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. 2020 Mar 5;9(3):217.
doi: 10.3390/antiox9030217.

Vitamin C Activates the Folate-Mediated One-Carbon Cycle in C2C12 Myoblasts

Armando Alcazar Magana  1   2 , Ralph L Reed  2   3 , Rony Koluda  1 , Cristobal L Miranda  2   3 , Claudia S Maier  1   2 , Jan F Stevens  2   3
Affiliations

Vitamin C Activates the Folate-Mediated One-Carbon Cycle in C2C12 Myoblasts

Armando Alcazar Magana et al. Antioxidants (Basel). .

Abstract

Vitamin C (L-ascorbic acid, AA) is an essential cellular antioxidant and cofactor for several α-ketoglutarate-dependent dioxygenases. As an antioxidant, AA interacts with vitamin E to control oxidative stress. While several reports suggest an interaction of AA with folate (vitamin B9) in animals and humans, little is known about the nature of the interaction and the underlying molecular mechanisms at the cellular level. We used an untargeted metabolomics approach to study the impact of AA on the metabolome of C2C12 myoblast cells. Compared to untreated cells, treatment of C2C12 cells with AA at 100 µM resulted in enhanced concentrations of folic acid (2.5-fold) and 5-methyl-tetrahydrofolate (5-methyl-THF, 10-fold increase) whereas the relative concentrations of 10-formyl-tetrahydrofolate decreased by >90% upon AA pretreatment, indicative of increased utilization for the biosynthesis of active THF metabolites. The impact of AA on the folate-mediated one-carbon cycle further manifested itself as an increase in the levels of methionine, whose formation from homocysteine is 5-methyl-THF dependent, and an increase in thymidine, whose formation from deoxyuridine monophosphate (dUMP) is dependent on 5,10-methylene-THF. These findings shed new light on the interaction of AA with the folate-mediated one-carbon cycle and partially explain clinical findings that AA supplementation enhances erythrocyte folate status and that it may decrease serum levels of homocysteine, which is considered as a biomarker of cardiovascular disease risk.

Keywords: C2C12 cells; ascorbic acid; folic acid; mass spectrometry; metabolomics; one-carbon metabolism; vitamin C.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Analysis of treated C2C12 cells with ascorbic acid and with ascorbate 100 µM. (a) Principal component analysis (PCA) scores plot, where each dot represents a sample analysis (thus, 6 dots per experiment resulting from biological triplicates and technical duplicates); (b) PCA biplot visualizes which metabolites contribute most to the separation of the experimental groups; (c) heatmap visualizing the top 40 most differentiating metabolites. Color coding indicates greater deviation from the mean of all samples for a particular metabolite; (d) dendrogram indicating the degree of similarity among samples, constructed on the basis of all 204 metabolites. The analysis was performed using MetaboAnalyst V4.0.
Figure 2
Figure 2
Positive ion mode MS/MS spectra of the two detected formyl-tetrahydrofolate (THF) isomers and the proposed structures of their fragment ions (CE 35 V; CES of 15 V). (a) 10-formyl-tetrahydrofolate (10-formyl-THF) and (b) 5-formyl-tetrahydrofolate (5-formyl-THF, authentic standard). Peak labels denote accurate masses and fragment ion m/z values denote exact masses.
Figure 3
Figure 3
Simplified one-carbon cycle and associated pathways. Relative cellular levels of indicated metabolites are presented in the bar graphs (y-axes indicate peak areas expressed as 103 counts per second). Enzyme abbreviations: DHFR, dihydrofolate reductase; Glu, glutamic acid; MTHFD1, 5,10-methylene-THF dehydrogenase 1; MTHFR, 5,10-methylene-THF reductase; SHMT, serine hydroxymethyl transferase; TS, thymidylate synthase. Asterisks (*) indicate statistical significance between the control and treatment (*: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001).
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