inhibition of Co57-CNCbl uptake was demonstrated for cold CNCbl and for desdemethyl B12, although statistical significance was achieved only for cold CNCbl at 40× and 100× the molar quantity of the radiolabeled CNCbl. However, a trend towards progressive inhibition of specific CNCbl binding was shown as increasing concentrations of CNCbl or desdemethyl B12 were used. No inhibition of specific CNCbl binding could be demonstrated for any of the naturally occurring cobamides tested.

Production of vitamin B 12 analogues in patients with small-bowel bacterial overgrowth.

We investigated the presence of vitamin B 12 analogues (cobamides) and the bacterial conversion of 57Co-B12 (vitamin B12 cyanocobalamin, [57Co]-CN-Cbl) into cobamides in the intestinal contents of four patients with bacterial overgrowth. The (57Co)-CN-Cbl bound to intrinsic factor was given orally. Jejunal contents were aspirated for 24 h and cultured aerobically and anaerobically. The CN-Cbl and cobamides were separated by electrophoresis and chromatography and identified by bioautography. Radioactivity of cobamide zones from duplicate chromatograms showed bacterial conversion of (57Co)-cn-cbl into cobamides. Cobamides ([Ade]CNCBA, [2-Me Ade] CNCba, [CN]2Cbi and factor E) were found in the intestinal contents in three of the four patients, and in two of three patients cobamides represented more than 25% of the administered CN-Cbl. Thus bacterial production of cobamides, both de novo and from ingested CN-Cbl bound to intrinsic factor, occurs in humans with bacterial overgrowth states and results in a significant loss of vitamin B12 to the host.

Reaction of Dicyanocobyrinic Heptamethyl Ester with Ascorbic Acid

Stable yellow corrinoids can arise as by‐products in reactions of vitamin B12 involving a change in the oxidation state of the cobalt. The structure of the pigment from a vitamin B12 analog and ascorbic acid has now been elucidated by X‐ray structure analysis.

Contributions to analytical chemistry of vitamin B12.

The possibility of determination of small amounts of cyanide (0.02–1.0μg) in Vitamin B12, without distillation of hydrogen cyanide, is demonstrated. Vitamin B12 samples (10−6−4×10−5M), acidified to pH 3–4 with sulphuric or nitric acid, are irradiated in 25-ml volumetric flasks with an ultraviolet lamp (without filter) at 35–40° for 30 min. After cooling, the samples are adjusted to pH 12–13 with sodium hydroxide, the ionic strength is adjusted to 0.1 with potassium nitrate, and cyanide is determined with an ionselective membrane electrode. The calibration curve is prepared with KCN or Vitamin B12. The total error is ±2 %. The practical advantage of the method is that 5–10 samples can be analysed simultaneously, and the equilibria between cyanocobalamine, analogues of Vitamin B12 and potassium cyanide can be studied. The thermolysis and photolysis data can serve for evaluation of the strength of the Co—CN bond in Vitamin B12 and analogous molecules, by comparison with other cyanide complexes which have accurately known stability constants and structures.