Wang Mengle, Asam Stefan, Chen Jianqi, Ehrmann Matthias, Rychlik Michael
Chair of Analytical Food Chemistry, Technical University of Munich, Freising, Germany.
Chair of Technical Microbiology, Technical University of Munich, Freising, Germany.
Front Microbiol. 2021 Aug 13;12:713321. doi: 10.3389/fmicb.2021.713321. eCollection 2021.
Cobalamins (vitamin B12) are required by humans for their essential roles as enzyme cofactors in diverse metabolic processes. The four most common cobalamin vitamers are hydroxocobalamin (OHCbl), adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), and cyanocobalamin (CNCbl). Humans are not able to synthesise cobalamins and thus must acquire them from external sources. Therefore, a reliable and robust analytical method to determine the cobalamins in dietary sources is highly required. For such a purpose, stable isotope dilution assays (SIDAs) with LC-MS/MS are most suited due to their superior sensitivity, specificity, and ability to compensate for matrix effects and analyte loss during sample work-up. However, a critical bottleneck for developing a SIDA method for cobalamins is the availability of stable isotope-labelled internal standards. In the present study, we harnessed the potential of for the biosynthesis of N-labelled cobalamins. First, we developed a chemically defined medium (CDM) containing ammonium sulphate as a single nitrogen source except three essential vitamins that supported long-term stable growth of throughout continuous transfers. The CDM was further optimised for cobalamin production under different incubation schemes. With the optimised CDM and incubation scheme, fully N-labelled cobalamins were obtained in with a final yield of 312 ± 29 μg/L and 635 ± 102 μg/L, respectively, for [N]-OHCbl and [N]-AdoCbl. Additionally, an optimised incubation process under anaerobic conditions was successfully employed to produce specifically labelled [N, N]-cobalamins, with a yield of 96 ± 18 μg/L and 990 ± 210 μg/L, respectively, for [N, N]-OHCbl and [N, N]-AdoCbl. The labelled substances were isolated and purified by solid phase extraction and semi-preparative HPLC. Chemical modifications were carried out to produce [N]-CNCbl and [N]-MeCbl. Eventually, N-labelled compounds were obtained for the four cobalamin vitamers in high chromatographic and isotopic purity with desired N-enrichment and labelling patterns, which are perfectly suited for future use in SIDAs or other applications that require isotopologues.
钴胺素（维生素B12）是人类必需的物质，在多种代谢过程中作为酶辅因子发挥重要作用。四种最常见的钴胺素维生素形式是羟钴胺（OHCbl）、腺苷钴胺（AdoCbl）、甲基钴胺（MeCbl）和氰钴胺（CNCbl）。人类无法合成钴胺素，因此必须从外部来源获取。因此，迫切需要一种可靠且强大的分析方法来测定膳食来源中的钴胺素。出于这一目的，采用液相色谱-串联质谱（LC-MS/MS）的稳定同位素稀释分析（SIDA）最为合适，因为其具有卓越的灵敏度、特异性，以及补偿样品处理过程中基质效应和分析物损失的能力。然而，开发钴胺素SIDA方法的一个关键瓶颈是稳定同位素标记内标的可用性。在本研究中，我们利用了[具体微生物名称]生物合成N标记钴胺素的潜力。首先，我们开发了一种化学成分明确的培养基（CDM），除了三种必需维生素外，以硫酸铵作为单一氮源，该培养基支持[具体微生物名称]在连续传代过程中的长期稳定生长。在不同的培养方案下，对CDM进行了进一步优化以提高钴胺素产量。通过优化的CDM和培养方案，分别获得了最终产量为312±29μg/L和635±102μg/L的全N标记的OHCbl和AdoCbl。此外，成功采用厌氧条件下的优化培养过程来生产特异性标记的[N, N]-钴胺素，[N, N]-OHCbl和[N, N]-AdoCbl的产量分别为96±18μg/L和990±210μg/L。通过固相萃取和半制备HPLC对标记物质进行分离和纯化。进行化学修饰以制备[N]-CNCbl和[N]-MeCbl。最终，获得了四种钴胺素维生素形式的N标记化合物，其具有高色谱纯度和同位素纯度，以及所需的N富集和标记模式，非常适合未来用于SIDA或其他需要同位素异构体的应用。
