Accurate assessment and identification of naturally occurring cellular cobalamins

Background: Accurate assessment of cobalamin profiles in human serum, cells, and tissues may have clinical diagnostic value. However, non-alkyl forms of cobalamin undergo beta-axial ligand exchange reactions during extraction, which leads to inaccurate profiles having little or no diagnostic value. Methods: Experiments were designed to: 1) assess beta-axial ligand exchange chemistry during the extraction and isolation of cobalamins from cultured bovine aortic endothelial cells, human foreskin fibroblasts, and human hepatoma HepG2 cells, and 2) to establish extraction conditions that would provide a more accurate assessment of endogenous forms containing both exchangeable and non-exchangeable beta-axial ligands. Results: The cobalamin profile of cells grown in the presence of [Co-57]-cyanocobalamin as a source of vitamin B-12 shows that the following derivatives are present: [Co-57]-aquacobalamin, [Co-57]-glutathionylcobalamin, [Co-57]-sulfitocobalamin, [Co-57]-cyanocobalamin, [Co-57]-adenosylcobalamin, [Co-57]-methylcobalamin, as well as other yet unidentified corrinoids. When the extraction is performed in the presence of excess cold aquacobalamin acting as a scavenger cobalamin (i.e., "cold trapping''), the recovery of both [Co-57]-glutathionylcobalamin and [Co-57]-sulfitocobalamin decreases to low but consistent levels. In contrast, the [Co-57]-nitrocobalamin observed in extracts prepared without excess aquacobalamin is undetectable in extracts prepared with cold trapping. Conclusions: This demonstrates that beta-ligand exchange occurs with non-covalently bound beta-ligands. The exception to this observation is cyanocobalamin with a non-covalent but non-exchangeable CN- group. It is now possible to obtain accurate profiles of cellular cobalamins. Clin Chem Lab Med 2008; 46: 1739-46.