Extraction cobalamins

Intestinal malabsorption of vitamin B12 may be caused by gastrectomy or ileal resection, with an inverse relationship between the length of ileum resected and the absorption of vitamin B12. Other causes of malabsorption are tropical sprue, inflammatory disease of the small intestine, intestinal stasis with overgrowth of colonic bacteria, which consume the vitamin 6,2 ingested by the host, and HIV infection. Another cause of vitamin B malabsorption is failure to extract cobalamin from food. Some patients fail to absorb cobalamin bound to food, whereas absorption of nonfood-bound cobalamin in the Schilling test is unimpaired. This is particularly a problem in patients with compromised gastric status or early in the course of development of pernicious anemia. 

The extracted cobalamins are mixed with excess cobalamin-binding protein of unknown origin and are allowed to bind. The reaction mixture is applied to a cobalamin-precoated microchip. The amount of unsaturated cobalamin-binding protein that binds to the chip is detected and is inversely proportional to the cobalamin concentration in the sample. The specificity of the method depends on the binding protein employed. If intrinsic factor is used as binding protein, only cobalamin will be quantified. If a haptocorrin-like protein is employed, the sum of cobalamin and analogues is measured. 

Radioisotope dilution assays are based on the principle of competition between radioactive labeled ( Co) vitamin B 2 and cobalamins extracted from matrices for binding sites on the intrinsic factor (a glycoprotein). Binding is in proportion to the concentration of the radioactive and nonradio active B 2 with the concentration of intrinsic factor as the limiting factor. Free cobalamins are separated from those bound on the intrinsic factor by absorption... 

Spectrophotometric deterrnination at 550 nm is relatively insensitive and is useful for the deterrnination of vitamin B 2 in high potency products such as premixes. Thin-layer chromatography and open-column chromatography have been appHed to both the direct assay of cobalamins and to the fractionation and removal of interfering substances from sample extracts prior to microbiological or radioassay. Atomic absorption spectrophotometry of cobalt has been proposed for the deterrnination of vitamin B 2 in dry feeds. Chemical methods based on the estimation of cyanide or the presence of 5,6-dimethylben2irnida2ole in the vitamin B 2 molecule have not been widely used. 

Beck and Brink [28] have described a sensitive method for the routine assay of cobalamins in activated sewage sludge. The method involves extraction with benzyl alcohol, removal of interfering substances using a combination of gel filtration and chromatography on alumina, concentration of the extract by lyophilization, and direct determination of total cobalamin by high-speed liquid chromatography, in comparison with cobalamin standards. 

The search for RNAs with new catalytic functions has been aided by the development of a method that rapidly searches pools of random polymers of RNA and extracts those with particular activities SELEX is nothing less than accelerated evolution in a test tube (Box 26-3). It has been used to generate RNA molecules that bind to amino acids, organic dyes, nucleotides, cyano-cobalamin, and other molecules. Researchers have isolated ribozymes that catalyze ester and amide bond formation, Sn2 reactions, metallation of (addition of metal ions to) porphyrins, and carbon-carbon bond formation. The evolution of enzymatic cofactors with nucleotide handles that facilitate their binding to ribozymes might have further expanded the repertoire of chemical processes available to primitive metabolic systems. 

Overton and coworkers discovered a leucine 2,3-aminomutase in plant tissue cultures of Andrographis paniculata that converts (S)-leucine in (R)-f-leucine [43] (Scheme 1.6.10). The enzyme activity was investigated in cell free extracts by incubation with (S)-[U-14C]leucine and by measuring the radioactivity of the methyl ester camphanamide derivatives of the reaction mixtures by radio-GC. The stereochemistry of the /i-am i no acid was determined by radio-GC comparison of the enzyme reaction product as methyl ester camphanamide derivative with an authentic sample. The enzyme is not dependent on cobalamin, because addition of intrinsic factor does not induce its inhibition. 

The first evidence for cobalamin involvement in the conversion of methanol to methane was provided by Blaylock and Stadtman [196,216-218] with extracts of methanol-grown M. barkeri they demonstrated enzymatic formation of methylcobalamin from methanol, and subsequent reduction of methylcobalamin to methane. Later Blaylock [196] showed that conversion of methanol to methylcobalamin requires a heat-stable cofactor and at least three proteins, a 100-200 kDa Bi2-enzyme (methyltransferase), a ferredoxin, and an unidentified protein. Blaylock speculated that the role of hydrogen and ferredoxin in the conversion of methanol to methylcobalamin was in the reduction of the Bi2-protein. This work led to the proposal that methylcobalamin was the direct precursor of methane in methanogenesis from various substrates [196,218]. 

Most reports pertinent to Reaction (18) describe experiments with extracts or membrane preparations [117,377-380]. Results indicate involvement of corrinoid as an intermediate methyl carrier [379], and an oxygen-labile enzyme [377], similar to the methanohCoM methyltransferase system (MT ) of M. barkeri[ 5A], that requires ATP-dependent reductive activation for activity. Since M bryantii, M. formicicum [380], and M thermoautotrophicum possess cobalamin CoM methyltransferase activities that resemble the analogous protein MT2 in the methanol to CH3-C0M conversion pathway (see sections 3 and 4.13) [152], a scheme similar to that shown in Reactions (19) and (20) has been envisaged, where CH3-H4MPT replaces methanol in Reaction (28). Three recent reports [117,157,195] provide more information about this system, and are reviewed below. 

The ratio of methylcobalamin to total vitamin derivatives of extractable B12 has been determined in liver from mice who were subjected to different types of injury (mechanical trauma, bums, and ionizing radiation) inflicted separately or in various combinations. A decrease in methylcobalamin was observed paralleling the severity of the damage. There may thus be a decreased synthesis of methycobalamin or an increased catabolism or leakage from the liver—or combination of these causes. The method used did not determine the nonextractable cobalamin, so that a disappearance into a nonextractable form could have been the cause (L9). 

Early work in fractionated extracts of both Escherichia coli and liver indicated the participation of a vitamin Bi2-containing protein in the reaction of homocysteine with 5-methyltetrahydrofolate to form methionine and tetrahydrofolate. ° The reaction was stimulated by SAM, although SAM was not the stoichiometric methyl donor. Methylcobalamin was not the primary methyl donor however, it could serve as a methyl donor in the absence of 5-methyltrahydrofolate. It was suggested that methylcobalamin could be a catalytic intermediate in methyl transfer from 5-methyltetrahydrofolate. The role of SAM remained obscure until recent years, when it was found to preserve enzyme activity by maintaining cobalamin as methylcobalamin in the Bi2-protein. 

Cobalamins are a family of cobalt-containing cofactors, also known as the vitamin B12 family (Scheme 1). It was observed in the early 1900s that raw liver extracts could cure an otherwise fatal disease, pernicious anemia. In 1948, a red crystalline compound was isolated from liver extracts (cyanocobalamin), which was structurally characterized by Dorothy Hodgkin in 1956. These discoveries were honored by Nobel prizes in 1934 (to Whipple, Minot, and... 

LAB are traditionally known to be auxotrophic for cobalamin. However, Taranto et al. [103] described for the first time the production of a cobalamin-like compound by the strain Lb. reuteri CRL 1098. Cobalamin concentrations of 50-500pgl were detected in cell extracts of this strain using different bioassays its cobalamin synthesis was confirmed by genetic analysis finding at least 30 genes involved in the novo synthesis [104]. Other if), reuteri strains (DCM 20016, JCM1112, and CRL 1324 and 1327) were reported also to synthesize this vitamin [105]. Cobalamin production by other species such as Lb. coryniformis has also been studied [241]. 

In quantitative immunoassays, e.g., enzyme-linked immunosorbent assay and radioimmunoassay, a known amount of labeled vitamin is mixed with sample extract in which the vitamin content should be determined. Methods of labeling include radioisotopes (e.g., cobalamine), fluorescence, or luminescence markers (e.g., folate). The mixture is subjected to binding agent, equally forming complexes with both labeled and unlabeled vitamin. This complex is then isolated, and the amount of labeled vitamin present is measured. Sample vitamin concentration can be deduced from the ratio of labeled vitamin added to labeled vitamin measured after isolation. Advantages of immunoassays are short analysis time, and the possibility of automating them on clinical analyzers. 

In practice, eluents of widely varying polarity have been used. Very weakly polar steroids (oestrenols) can be quantitatively eluted with hydrophobic solvents like methylene dichloride (cf Table 14) [217], whereas difficulties are reported in the elution of the more polar progesterone under the same conditions [691]. Chloroform has been used to elute dinitrophenylhydrazones from silica gel and opium alkaloids from alumina [470, 535]. Methanol and ethanol are often used for elution of substances of all types of compound class from silica gel [213, 215, 259, 434] or alumina [122, 437]. Butyl acetate has been chosen as the most suitable eluent for penicillin V [486]. Acetone has proved suitable for recovering ubiquinones [733]. Polar neutral, acid and basic aqueous eluents have also been employed, e.g., water for mucic acid derivatives [451] (cf Table 14), 1% Tween 80 solution for cobalamin [128], 0.2 N sulphuric acid for Vitamin Bg factors [702], 34% ammonium persulphate solution for nicotinic acid [702] and ammonium hydroxide for azo dyes [638]. Nitro-4-acetaminophenetoles have been extracted from alumina with the highly polar dimethylformamide [489]. 

Coverage includes B vitamins and folate in the context of a historical background, disease, cardiovascular effects and the importance of vitamins in biochemistry as illustrated by a single vitamin. Thereafter there are chapters on the chemistry and biochemistry of thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, folate and cobalamin. Methodical aspects include characterization and assays of B vitamins and folate in foods of all kinds, dietary supplements, biological fluids and tissues. The techniques cover solid-phase extraction, spectrofluorimetry, mass spectrometry, HPLC, enzymatic assay, biosensor and chemiluminescence. In terms of fimction and effects or... 

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