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1.
Food Chem ; 298: 125010, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31284091

RESUMO

Vitamin B12 dietary supplement can be critical to the alleviation strategies against micronutrient malnutrition and food insecurity. An HPLC-DAD method has been developed and validated, per AOAC SMPR 2016.017 (Standard Method Performance Requirements), for the quantitation of four bioactive forms of vitamin B12 (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, methylcobalamin) from dietary ingredients and supplements. The method achieves chromatographic baseline resolution of vitamin B12 forms on a modern column platform without the expensive requirement of an ultra-high pressure liquid chromatography and/or mass spectrometry. The method has a wide analytical range (0.0005%w/w-85%w/w), high precision (reproducibility relative standard deviations ranged from 1.43% to 4.67%), and high accuracy (>96% spike recovery rate for 11 out of 12 accuracy testing data points). The method detection and quantification limits are less than 0.16 and 0.52 µg/mL, respectively. To our best knowledge, it is simpler, less time-consuming, and more economical than other published methods for its intended uses.


Assuntos
Cromatografia de Fase Reversa/métodos , Suplementos Nutricionais/análise , Vitamina B 12/análise , Cobamidas/análise , Laboratórios , Limite de Detecção , Vitamina B 12/análogos & derivados , Complexo Vitamínico B/análise , Complexo Vitamínico B/química
2.
Biotechnol J ; 9(12): 1526-35, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25146562

RESUMO

Coenzyme B12 (Vitamin B12 ) is one of the most complex biomolecules and an essential cofactor required for the catalytic activity of many enzymes. Pseudomonas denitrificans synthesizes coenzyme B12 in an oxygen-dependent manner using a pathway encoded by more than 25 genes that are located in six different operons. Escherichia coli, a robust and suitable host for metabolic engineering was used to produce coenzyme B12 . These genes were cloned into three compatible plasmids and expressed heterologously in E. coli BL21 (DE3). Real-time PCR, SDS-PAGE analysis and bioassay showed that the recombinant E. coli expressed the coenzyme B12 synthetic genes and successfully produced coenzyme B12 . However, according to the quantitative determination by inductively coupled plasma-mass spectrometry, the amount of coenzyme B12 produced by the recombinant E. coli (0.21 ± 0.02 µg/g cdw) was approximately 13-fold lower than that by P. denitrificans (2.75 ± 0.22 µg/g cdw). Optimization of the culture conditions to improve the production of coenzyme B12 by the recombinant E. coli was successful, and the highest titer (0.65 ± 0.03 µg/g cdw) of coenzyme B12 was obtained. Interestingly, although the synthesis of coenzyme B12 in P. denitrificans is strictly oxygen-dependent, the recombinant E. coli could produce coenzyme B12 under anaerobic conditions.


Assuntos
Cobamidas/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Pseudomonas/genética , Aerobiose , Anaerobiose , Reatores Biológicos , Cobamidas/análise , Meios de Cultura , Genes Bacterianos , Ácido Láctico/análogos & derivados , Ácido Láctico/metabolismo , Propilenoglicóis/metabolismo , Pseudomonas/enzimologia
3.
Chem Commun (Camb) ; 50(36): 4674-6, 2014 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-24623153

RESUMO

We performed the reaction of vitamin B12 with N,N-dimethylformamide dimethyl acetal for primary amide activation, and added MeOH as a nucleophile, to afford cobalester, the first amphiphilic cobalamin derivative. The unique combination of redox properties and solubility represents an asset for its use as a catalyst in C-C bond forming reactions.


Assuntos
Tensoativos/química , Vitamina B 12/química , Catálise , Cobamidas/análise , Cobamidas/química , Cobamidas/metabolismo , Dimetilformamida/análogos & derivados , Dimetilformamida/análise , Dimetilformamida/química , Dimetilformamida/metabolismo , Oxirredução , Tensoativos/análise , Tensoativos/metabolismo , Vitamina B 12/análise , Vitamina B 12/metabolismo , Difração de Raios X
4.
Biomed Chromatogr ; 20(8): 806-14, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16345011

RESUMO

Corrinoids from various ovine tissue samples (liver, blood, small intestinal fluid and faeces) were analysed using a combination of high-performance liquid chromatography (HPLC) and a radioisotope dilution assay (RIDA) to estimate the distribution of corrinoids--the cobalamins hydroxocobalamin (OH-cbl), methylcobalamin (me-cbl) and 5'-deoxyadenosylcobalamin (ado-cbl), and cobalamin analogues--in these tissues. Samples were taken from either cobalt-deficient or cobalt-replete ewes, and ruminant and pre-ruminant lambs. In liver, ado-cbl predominated, followed by analogues, OH-cbl and me-cbl. Supplementation with either cobalt (ruminant) or vitamin B12 injections (pre-ruminant) increased the amount of ado-cbl and decreased analogues. In blood, OH-cbl predominated, followed by ado-cbl, analogues and me-cbl, respectively. In small intestinal fluid, the distribution from largest to smallest percentage was analogues, ado-cbl, OH-cbl and me-cbl. In faeces, analogues constituted the greatest proportion, followed by OH-cbl, ado-cbl and me-cbl, respectively. Owing to the small sample sizes only cautionary interpretations can be made. In contrast to humans, where me-cbl constitutes the highest proportion of corrinoids in plasma and ado-cbl in the liver, in sheep the amount of ado-cbl was consistently higher than me-cbl in all tissues. This may be due to the higher metabolic need of sheep for ado-cbl due to gluconeogenesis. Analogues and OH-cbl were found in each tissue, contrary to previous postulations. The much higher amount of vitamin B12 in small intestinal fluid compared with faeces indicates that a large proportion of the vitamin is absorbed by the gastro-intestinal tract.


Assuntos
Corrinoides/análise , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cobalto/deficiência , Cobalto/fisiologia , Cobamidas/análise , Fezes/química , Feminino , Conteúdo Gastrointestinal/química , Humanos , Hidroxocobalamina/análise , Intestino Delgado/química , Fígado/química , Técnica de Diluição de Radioisótopos , Ovinos , Vitamina B 12/análogos & derivados , Vitamina B 12/análise
5.
J Bacteriol ; 182(17): 4773-82, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10940017

RESUMO

The corrinoids from the obligate anaerobe Clostridium cochlearium were extracted as a mixture of Co(beta)-cyano derivatives. From 50 g of frozen cells, approximately 2 mg (1.5 micromol) of B(12) derivatives was obtained as a crystalline sample. Analysis of the corrinoid sample of C. cochlearium by a combination of high-pressure liquid chromatography and UV-Vis absorbance spectroscopy revealed the presence of three cyano corrinoids in a ratio of about 3:1:1. The spectroscopic data acquired for the sample indicated the main components to be pseudovitamin B(12) (Co(beta)-cyano-7"-adeninylcobamide) (60%) and factor A (Co(beta)-cyano-7"-[2-methyl]adeninylcobamide) (20%). Authentic pseudovitamin B(12) was prepared by guided biosynthesis from cobinamide and adenine. Both pseudovitamin B(12) and its homologue, factor A, were subjected to complete spectroscopic analysis by UV-Vis, circular dichroism, mass spectrometry, and by one- and two-dimensional (1)H, (13)C-, and (15)N nuclear magnetic resonance (NMR) spectroscopy. The third component was indicated by the mass spectra to be an isomer of factor A and is likely (according to NMR) to be 7"-[N(6)-methyl]-adeninylcobamide, a previously unknown corrinoid. C. cochlearium thus biosynthesizes as its native "complete" B(12) cofactors the 7"-adeninylcobamides and two homologous corrinoids, in which the nucleotide base is a methylated adenine.


Assuntos
Clostridium/química , Cobamidas/análise , Porfirinas/análise , Vitamina B 12/análogos & derivados , Dicroísmo Circular , Clostridium/metabolismo , Corrinoides , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Espectrofotometria Ultravioleta/métodos , Vitamina B 12/análise , Vitamina B 12/biossíntese
6.
Biochem J ; 341 ( Pt 1): 133-8, 1999 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-10377254

RESUMO

We investigated the co-ordinate variations of the two cobalamin (Cbl)-dependent enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MCM), and measured the levels of their respective cofactors, methylcobalamin (CH3Cbl) and adenosylcobalamin (AdoCbl) in cultured human glioma cells during nitrous oxide exposure and during a subsequent recovery period of culture in a nitrous oxide-free atmosphere (air). In agreement with published data, MS as the primary target of nitrous oxide was inactivated rapidly (initial rate of 0.06 h(-1)), followed by reduction of CH3Cbl (to <20%). Both enzyme activity and cofactor levels recovered rapidly when the cells were subsequently cultured in air, but the recovery was completely blocked by the protein-synthesis inhibitor, cycloheximide. During MS inactivation, there was a reduction of cellular AdoCbl and holo-MCM activity (measured in the absence of exogenous AdoCbl) to about 50% of pre-treatment levels. When the cells were transferred to air, both AdoCbl and holo-MCM activity recovered, albeit more slowly than the MS system. Notably, the regain of the holo-MCM and AdoCbl was enhanced rather than inhibited by cycloheximide. These findings confirm irreversible damage of MS by nitrous oxide; hence, synthesis of the enzyme is required to restore its activity. In contrast, restoration of holo-MCM activity is only dependent on repletion of the AdoCbl cofactor. We also observed a synchronous fluctuation in AdoCbl and the much larger hydroxycobalamin pool during the inactivation and recovery phase, suggesting that the loss and repletion of AdoCbl reflect changes in intracellular Cbl homoeostasis. Our data demonstrate that the nitrous oxide-induced changes in MS and CH3Cbl are associated with reversible changes in both MCM holoactivity and the AdoCbl level, suggesting co-ordinate distribution of Cbl cofactors during depletion and repletion.


Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/efeitos dos fármacos , Cobamidas/análise , Metilmalonil-CoA Mutase/efeitos dos fármacos , Óxido Nitroso/farmacologia , Vitamina B 12/análogos & derivados , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/análise , Aerobiose , Anestésicos Inalatórios/farmacologia , Feminino , Glioma , Humanos , Metilmalonil-CoA Mutase/análise , Tecido Nervoso/efeitos dos fármacos , Oxirredução , Células Tumorais Cultivadas , Vitamina B 12/análise
7.
Przegl Lek ; 55(4): 164-7, 1998.
Artigo em Polonês | MEDLINE | ID: mdl-9656740

RESUMO

The present paper illustrates the authors 25-year experience in step by step approach to the definition of environmental and genetic background of neural tube defects. Based on the birth defects registry, a complete ascertainment of all deliveries was performed in Southern Poland during two period: 1970-1972, and 1979-1981. The birth prevalence of neural tube defects (NTD), as well as other CNS malformations was determined. The empiric recurrence risk was calculated as 3.2% +/- 1.6. Based on this figure, the relative risk (RR = 37.6 p < 0.001) and heritability (h2 = 74.7 +/- 6.7) were estimated. Our own modification of Morton's complex segregation analysis was applied. Three Mendelian (dominant, additive and recessive) and one multifactorial model were tested. The results did not provide a clear cut discrimination between different models; however the lowest 2 value was obtained for additive inheritance with 61% of penetrance and the frequency of sporadic cases equaled 55%. A search for genetic markers did not support the hypothesis that HLA-A,B,C loci are equivalents of T/t like locus in mice. The results of the study on transcobalamine levels in amniotic fluid may suggests that different transcobalamine metabolism reflects phenotypic expression of genetic susceptibility to NTD development. Current research and future perspectives on genetic and environmental background of NTD are also presented.


Assuntos
Defeitos do Tubo Neural/epidemiologia , Líquido Amniótico/química , Animais , Cobamidas/análise , Cobamidas/genética , Suscetibilidade a Doenças , Marcadores Genéticos , Humanos , Camundongos , Defeitos do Tubo Neural/genética , Fenótipo , Polônia/epidemiologia , Prevalência , Medição de Risco
8.
Biol Chem Hoppe Seyler ; 374(1): 85-90, 1993 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8382495

RESUMO

Purified 2-methyleneglutarate mutase from Clostridium barkeri contains adenosylcobalamin (coenzyme B12) and varying amounts of oxygen-stable cob(II)alamin. The content of the latter was estimated by EPR spectroscopy at 6-11% of the total cobalamin (2-4 mol/mol enzyme). Tryptic digestion of the enzyme liberated the prosthetic groups, cob(II)alamin being oxidized by air to aquocobalamin. HPLC analysis of the released cobamides from several preparations revealed > 90% adenosylcobalamin and < 10% aquocobalamin. Treatment of active 2-methyleneglutarate mutase with 8M urea followed by gelfiltration yielded an inactive enzyme from which 50% of the adenosylcobalamin and up to 70% of the cob(II)alamin was removed. Addition of adenosylcobalamin to the urea-treated enzyme resulted in complete reactivation, but the content of cob(II)alamin was not increased. These data suggest that the oxygen-stable cob(II)alamin is not involved in catalysis. In the presence of the competitive inhibitor itaconate (methylenesuccinate, Ki = 0.7mM), an alteration of the UV/visible spectrum at 470 nm as well as a new line in the EPR spectrum of the enzyme (around g = 2.1) was observed. The results indicate the formation of an unusual, oxygen sensitive Co(II) species during catalysis. The EPR signal of the oxygen-stable cob(II)alamin (gx,y = 2.24) remained unchanged under those conditions.


Assuntos
Clostridium/enzimologia , Cobalto/química , Cobamidas/metabolismo , Transferases Intramoleculares , Isomerases/metabolismo , Catálise , Cromatografia Líquida de Alta Pressão , Cobalto/análise , Cobamidas/análise , Espectroscopia de Ressonância de Spin Eletrônica , Isomerases/química , Isomerases/isolamento & purificação , Espectrofotometria Ultravioleta
9.
Eur J Biochem ; 205(2): 759-65, 1992 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-1315276

RESUMO

Both components, E and S, of the adenosylcobalamin-(coenzyme B12)-dependent glutamate mutase from Clostridium cochlearium were purified. Component S (16 kDa) must be added to component E to obtain activity, although the latter contains substoichiometric amounts of component S besides the major 50-kDa subunit. The enzyme proved to be very similar to that of C. tetanomorphum as described by Barker et al. [Barker, H. A., Rooze, V., Suzuki, F. & Iodice, A. A. (1964) J. Biol. Chem. 239, 3260-3266] but component E of C. cochlearium was more stable and led to the first pure preparation. The pink component E showed a cobamide-like absorbance spectrum with a characteristic maximum at 470 nm indicating the presence of a cob(II)amide, probably Co alpha-[alpha-(aden-9-yl)]-cob(II)amide. A typical cob(II)amide signal at g = 2.23 with hyperfine and superhyperfine splitting was observed by EPR spectroscopy. A cobamide content of about 0.43 mol/mol 50-kDa subunit was determined by cyanolysis. Substitution of the migrating hydrogen at C-4 of glutamate by fluorine yielded the potent competitive inhibitor (2S,4S)-4-fluoroglutamate (Ki = 70 microM). (2R,3RS)-3-Fluoroglutamate (Ki = 600 microM) was also inhibitory. The competitive inhibition by 2-methyleneglutarate (Ki = 400 microM) and (S)-3-methylitaconate (Ki = 100 microM) but not by (RS)-2-methylglutarate suggested the transient formation of an sp2 center during catalysis. However, the presence of an N-terminal pyruvoyl residue was excluded and no evidence for the participation of another electrophilic center in the reaction was obtained.


Assuntos
Isomerases de Aminoácido/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Clostridium/enzimologia , Cobamidas/análise , Transferases Intramoleculares , Isomerases de Aminoácido/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Cromatografia em Gel , Cromatografia por Troca Iônica , Espectroscopia de Ressonância de Spin Eletrônica , Eletroforese em Gel de Poliacrilamida , Glutamatos/farmacologia , Cinética , Dados de Sequência Molecular , Conformação Proteica , Espectrofotometria , Relação Estrutura-Atividade
10.
Eur J Biochem ; 205(2): 767-73, 1992 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-1315277

RESUMO

The ultraviolet/visible spectrum of the pure pink-orange 2-methyleneglutarate mutase from Clostridium barkeri between 300-600 nm showed the presence of cobalamins; notably the peaks at 470 and 528 nm were indicative of oxygen-stable cob(II)alamin and adenosylcobalamin (coenzyme B12), respectively. Using the absorption coefficients of the isosbestic points at 340, 393 and 489 nm, the total cobalamin content was estimated as 3.7 +/- 0.3 mol/mol tetrameric enzyme (m = 300 kDa). Denaturation with 8 M urea in the presence of 2 mM dithiothreitol followed by gel chromatography and renaturation afforded an inactive enzyme which contained 40-50% of the initially bound cobalamin. This preparation could be reactivated to 95-100% by addition of adenosylcobalamin. The cobalamins were removed to 85% from the mutase by denaturation with 8 M urea in the presence of 1 M cyanide (pH 12) with irreversible loss of activity. 2-Methyleneglutarate mutase was inactivated by incubation with aquo-, cyano- or methylcobalamin; up to 50% of the activity was recovered by addition of adenosylcobalamin. Upon incubation of the mutase with [5'-3H]adenosylcobalamin about 30% of the total cobalamin was exchanged by the tritium-labelled cofactor without loss of activity. During aerobic catalysis the enzyme became sensitive towards oxygen which was accompanied by loss of activity and formation of aquocobalamin from adenosylcobalamin. EPR spectroscopy demonstrated the presence of 0.8 mol base-on cob(II)alamin/mol enzyme. Upon addition of 2-methyleneglutarate a second EPR signal of about equal intensity at g = 2.13 arose. The question of whether the oxygen-stable cob(II)alamin participates in catalysis or its complex with the enzyme represents an inactive form is currently under investigation.


Assuntos
Clostridium/enzimologia , Cobamidas/análise , Transferases Intramoleculares , Isomerases/química , Vitamina B 12/análise , Ditiotreitol/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Isomerases/metabolismo , Cinética , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Espectrofotometria
11.
Arch Microbiol ; 158(5): 370-3, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1444720

RESUMO

The major cobamide-containing protein from methanol-utilizing Sporomusa ovata was 8-fold enriched to apparent homogeneity. The protein exhibited a molecular mass of 40 kDa and of 38 kDa determined by gel filtration and by SDS-polyacrylamide gel electrophoresis, respectively. This finding indicates a monomeric protein structure. Monospecific polyclonal antisera raised against the protein did not cross react with another cobamide-containing protein from Sporomusa cells. Only the 40 kDa cobamide-containing protein was induced by methanol, since proteins from cells grown on 3,4-dimethoxybenzoate, betaine H2/CO2, or fructose showed faint or no cross reaction. Hence, the 40 kDa cobamide-containing protein is presumably involved in the methyl-transfer reaction of the methanol metabolism. The purified enzyme revealed 1.1 mol of p-cresolyl cobamide per mol of protein, but it lacked of iron-sulfur centers. Remarkably, the cofactor was firmly bound to its protein.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Bactérias Anaeróbias Gram-Negativas/química , Metanol/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Cromatografia em Gel , Cobamidas/análise , Coenzimas/isolamento & purificação , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Bactérias Anaeróbias Gram-Negativas/metabolismo , Dados de Sequência Molecular , Peso Molecular
12.
J Biol Chem ; 266(12): 7656-60, 1991 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-1850415

RESUMO

Lysine-2,3-aminomutase from Clostridium SB4 contains iron and sulfide in equimolar amounts, as well as cobalt, zinc, and copper. The iron and sulfide apparently constitute an Fe-S cluster that is required as a cofactor of the enzyme. Although no B12 derivative can be detected, enzyme-bound cobalt is a cofactor; however, the zinc and copper bound to the enzyme do not appear to play a role in its catalytic activity. These conclusions are supported by the following facts reported in this paper. Purification of the enzyme under anaerobic conditions increases the iron and sulfide content. Lysine-2,3-aminomutase purified from cells grown in media supplemented with added CoCl2 contains higher levels of cobalt and correspondingly lower levels of zinc and copper relative to enzyme from cells grown in media not supplemented with cobalt. The specific activity of the purified enzyme increases with increasing iron and sulfide content, and it also increases with increasing cobalt and with decreasing zinc and copper content. The zinc and copper appear to occupy cobalt sites under conditions of insufficient cobalt in the growth medium, and they do not support the activity of the enzyme. The best preparations of lysine-2,3-aminomutase obtained to date exhibit a specific activity of approximately 23 units/mg of protein and contain about 12 g atoms of iron and of sulfide per mol of hexameric enzyme. These preparations also contain 3.5 g atoms of cobalt per mol, but even the best preparations contain small amounts of zinc and copper. The sum of cobalt, zinc, and copper in all preparations analyzed to date corresponds to 5.22 +/- 0.75 g atoms per mol of enzyme. An EPR spectrum of the enzyme as isolated reveals a signal corresponding to high spin Co(II) at temperatures below 20 K. The signal appears as a partially resolved 59Co octet centered at an apparent g value of 7. The 59Co hyperfine splitting (approximately 35 G) is prominent at 4.2 K. These findings show that lysine-2,3-aminomutase requires Fe-S clusters and cobalt as cofactors, in addition to the known requirement for pyridoxal 5'-phosphate and S-adenosylmethionine.


Assuntos
Isomerases de Aminoácido/química , Clostridium/enzimologia , Cobalto/química , Transferases Intramoleculares , Ferro/química , Cobamidas/análise , Espectroscopia de Ressonância de Spin Eletrônica , Sulfetos/análise
13.
J Chromatogr ; 537(1-2): 235-47, 1991 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-2050781

RESUMO

The cyanoaquo and aquocyano stereoisomers of several putative vitamin B12 precursors are reversibly formed and can be separated using analytical high-performance liquid chromatographic methods. The behavior of these stereoisomers varies somewhat depending on the type of column used and the chromatographic conditions employed. Both reversed-phase and ion-exchange columns can be used to observe the reversible formation and separation of the stereoisomers of (H2O,CN)cobyric acid, cobinamide and the cobinic acid pentaamide-1, -2 and -3 structural isomers. The greatest differences in retention times are seen when the pH of the eluting buffer is less than 4.0 and the buffer contains no KCN.


Assuntos
Cobamidas/análise , Vitamina B 12/análogos & derivados , Vitamina B 12/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Corrinoides , Concentração de Íons de Hidrogênio , Espectrofotometria Ultravioleta , Estereoisomerismo , Vitamina B 12/análise
15.
Am J Gastroenterol ; 80(11): 841-52, 1985 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2996328

RESUMO

We found in four of five pernicious anemia gastric juices a partly degraded R binder which was cobalamin specific and has an apparent molecular weight of 60-70,000 daltons. Twenty-nine to 74% (4.8-27.0 ng/ml) of the corrinoid binding capacity could not be blocked by cobinamide (a noncobalamin corrinoid). The fifth pernicious anemia gastric juice and five nonpernicious anemia gastric juices had minimal amounts of this binder (2.5 and 2.2 +/- 1.4 ng/ml). Scatchard analysis revealed that cobalamin-specific R binder has 1000-fold lower affinity for cobinamide than cobalamin. Increasing quantities of trypsin and/or chymotrypsin digested increasing amounts of saliva R binder and an increasing percentage of the remaining digest-resistant R binder acquired cobalamin specificity. Partly degraded R binder in pernicious anemia gastric juice was resistant to further proteolysis. Cobalamin-specific R binder, perhaps produced in vivo by the action of refluxed pancreatic enzymes on swallowed R would preferentially bind ingested and/or biliary cobalamin rather than analogue and thereby could play a role in hastening the development of cobalamin deficiency in pernicious anemia.


Assuntos
Anemia Perniciosa/metabolismo , Suco Gástrico/análise , Fator Intrínseco/análise , Receptores de Superfície Celular/análise , Saliva/análise , Vitamina B 12/análise , Anemia/metabolismo , Cromatografia em Gel , Cobamidas/análise , Humanos , Peso Molecular , Pâncreas/enzimologia
16.
Prikl Biokhim Mikrobiol ; 19(6): 840-3, 1983.
Artigo em Russo | MEDLINE | ID: mdl-6664963

RESUMO

AdoCbl, MeCbl and OH-Cbl were studied by methods of polarography and three-angle scan voltammetry. Cobalamines studied show a specific electrochemical activity that can be taken as a basis for their identification after separation and purification. The high sensitivity of the method allows to carry out the quantitative analysis of AdoCbl at concentrations ranging from 10(-6) M to 10(-4) M.


Assuntos
Cobamidas/análise , Polarografia/métodos , Vitamina B 12/análise , Eletroquímica , Polarografia/instrumentação , Soluções
18.
Ann Intern Med ; 87(5): 546-51, 1977 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-921081

RESUMO

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.


Assuntos
Cobamidas/análise , Intestino Delgado/metabolismo , Síndromes de Malabsorção/metabolismo , Vitamina B 12/metabolismo , Anemia Megaloblástica/microbiologia , Bactérias/metabolismo , Cobamidas/biossíntese , Diverticulite/metabolismo , Humanos , Obstrução Intestinal/metabolismo , Intestino Delgado/análise , Intestino Delgado/microbiologia , Síndromes de Malabsorção/etiologia
19.
Vopr Med Khim ; 23(5): 681-4, 1977.
Artigo em Russo | MEDLINE | ID: mdl-339530

RESUMO

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.


Assuntos
Cobamidas/análise , Neoplasias Esplênicas/análise , Animais , Cromatografia em Camada Fina/métodos , Escherichia coli , Leucemia Experimental/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana/métodos , Transplante de Neoplasias , Vitamina B 12/isolamento & purificação
20.
J Parasitol ; 62(6): 948-50, 1976 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1003284

RESUMO

A light-sensitive vitamin B12 derivative has been extracted from the adult cestode, Spirometra mansonoides. This corrinoid was identified as the cobamide coenzyme, adenosylcobalamin, by its chromatographic, chemical, and spectral properties.


Assuntos
Cestoides/enzimologia , Cobamidas/isolamento & purificação , Plerocercoide/enzimologia , Animais , Gatos , Cobamidas/análise
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