Cobalamin, determination

The organo-corrinoids show similar behavior, but also additional complications. Rate constants have been determined 84) for the attack of mercury(II) acetate on various organocobalt cobinamides (X = H2O or absent) and cobalamins (X = 5,6-dimethylbenziminazole). The first complication, which has to be born in mind when comparing the cobinamides with the cobalamins or DMG complexes, is that the organocobin-amides are partly (R = vinyl and methyl) or wholly (R = Et, -Pr, t-Pr,... 

Sharama et al. [479] compared results obtained in the determination of cobal-amins in ocean waters by radioisotope dilution and bioassay techniques. These workers showed that the isotopic methods measured both biologically active and inactive cobalamins indiscriminately when porcine factor was used as the B12-specific binder. 

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. 

P Gimsing, E Nexo, E Hippe. Determination of cobalamins in biological material. II. The cobalamins in human plasma and erythrocytes after desalting on nonpolar adsorbent material, and separation by one-dimensional thin-layer chromatography. Anal Biochem 129 296-304, 1983. 

As will be noted later, it is commonly thought that homolytic cleavage of the Co—C bond is an important initial stage in the reactions of cobalamins. Accordingly, there has been much interest in the formation of Co—C bonds, the factors that determine their stability, and the cleavage of these Co—C bonds. 

Hall, M Gamble, M Slavkovich, V. et al. (2007) Determinants of arsenic metabolism Blood arsenic metabolites, plasma folate, cobalamin, and homocysteine concentrations in maternal-newborn pairs. Environmental Health Perspectives, 115 (10), 1503-9. 

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. 

S-Methylmalonyl-CoA mutase (EC 5.4.99.2) is a deoxyadenoxyladen-osylcobalamin-dependent enzyme of mitochondria required to catalyze the conversion of methylmalonyl-CoA to succinyl-CoA. A decrease in the activity of methylmalonyl-CoA mutase leads to the urinary excretion of large amounts of methylmalonic acid (C22). The biochemical lesion may be at the mutase level due to an abnormality of apoenzyme protein or an inability to elaborate the required coenzyme form of vitamin B12> i.e., adenosyl-cobalamin. In rare cases the abnormality may be due to an inability to convert the d form of methylmalonyl-CoA mutase to the l form as a result of a defective racemase (EC 5.1.99.1) (Kll). In patients, the nature of the abnormality can be determined by tissue culture studies (D13) and by clinical trial, since patients with a defect in adenosylcobalamin production will show clinical improvement when treated with very large doses of vitamin B12 (Mil). 

Giese and coworkers determined on the basis of experimental results obtained by Scheffold et al. [268, 299] that addition reactions of alkyl bromides 249 to a,p-unsaturated nitriles or esters 248 catalyzed by cobalamine 247 are free radical reactions (Fig. 61) [300], This conclusion was based on the similar cis/trans-selectivities in addition reactions of the 4-tert-butylcyclohexyl radical to different electron-poor alkenes 248 using 247 as a catalyst on one hand and classical tributyltin hydride conditions on the other. The kinetics of the radical addition was determined. 

Thermochemical. Application to the estimation of the enthalpy of a process such as that depicted by Equation 15 requires determination of the heats of formation of LnM—R, R , and LnM . The latter usually is not accessible to measurement although it is in the case of alkyl-cobalamins (where LnM- corresponds to vitamin B12r, a stable and accessible compound). Thus, thermochemical approaches, in principle, are potentially applicable to the estimation of the Co-C bond dissociation energy in coenzyme B12. However, the practical difficulties are considerable and the probable accuracy of the result is questionable. 

The ligand attached to the cobalt atom determines the activity of vitamin B12 in human enzymatic reactions. The two active coenzyme forms are methyl-cobalamin and 5 -adenosylcobalamin, the primary form of vitamin B12 in tissues. Cyanocobalamin, the therapeutic form of vitamin B12 contained in vitamin supplements, is produced by the cleavage of the unstable fink... 

Baker, S.A., Miller-Ihli, N.J. Determination of cobalamins using capillary electrophoresis inductively coupled plasma mass spectrometry. Spectrochim. Acta B 55, 1823-1832 (2000)... 

Spectrophotometric determination at 550 nm is relatively insensitive and is usehil for the determination of vitamin B 2 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 feeds. Chemical methods based on the estimation of cyanide or the presence of 5,6-dimethylben2iniida2ole in the vitamin B 2 molecule have not been widely used. 

The amount of vitamin B12 in the serum sample is determined as follows. The amount of radioactive [ Co]cobalamin bound to the beads is measured. The purpose of using the beads is to facilitate the separation of bound P CoJcobalamin from the nonbound [ Co]cobalamin remaining in solution (floating around versus sinking to the bottom of the test tube). High levels of bound radioactivity indicate that the scrum sample contains low vitamin levels. Low levels of bound radioactivity indicate that the scrum contains high levels of vitamin. Plasma folate is... 

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