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

• Published in: Biochimica et biophysica acta. General subjects Vol. 1867; no. 4; p. 130315
• Main Authors: Fedosov, Sergey N., Nexo, Ebba, Heegaard, Christian W.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2023
• Subjects: ammonium acetate; Animals; cobalt; coenzymes; demethylation; Extraction; homogenization; HPLC; isotope dilution technique; ligands; Liver; Liver - metabolism; Methylcobalamin; Rat; Rats; thiols; Vitamin B 12 - metabolism; vitamin B12; Vitamins; GSSG; Rat; Liver; EtOH; Methylcobalamin; Cbl; GS; GSCbl; MMCM; CN[57Co]Cbl; MetS; CNCbl; Cbx; DMB; MeCbl; Extraction; HC; HOCbl; GSH; IF; HPLC; AdoCbl
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2023.130315

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. 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. 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. Our procedure reveals a high content of MeCbl in rat liver. This result challenges previous data and pinpoints the need for new studies to characterize the endogenous Cbl-forms in health and disease. [Display omitted] •Low methylcobalamin (MeCbl) in liver tissues is an artifact of extraction.•The artificial decrease in MeCbl is caused by its conversion to hydroxocobalamin.•A harsh heating of a tissue sample at pH < 4 or pH > 7 accelerates this conversion.•Thiols are possible agents that demethylate MeCbl during its harsh extraction.•Milder extraction methods reveal a high level of MeCbl in rat liver.