Related Experiment Video
Updated: Aug 11, 2025
Quantification of Coenzyme A in Cells and Tissues
Published on: September 27, 2019
Low methylcobalamin in liver tissues is an artifact as shown by a revised extraction procedure
Sergey N Fedosov1, Ebba Nexo2, Christian W Heegaard3
1Department of Molecular Biology and Genetics, Aarhus University, Universitetsbyen 81, 8000 Aarhus C, Denmark; Department of Clinical Biochemistry, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark.
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 °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 °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.
More Related Videos
11:25Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis
Published on: July 11, 2014
07:56Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies
Published on: September 3, 2012