The IIOAB Journal 01/2011; 2(2):23-32.


Vitamin B12 is a water-soluble vitamin. It is one of the eight vitamins of vitamin B complex, needed for blood and cell maturation. It helps maintain healthy nerve cells and red blood cells, and it is needed in DNA replication. Its deficiency may cause megaloblastic anemia (amidst others health issues). For these and many similar reasons, it sometimes becomes necessary to measure its concentration. This article has carefully reviewed the different methods used for measuring vitamin B12 concentration, and the unique principles involved. The principles, basically, depend on the molecular structure of Vitamin B12 and its reactions with other substances. The methods include microbiological assay and spectrophotometric methods – these are old methods: they were the first available methods, but they are still in use for reference purposes. Another method is electroluminescent (ECL) which involves highly reactive materials. However, inductive-coupled plasma-mass spectrometry (ICP-MS) is a very important method, which is used routinely, even in many research. On the other hand, atomic absorption spectroscopy depends on measuring the amount of energy involved in the reaction; while radioimmunoassay (RIA) is a highly sensitive immunoassay technique. In addition, there are different techniques for separating and preparing samples to be used in the various measurement methods. High-performance liquid chromatography (HPLC) is used for non-validate analyst, while capillary-electrophoresis (CE) that have high resolving power than traditional electrophoresis, which when they are coupled with certain detectors they afford us another principle for measuring this vitamin. Choosing the best method for measuring vitamin B12 concentration depends on many factors – including the type of sample, purpose of the test, necessity of pre-processing, time limitations, cost, sensitivity, specificity.

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    ABSTRACT: 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 μ g/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 μ g/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.
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