Methylcobalamin Affects Thiol Metabolism and Bioenergetic Status Across a Broad Range of Concentrations
Authors: Schrier Matthew Scott , Irausquin Andres , Dong Jianan , Trivedi Malav Suchin , Deth Richard Carlton 
Published: April 2020

Objective

To assess metabolic responses of SH‐SY5Y human neuronal cells to a broad range of cobalamin concentrations.

Background

Cobalamin (Vitamin B12) consists of a heme‐like corrin ring with a cobalt atom that coordinates both a lower axial dimethylbenzimidazole ligand and a variable upper ligand. The upper axial ligand determines the biological activity of the molecule, and upon uptake into cells, the pre‐existing upper axial ligand is enzymatically cleaved and replaced by either a methyl group or deoxyadenosine group. Methylcobalamin (MeCbl) serves as a cofactor for methionine synthase, facilitating conversion of homocysteine to methionine. Plasma cobalamin concentrations are typically in the picomolar range. However, much higher concentrations have been employed pharmacologically to reverse post‐operative shock or treat cyanide poisoning. The role of cobalamin in these settings extends beyond its role as a cofactor and likely reflects the ability of corrin rings to interact with endogenous gasotransmitters, such as nitric oxide and hydrogen sulfide. In these high‐dose clinical applications, the underlying mechanisms leading to clinical benefits are incompletely understood, but may involve modulation of cellular metabolism.

Methods

SH‐SY5Y cells were plated at 2.5 × 10^5 cells/well in 6‐well plates containing DMEM, 10% FBS, and penicillin‐streptomycin‐fungizone. After 48h of growth, media was replaced for a further 24h. Cells were maintained in media containing increasing concentrations (10 pM to 1 mM) of MeCbl for either the final hour or the final 24 hours of experiments. Lysates were processed for targeted metabolomic profiling of the methionine cycle and associated pathways via LC/MS. Bioenergetic parameters (oxygen consumption rate and glycolysis‐related extracellular acidification) were evaluated with a Seahorse XFp apparatus following MeCbl dose‐escalation experiments.

Results

We observed significant changes in several methionine cycle and transsulfuration pathway metabolites as a function of MeCbl concentration. S‐adenosylmethionine (SAM) was decreased at 1h starting with 1 nM MeCbl. At 24h SAM was decreased at 10 uM MeCbl and higher. The SAM to S‐adenosylhomocysteine (SAH) ratio (SAM/SAH) decreased in a similar fashion by approximately 25%. Methionine and SAH did not change. 1 uM MeCbl and higher decreased cystathionine at 24h in a dose‐dependent manner, with 1 mM decreasing cystathionine by 45%. Lanthionine was decreased at both 1h and 24h starting at low nanomolar MeCbl concentrations. Cysteine was affected by the highest MeCbl concentrations. MeCbl increased oxygen consumption rate (OCR) at 1 mM. In the micromolar range MeCbl increased ECAR, but 1 mM dramatically decreased ECAR.

Conclusion

MeCbl exerts significant changes across a wide range of concentrations, likely reflecting its non‐cofactor role. While further investigation is needed, effects may involve the ability of corrin rings to modulate gasotransmitters. Understanding these mechanisms may reveal the origin of clinical benefits observed with high‐dose cobalamin treatment.

Support or Funding Information

This research was supported with a fellowship from the American Foundation for Pharmaceutical Education (AFPE).