Cobamide extraction procedures for analysis of tissue and microbiome samples

The structure of cobalamin has an upper β- axial ligand group and a lower α-axial ligand base. Different groups can attach to the upper ligand to form different forms of cobalamin. When the lower ligand base is changed, the molecule becomes unusable for humans. These are called the lower ligand analogues and are found most prominently in faecal samples. This study sets out to use a cobamide extraction procedure to identify the types of analogues present in faecal samples. It aims to investigate the effect of supplementation with B12 on the cobamides detected in racehorses' microbiome. Additionally, it aims to identify possible relations between treatment responses and cobamides detected in faecal samples of pernicious anaemia patients. Purification of cobamides is done with a His-tagged cobalamin binding protein and identification of them are done by HPLC-MS. The results show that supplementation does not have an effect on racehorses' cobamides detected and patients who take B12 sublingual spray or tablets have higher amounts of cyanocobalamin in their microbiome. Lastly, this procedure can successfully be adapted to study cobamides in tissue samples.