Low methylcobalamin in liver tissues is an artifact as shown by a revised extraction procedure

Background: Vitamin B12 (cobalamin, Cbl) is represented by several molecular variants distinguished by the exchangeable ligand X coordinated to cobalt ion (XCbl). The most typical XCbl-forms are cyanocobalamin (CNCbl), hydroxocobalamin (HOCbl), methylcobalamin (MeCbl) and 5 '-deoxydeoxyadenosylcobalamin (AdoCbl). Cells convert the inactive vitamins CNCbl and HOCbl to the two critically important coenzymes AdoCbl or MeCbl. Surprisingly, little or no MeCbl is usually uncovered in the tissue samples, as compared to AdoCbl and HOCbl. We hypothesized that a low level of MeCbl is an artifact of harsh extractions, leading to degradation of MeCbl and/or its conversion to other XCbl-forms. Methods: We designed a mild extraction protocol, including homogenization of rat liver in ammonium acetate (pH 4.6), dilution with EtOH (final 60%) and heating for 10 min at 70 degrees C. The XCbls were separated by HPLC and quantified by isotope dilution assays. Results: A mild extraction revealed the following composition of Cbls: 37% AdoCbl, 35% MeCbl, 15% HOCbl and 13% CNCbl. The usual harsh protocol (pH 7, 20 min at 80 degrees C) changed this balance to 33%, 5%, 43% and 17%, respectively. A model assay revealed that MeCbl underwent demethylation and conversion to HOCbl at pH 3 and pH > 7, when heated with thiols. Other changes included decyanation of CNCbl and destruction of HOCbl.Conclusions: Our procedure reveals a high content of MeCbl in rat liver. General significance: This result challenges previous data and pinpoints the need for new studies to characterize the endogenous Cbl-forms in health and disease.

Automated summary

Vitamin B12 exists in different molecular variants, among which MeCbl is usually not detected in tissue samples. We hypothesized that this is due to degradation or conversion caused by harsh extraction methods. By using a mild extraction protocol, we found a relatively high content of MeCbl in rat liver.