Evaluation of oxidized buckypaper as material for the solid phase extraction of cobalamins from milk: Its efficacy as individual and support sorbent of a hydrophilic-lipophilic balance copolymer.

This work describes a new analytical method for the determination of four cobalamins (adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), hydroxocobalamin (OHCbl) and cyanocobalamin (CNCbl)) in cow's milk. The extraction procedure is fast and based on dilution/protein precipitation of a milk sample with 50mM sodium acetate buffer (pH 4.6), followed by solid phase extraction (SPE) of the filtered supernatant. Relative recoveries higher than 60% have been obtained for all the cobalamins by combining two different types of sorbents in the same SPE cartridge: two disks of buckypaper (BP), a nanoporous felt composed of oxidized multiwalled carbon nanotubes (MWCNTs), separated by a Teflon frit from OASIS HLB (500mg), a hydrophilic-lipophilic balance copolymer. Before its use as sorbent, BP was characterized in terms of porosity, permeability, surface area, specific adsorption capacity and tested for a potential reuse after adequate chemical regeneration. The analysis of the extracts was performed by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) on an analytical C18 column in less than 10min. After validation, the method was applied to the determination of the natural content of the four B12 homologues in cow's milk samples, providing data lacking in the literature.

Thin-layer chromatography combined with diode laser thermal vaporization inductively coupled plasma mass spectrometry.

Here we present a novel coupling of thin-layer chromatography (TLC) to diode laser thermal vaporization inductively coupled plasma mass spectrometry (DLTV ICP MS). DLTV is a new technique of aerosol generation which uses a diode laser to induce pyrolysis of a substrate. In this case the cellulose stationary phase on aluminum-backed TLC sheets overprinted with black ink to absorb laser light. The experimental arrangement relies on economic instrumentation: an 808-nm 1.2-W continuous-wave infrared diode laser attached to a syringe pump serving as the movable stage. Using a glass tubular cell, the entire length of a TLC separation channel is scanned. The 8-cm long lanes were scanned in ∼35 s. The TLC - DLTV ICP MS coupling is demonstrated on the separation of four cobalamins (hydroxo-; adenosyl-; cyano-; and methylcobalamin) with limits of detection ∼2 pg and repeatability ∼15% for each individual species.

The discovery of vitamin B(12).

The discovery of vitamin B(12), the elucidation of its role in metabolism, and the effects and treatment of its deficiency occurred in distinct phases over more than 100 years, and it was the subject of two separate Nobel Prizes. The valuable contribution of clinical reports and studies of patients with pernicious anemia throughout the 19th century resulted in enough clinical definition to allow Minot and Murphy to put together the first hallmark study on treatment of the condition, leading them to a Nobel Prize. These researchers were not the first to suggest that an inadequacy of nutrients was the cause of pernicious anemia, but their particular input was a carefully designed intervention in well-characterized pernicious anemia patients, of a special diet containing large amounts of liver. They found consistent improvement in the clinical and blood status of all subjects, most of whom remained on remission indefinitely. After the successful intervention studies, the next advance was made by Castle who discovered that a gastric component, which he called intrinsic factor, was missing in pernicious anemia. Many years later, intrinsic factor was found to be a glycoprotein that formed a complex with vitamin B(12), promoting its absorption through ileal receptors. The vitamin was isolated by two groups simultaneously and was crystallized and characterized in the laboratory of Dorothy Hodgkin, contributing to her Nobel Prize in 1964. Subsequently, the various biochemical roles of vitamin B(12) were elucidated, including its important interaction with folate and their common link with megaloblastic anemia. Many of the early clinical studies recognized that vitamin B(12) deficiency also caused a severe neuropathy leading to paralysis and death, while post mortem analysis demonstrated spinal cord demyelination. Vitamin B(12) is still the subject of intense research and, in particular, its role in preventing these irreversible neurological lesions remains unclear.

The reaction of HOCl and cyanocobalamin: corrin destruction and the liberation of cyanogen chloride.

Overproduction of hypochlorous acid (HOCl) has been associated with the development of a variety of disorders such as inflammation, heart disease, pulmonary fibrosis, and cancer through its ability to modify various biomolecules. HOCl is a potent oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system. Recently, we have provided evidence to support the important link between higher levels of HOCl and heme destruction and free iron release from hemoglobin and RBCs. Our current findings extend this work and show the ability of HOCl to mediate the destruction of metal-ion derivatives of tetrapyrrole macrocyclic rings, such as cyanocobalamin (Cobl), a common pharmacological form of vitamin B12. Cyanocobalamin is a water-soluble vitamin that plays an essential role as an enzyme cofactor and antioxidant, modulating nucleic acid metabolism and gene regulation. It is widely used as a therapeutic agent and supplement, because of its efficacy and stability. In this report, we demonstrate that although Cobl can be an excellent antioxidant, exposure to high levels of HOCl can overcome the beneficial effects of Cobl and generate proinflammatory reaction products. Our rapid kinetic, HPLC, and mass spectrometric analyses showed that HOCl can mediate corrin ring destruction and liberate cyanogen chloride (CNCl) through a mechanism that initially involves α-axial ligand replacement in Cobl to form a chlorinated derivative, hydrolysis, and cleavage of the phosphonucleotide moiety. Additionally, it can liberate free Co, which can perpetuate metal-ion-induced oxidant stress. Taken together, these results are the first report of the generation of toxic molecular products through the interaction of Cobl with HOCl.

Determination of cobalamins (hydroxo-, cyano-, adenosyl- and methyl-cobalamins) in seawater using reversed-phase liquid chromatography with diode-array detection.

A high-performance liquid chromatography method was developed for the separation and determination of four cobalamins in seawater. Chromatographic separation was performed on a reversed-phase discovery RP-amide C(16) column with buffer potassium dihydrogenphosphate and acetonitrile as the mobile phases in linear gradients elution mode. Cobalamins were previously preconcentrated in C(18) resins cartridges. Detection was performed using UV-diode array detector in a range of λ of 200-400 nm. The method showed to be linear over a range of 1-300 ng mL(-1) with acceptable precision and accuracy. The detection limits ranged between 0.07 pg mL(-1) for 5'-deoxyadenosylcobalamin and 0.5 pg mL(-1) for hydroxocobalamin. The mean cobalamins recoveries for direct determination ranged between 76 and 93% for hydroxo-, cyano- and methylcobalamin, while the recovery for 5'-deoxyadenosylcobalamin was only 31% suggesting that the preconcentration method was not valid for this cobalamin. The method was successfully applied to coastal seawater where the concentrations ranged from 4.2 to 7.3 ng L(-1) for hydroxo-, 1.4-3.9 ng L(-1) for cyano-, 2.1-4.6 ng L(-1) for 5'-deoxyadenosyl- and 33-83.5 ng L(-1) for methylcobalamin.

Lactobacillus reuteri CRL1098 produces cobalamin.

We found that Lactobacillus reuteri CRL1098, a lactic acid bacterium isolated from sourdough, is able to produce cobalamin. The sugar-glycerol cofermentation in vitamin B(12)-free medium showed that this strain was able to reduce glycerol through a well-known cobalamin-dependent reaction with the formation of 1,3-propanediol as a final product. The cell extract of L. reuteri corrected the coenzyme B12 requirement of Lactobacillus delbrueckii subsp. lactis ATCC 7830 and allowed the growth of Salmonella enterica serovar Typhimurium (metE cbiB) and Escherichia coli (metE) in minimal medium. Preliminary genetic studies of cobalamin biosynthesis genes from L. reuteri allowed the identification of cob genes which encode the CobA, CbiJ, and CbiK enzymes involved in the cobalamin pathway. The cobamide produced by L. reuteri, isolated in its cyanide form by using reverse-phase high-pressure liquid chromatography, showed a UV-visible spectrum identical to that of standard cyanocobalamin (vitamin B12).

(Immuno)affinity chromatography: a versatile tool for fast and selective purification, concentration, isolation and analysis.

Today, thanks to the availability of tailor made biomolecules with the desired biological functions, separations based upon (immuno)affinity techniques are more and more common in a large field of applications. By using the high selectivity of biomolecules (antibodies, receptors, specific proteins), this technique offers the possibility of isolating compounds from complex samples with a selectivity which cannot be achieved by other chromatographic methods. In order to succeed, however, the solid phase support for the immobilisation of the ligand of interest plays a prominent role. For this reason, numerous supports have already been introduced while research on new materials with additional advantages is continued. Here, a new solid phase support will be discussed for (immuno)affinity applications. This material demonstrates low non-specific adsorption and high ligand accessibility, which enables an enhanced selectivity and capacity. Because the material is available in large quantities and exhibits superb mechanical and physical stability, selective isolations have been performed on analytical as well as preparative scale. To demonstrate the potential of this new support, several applications will be presented. Based upon immunoaffinity, two applications for the determination of oestradiol in serum respectively vitamin B12 in fermentation broth will be presented. Regarding affinity chromatography, an enzyme reactor in which the enzyme glucose oxidase is immobilised on the new material, is made for the detection of glucose by Flow Injection Analysis and electrochemical detection. Next, to isolate, identify and test components on their xeno-oestrogenic activity, an affinity column is produced in which human oestrogen receptor is covalently coupled. Several components are screened on their biological activity and the results obtained will be presented here.

Substrate and cofactor reactivity of a carbon monoxide dehydrogenase-corrinoid enzyme complex: stepwise reduction of iron-sulfur and corrinoid centers, the corrinoid Co2+/1+ redox midpoint potential, and overall synthesis of acetyl-CoA.

Cleavage of the acetyl carbon-carbon bond of acetyl-CoA in Methanosarcina barkeri is catalyzed by a high molecular mass multienzyme complex. The complex contains a corrinoid protein and carbon monoxide dehydrogenase and requires tetrahydrosarcinapterin (H4SPt) as methyl group acceptor. Reactions of the enzyme complex with carbon monoxide and with the methyl group donor N5-methyltetrahydrosarcinapterin (CH3-H4SPt) have been analyzed by UV-visible spectroscopy. Reduction of the enzyme complex by CO occurred in two steps. In the first step, difference spectra exhibited peaks of maximal absorbance decrease at 426 nm (major) and 324 nm (minor), characteristic of Fe-S cluster reduction. In the second step, corrinoid reduction to the Co1+ level was indicated by a prominent peak of increased absorbance at 394 nm. Spectrophotometric analyses of the corrinoid redox state were performed on the intact complex at potentials poised by equilibration with gas mixtures containing different [CO2]/[CO] ratios or by variation of the [H+]/[H2] ratio. The corrinoid Co2+/1+ midpoint potential was -426 mV (+/- 4 mV, n = 1.16 electrons, 24 degrees C), independent of pH (pH 6.4-8.0). The results indicated a significant fraction of Co1+ corrinoid at potentials existing in vivo. The reduced corrinoid reacted very rapidly with CH3-H4SPt. Reaction with methyl iodide was slow, and methylation by S-adenosylmethionine was not observed. Tne rate of methyl group transfer from CH3-H4SPt greatly exceeded the rate of CO reduction of enzyme centers. The enzyme complex catalyzed efficient synthesis of acetyl-CoA from coenzyme A, CO, and CH3-H4SPt. During acetyl-CoA synthesis, demethylation of CH3-H4SPt was monitored by the absorbance increase at 312 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and some properties of the corrinoid-containing membrane protein from Methanobacterium thermoautotrophicum.

The cytoplasmic membrane of the methanogenic archaebacterium Methanobacterium thermoautotrophicum does not contain cytochromes, but did contain a corrinoid protein of molecular mass about 33 kDa which, after treatment with 10 mg Triton X-100/mg protein, was contained in a protein complex of about 500 kDa. Washed membranes from 1 g dry cells contained about 70 nmol of the cobamide factor III (5-hydroxybenzimidazolyl cobamide) as the sole corrinoid. The corrinoid-containing protein complex was purified and some of its properties were studied. According to several criteria it is an integral membrane protein complex. The corrinoid-protein complex, after about 100-fold purification, gave a single band on native PAGE and still had molecular mass of about 500 kDa. In SDS-PAGE several subunits were observed: in addition to the corrinoid-carrying subunit of about 33 kDa, other polypeptides of approximately 28 kDa, 26 kDa, and possibly 23 kDa were present. One mole of the purified 500-kDa protein complex contained greater than or equal to eight moles of the cobamide factor III. It was estimated that the corrinoid-protein complex accounts for 8% of the membrane protein of M. thermoautotrophicum. The visible spectrum of the oxidized protein exhibited absorbance maxima at 547 nm, 511 nm, and a shoulder at 468 nm, which disappeared upon reduction with dithionite. The midpoint potential of this transition was around -145 mV (pH 7). With EPR a Co2+ signal was observed within -50 mV and -350 mV with a maximum around -200 mV. Possible reasons for the disappearance of the Co2+ signal at low redox potentials are discussed. The line shape of the Co2+ signal was similar to that of Co2+ in free corrinoids. The signal of Co2+ could also be evoked by reduction with 5 mM dithiothreitol. From the redox properties of the corrinoid membrane protein it may be expected that in vivo the cobalt may become reduced and reoxidized. Its possible function as an electron-mediating membrane protein in the metabolism of methanogenic bacteria is discussed.

Effect of pancreatic extracts on the faecal excretion and on the serum concentration of cobalamin and cobalamin analogues in cystic fibrosis.

A malabsorption of crystalline labelled cobalamin is observed in 100% of cystic fibrosis patients. Using radioisotope dilution assays and molecular sieve gel chromatography, we determined the serum concentration and the faecal excretion of cobalamin and cobalamin analogues in nine cystic fibrosis children before and after 4 days' interruption of pancreatic extract treatment. On chromatography, the unsaturated cobalamin binders of the faecal extracts eluted in two positions with molecular masses of 44 300 and 20 300, corresponding mostly to partially degraded R binders. The amounts of the less degraded form of R binder (molecular mass 44 300) increased significantly after interruption of the treatment. The cobalamin concentration in the serum remained normal after interruption of the treatment but the analogue concentrations in the serum decreased and faecal excretion of cobalamin and analogues increased significantly. These results allowed us to suggest that (1) pancreatic insufficiency in cystic fibrosis is responsible for a decrease in the absorption of digestive analogues induced by a defective degradation of R binders, and (2) cobalamin analogues have a short half-life in blood.

Defective adenosylcobalamin synthesis in a case of transcobalamin II deficiency.

Cobalamin metabolism has been investigated in a new case of transcobalamin II (TC II) deficiency. Using the chromatobioautographic technique, an abnormal distribution of cobalamins was detected in the child's erythrocytes and reduced synthesis of adenosylcobalamin but not of methylcobalamin in cultured fibroblasts. These results suggest that there may be a close link between TC II-mediated cobalamin transport and intracellular synthesis of adenosylcobalamin (Ado-Cbl).

High-performance liquid chromatographic separation of cobalamins.

Physiological cobalamins were separated by means of high-performance liquid chromatography (HPLC). Optimal conditions for elution of methylcobalamin, adenosylcobalamin, hydroxycobalamin and cyanocobalamin were determined. Excellent separation and resolution of these physiological cobalamins by HPLC were achieved. In addition, several cobalamin analogues were also studied and shown to be separable from the physiological forms. HPLC provides a rapid, sensitive, reproducible means of characterizing physiological cobalamins.

A novel form of vitamin B-12 and its derivatives.

A new isomeric form of cobalamins is reported. The conversion of cobalamin to cobalamin (the new form) is achieved by substituting the benzimidazole base by a less bulky group like H2O or CN- and modest thermal treatment. The back conversion of adenosylcobalamin to the corresponding regular form occurs in the "base-off" form at room temperature. It seems that the corrin ring becomes quite flexible in the "base-off" form and the freer axial movement of the cobalt atom flips the corrin ring into a different conformation. The change in conformation is borne out by subtle changes in the proton magnetic resonances on the corrin ring and the base, and very marked variation in the emission Mössbauer spectra. The latter is indicative of appreciable changes in the spatial conformation in the immediate vicinity of the central metal atom. The ultraviolet-visible and infrared spectra of cobalamin are indistinguishable from those of its corresponding regular form. The new conformational isomeric species is present as an impurity in all commercially available cobalamins (including pharmaceutical preparations). It raises the question whether the cobalamins' constitute the real biologically active anti-anemic factor in humans.

[Analysis of the cobalamin coenzymes in mouse splenic tumor cells]. / Analiz kobalaminovykh kofermentov v opukholevykh kletkakh selezenki myshei.

Coabalamine coenzymes were studied in tumor spleen cells of mice with La leukosis. Endogenous cobalamines in the cell extracts were separated by two-dimensional thin-layer chromatography and by bioautography. Analysis of the cobalamines ratio was carried out using bioautochromatographic technique. 5-deoxyladenosyl- and methyl cobalamines were found in extracts of the tumor cells.

[On cobyrinic acid biosynthesis. Novel methylated hydroporphyrins and their role in cobyrinis acid formation (author's transl)]. / Zur Cobyrinsäure-Biosynthese. Neuartige, methylierte Hydroporphyrine und deren Bedeutung bei der Cobyrinsäure-Bildung

Clostridium tetanomorphum and Propionibacterium shermanii were examined for intermediates in the synthetic pathway uroporphyrinogen III leads to cobyrinic acid. The isolation of two novel methylated hydroporphyrins, whose methyl groups are derived from S-adenosyl-L-methionine, is described. Spectroscopic (field desorption-mass, visible absorption) und electrophoretic studies as well as incorporation of labelled substrates indicate that they are analogues of a dihyrouroporphyrin and a tetrahydrouroporphyrin with adjacent reduced rings. Field desorption spectra of the [C2H3]- and [CH3]-tetrahydrouroporphyrin analogues show that the compound contains two methyl groups; it is concluded that the chlorine-like compound has one methyl group. Dehydrogenation experiments indicate that the methyl groups are located at one beta-carbon of the reduced rings. Incorporation experiments suggest that the tetrahydrouroporphyrin-like compound is an intermediate in cobyrinic acid biosynthesis. Studies on the utilization of a heptacarboxyporphyrinogen from C. tetanomorphum for cobyrinic acid formation are also described.

Protein-mediated uptake of vitamin B12 by isolated mitochondria.

Protein-mediated B12 uptake by isolated rat liver mitochondria has been shown to be enhanced by plasma transcobalamin (TC-II) but not by salivary R binder in vitro. The process is enhanced by calcium and depends on active mitochondrial respiration. Following uptake, cyanocobalamin is converted to adenosyl and methylcobalamins and released from the mitochondria. TC-II appears to be required for both cellular and mitochondrial uptake of vitamin B12.

Extraction of serum vitamin B12 for radio-isotopic and Lactobacillus leichmannii assay.

The protein precipitates discarded during the extraction process of the Lactobacillus leichmannii vitamin B12 assay have been shown to contain significant amounts of vitamin B12. This loss of vitamin B12 provide a satisfactory explanation for many of the descrepancies between the serum vitamin B12 values obtained by the L. leichmannii method and the radio-isotopic method of Raven et al (1969). It is possible to produce lower results by the method of Raven et al (1969)by incorporating into that method the L. leichmannii extraction process; it is also possible to produce higher results by the L. leichmannii method using a papain extraction process.