Development of a stable isotope dilution assay for vitamers of the B12 group after biosynthesis of stable isotope-labelled isotopologues
Final Report Abstract
Vitamin B12, or cobalamin, is the vitamin with the most complex chemical structure. It consists of a central cobalt atom coordinated with a tetracyclic corrin system, an upper ligand (cyanide, methyl, hydroxyl, or adenosyl), and 5,6-dimethylbenzimidazole as the lower ligand. Nutritionally, vitamin B12 is one of the most critical vitamins because it is naturally produced only by certain bacteria. Due to its accumulation in the food chain, animal-based foods (i.e., meat, milk, eggs, fish) and fermented foods are considered sources of vitamin B12, whereas plant-based foods are not. Therefore, B12 deficiency is common worldwide, especially among vegans and the elderly. To prevent this deficiency, it is essential to know the B12 content in foods. However, analysing B12 is challenging due to (a) low levels in non-fortified foods, with natural vitamers having even lower levels, and (b) the chemical lability of cobalamins, leading to the degradation and interconversion of vitamers. The usual B12 analysis is based on either microbiological methods or HPLC- UV after converting all cobalamins to cyanocobalamin. However, these methods do not provide information on the natural distribution of B12 vitamers, which may have different bioavailabilities. Therefore, we have developed an LC-MS/MS-based method using stable isotopologues of the natural B12 vitamers as internal standards. The stable isotope-labelled compounds were biosynthesised in our project by culturing Propionibacterium freudenreichii on N-15 labelled ammonium sulphate as the sole nitrogen source. The biosynthesised compounds were purified using chromatographic methods and fully characterised using spectrometric methods. These substances were then used to optimise and validate an LC-MS/MS method for natural forms of cobalamin. A comparison of the method with the standard cyanocobalamin method revealed that the cyanidation process is neither complete nor reproducible in all matrices as previously assumed. Therefore, the reliability of the content data based on this method must be questioned. Finally, the developed method was applied to meat and other foods containing vitamin B12. It was found that meat from ruminants such as sheep or cattle is the best source of B12, and the vitamers were also measured in algal supplements (Chlorella) and various types of milk. The newly developed method was also applied to prospect the potential of lactic acid bacteria to synthesize B12 as foods fermented with lactic acid bacteria could be also potential providers for B12. Whereas a microbiological assay detected for many strains the capability for producing B12, the more specific LC- MS/MS revealed that mostly pseudovitamin B12 is generated along with some other cobamides containing other lower ligandes.
Publications
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Development of Stable Isotope Dilution Assays for the Analysis of Natural Forms of Vitamin B12 in Meat. Journal of Agricultural and Food Chemistry, 69(36), 10722-10730.
Wang, Mengle; Asam, Stefan; Chen, Jianqi & Rychlik, Michael
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Production of Four 15N-Labelled Cobalamins via Biosynthesis Using Propionibacterium freudenreichii. Frontiers in Microbiology, 12.
Wang, Mengle; Asam, Stefan; Chen, Jianqi; Ehrmann, Matthias & Rychlik, Michael
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Challenges in the determination of total vitamin B12 by cyanidation conversion: insights from stable isotope dilution assays. Analytical and Bioanalytical Chemistry, 415(23), 5797-5807.
Wang, Mengle; Schuster, Kathrin; Asam, Stefan & Rychlik, Michael
