Introduction

Cellular and organismal functions critically depend on the continuous generation of adenosine triphosphate (ATP), the universal energy currency that sustains virtually all biological processes. In plants, ATP production is primarily driven by photosynthesis within chloroplasts, whereas in mammalian cells, mitochondria function as the principal centers of energy metabolism through oxidative phosphorylation (OXPHOS).¹ Mitochondria are highly specialized, double-membrane cellular organelles that coordinate the oxidation of pyruvate and fatty acids via the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC), coupling nutrient oxidation to ATP synthesis through ATP synthase activity^2,3. Beyond supporting basal cellular maintenance, mitochondrial ATP production is indispensable for energetically demanding processes, including ion transport, muscle contraction, neurotransmission, biosynthesis, and regulated cell death, making mitochondrial function a central determinant of tissue integrity and organismal health⁴.

Among metabolic regulators, vitamin C (ascorbic acid) serves as a part of mitochondrial Complex III (cytochrome b/c1) – an essential part in cellular energy production. Its multiple functions include context-dependent modulation of mitochondrial function and cellular bioenergetics.^6,7 Following cellular uptake, vitamin C and its oxidized form, dehydroascorbic acid, affect mitochondrial metabolism through redox-sensitive mechanisms that affect ROS balance, mitochondrial membrane potential, and respiratory chain activity.^7,8 Vitamin C has also been implicated in the regulation of mitochondrial biogenesis through signaling pathways involving AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), nuclear respiratory factors (NRFs), and mitochondrial transcription factor A (TFAM).^9,10

In several experimental systems, pharmacological concentrations of vitamin C enhance oxidative phosphorylation capacity and increase mitochondrial ATP production under conditions of oxidative or metabolic stress.^8–10 Conversely, other studies demonstrated that vitamin C can suppress glycolytic metabolism and induce energetic stress, particularly in cancer cells, where a shift toward mitochondrial respiration may fail to compensate for diminished glycolytic ATP production, ultimately leading to growth inhibition and cell death.^11–14 These apparently divergent effects suggest that vitamin C-mediated metabolic reprogramming is highly dependent on cellular context, redox state, and mitochondrial functional reserve.

In this study, we investigated the effects of a combination of vitamin C derivatives, applied at physiological, oral, and intravenous-equivalent concentrations on mitochondrial ATP production across distinct cell types. Better understanding of the bioenergetic effects of different forms of vitamin C used as a combination is important in evaluating the therapeutic potential of a vitamin C complex in restoring or optimizing cellular bioenergy in cells originating from different organs, and in a wider context, as a potential measure in natural control of metabolic and mitochondrial disorders.

Materials and methods

Cell Culture. Human normal dermal fibroblasts (HNDF), human hepatocellular carcinoma cells (HepG2), immortalized rat skeletal myoblasts (L6), human ventricular cardiomyocyte-derived cells (AC16), human epithelial kidney cell line (HEK), and primary human skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HNDF, HepG2, L6, HEK, and AC16 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin-streptomycin (PS; Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained at 37°C in a humidified incubator containing 5% CO₂. Primary human skeletal muscle cells were cultured in Mesenchymal Stem Cell Basal Medium supplemented with the Primary Skeletal Muscle Growth Kit (ATCC) according to the manufacturer’s instructions. For myogenic differentiation, cells were cultured in Skeletal Muscle Differentiation Medium (ATCC) for 15 days prior to experimentation. Immortalized microglial cells (IMG) were obtained from Kerafast (Boston, MA, USA). These cells were originally isolated from adult murine brain tissue and immortalized for long-term propagation. IMG cells were maintained in DMEM supplemented with 10% FBS and 1% PS (MilliporeSigma, Burlington, MA, USA) under standard culture conditions.

Vitamin C Derivatives and Fatty Acids. All vitamin C derivatives, except for dehydroascorbic acid (DHA), were purchased from MilliporeSigma (St. Louis, MO, USA). DHA was obtained from Cayman Chemical (Ann Arbor, MI, USA). Stock solutions were prepared at a concentration of 6.0 mM in sterile 1× phosphate-buffered saline (PBS), except for ascorbic acid-6-phosphate (A6P), which was dissolved in 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich). All solutions were sterilized using 0.2 µm syringe filters prior to use.

ATP Production Assay. Intracellular ATP production was quantified using a bioluminescence-based ATP assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. Cells were seeded into 96-well plates at a density of 1.0 × 10⁵ cells per well in Minimum Essential Medium Eagle (MEM) lacking vitamin supplementation and containing 10% FBS. Cells were allowed to attach for 8 h at 37°C. Following attachment, the culture medium was replaced with serum-free MEM, and cells were treated with different forms of vitamin C at final concentrations of 60 µM, 90 µM, 120 µM, 250 µM, or 500 µM for 12 h at 37°C. After treatment, conditioned media were removed and ATP levels were immediately measured using ATP detection reagent diluted 1:4 in assay buffer. Luminescence was quantified as relative luminescence units (RLU) using a multimode microplate reader (Tecan Group Ltd., Männedorf, Switzerland). ATP levels were normalized to untreated control cells and expressed as percentage of control values. Data are presented as mean ± standard deviation (SD) from quadruplicate measurements.

Statistical Analysis. All experiments were independently repeated at least three times. Data are presented as mean ± standard deviation (SD). Statistical comparisons between groups were performed using Student’s t-test with GraphPad Prism software (GraphPad Software, Boston, MA, USA). Differences were considered statistically significant at p < 0.05.

Results

The effects of a commercially available vitamin C formulation consisting of vitamin C (ascorbic acid), lipid-soluble palmitoyl-6-ascorbate, calcium and magnesium forms of vitamin C, together with a bioflavonoid complex and L-leucine (VCF formula), as well as an in-house, laboratory-prepared mixture of these four vitamin C compounds (VCF mix), were evaluated across multiple human and rodent cell types at concentrations ranging from 60–500 µM.

Figure 1. Effect of different forms of vitamin C on ATP production in human cells in vitro, evaluated in normal dermal fibroblasts (HNDF), the cardiomyocyte cell line AC16, the hepatoma cell line HepG2, the kidney cell line HEK, and differentiated human skeletal muscle cells (hSkM). Cells were treated with a commercial VCF (formula) and laboratory combined VCF (mix) at concentrations raging between 60–500 µM for 12h. ATP production was evaluated by ELISA assay. *p<0.05, #p<0.001 compared to control; control – 0.02% DMSO, positive control – 60 µM of taurine, negative control – 100% dead cells.

As shown in Figure 1, treatment of HNDF with both formulations significantly increased ATP production at the concentrations of 250–500 µM. In human cardiomyocytes and human hepatoma (HepG2) cell lines, both formulations significantly increased ATP production starting at 120 and up to 500 µM. In human cardiomyocytes, the ATP levels increased by 21% compared to control, while in HepG2 cells, the ATP increase was 79% at 500 µM. HEK cells significantly increased ATP production following treatment with both formulations, starting at 90 µM, which remained at fairly constant levels across higher concentrations used. Human differentiated skeletal muscle cells also exhibited significantly elevated ATP production in response to both formulations. The highest increase was 48%, at a concentration of 60 µM. At the highest concentration of 500 µM, the ATP level in these cells decreased to the control value.

As presented in Figure 2, treatment of mouse microglial cells with both formulations resulted in a significant 25% increase in ATP production at concentrations of 250 and 500 µM. In rat skeletal myoblasts, a significant increase in ATP production of 38% was observed only at the highest concentration tested (500 µM). The ATP increase in these cells was similar for both test formulations.

Figure 2. Effect of different forms of vitamin C on ATP production in rodent cells in vitro evaluated in immortalized mouse microglial cells (IMG) and differentiated rat skeletal muscle cells myoblasts (L6). Cells were treated with commercial VCF (formula) and laboratory composed VCF (mix) combinations of vitamin C at concentrations ranging between 60-500 µM for 12h. ATP production was evaluated by ELISA assay. *p<0.05, #p<0.001 compared to control; control – 0.02% DMSO, positive control – 60 µM of taurine, negative control – 100% dead cells.

Discussion

In the present study, we evaluated the effects of a commercially available vitamin C formulation containing vitamin C, its fat soluble palmitoyl-6-ascorbate, and mineral forms of vitamin C (calcium and magnesium ascorbates), together with a citrus bioflavonoid complex and a manufacturing-required natural filler – the amino acid L-leucine. The effects of this formulation and an in-house, laboratory-prepared mixture of the same four vitamin C derivatives were tested on cellular ATP production across multiple human and rodent cell models at concentrations ranging from 60–500 µM. Our findings demonstrate that structurally distinct vitamin C derivatives applied together can significantly enhance ATP generation in a cell type-and concentration-dependent manner.

There were no distinct differences in the efficacy of a commercial and laboratory composed mix of vitamin C derivatives in all cell lines tested on ATP production.

The most significant ATP increases were observed at concentrations between 90–500 µM, although human fibroblasts and mouse microglial cells showed a statistically significant ATP increase when vitamin C formulations were applied at 250 and 500 µM, while rat skeletal myoblasts exhibited significant ATP elevation only at 500 µM. Interestingly, the highest ATP stimulation of 79% was recorded in highly metabolically active liver cells (HepG2) and human differentiated skeletal cells (48%). However, while the highest ATP increase in HepG2 cells was obtained when using higher concentrations (250–500 µM) of vitamin C (achievable by i.v. infusion), the highest ATP stimulation in human skeletal cells was achievable at 60 µM (a physiological concentration of vitamin C). These observations suggest that metabolic responsiveness to vitamin C derivatives may vary according to tissue origin, mitochondrial demand, and intrinsic cellular redox capacity.

Our observed ATP-enhancing effects of vitamin C formulations challenge a substantial body of literature indicating that vitamin C primarily preserves, rather than markedly increases, ATP production and mitochondrial content in healthy cell.^6,7 In normal physiological settings, vitamin C functions predominantly as an antioxidant and enzymatic cofactor that maintains mitochondrial integrity, supports collagen and carnitine synthesis, and stabilizes intracellular redox homeostasis.^8,15 Previous studies have shown that vitamin C does not consistently elevate ATP levels above physiological baseline in healthy cells, and in some contexts, high concentrations may even impair ATP generation by disrupting glycolysis or mitochondrial function, particularly in cancer cells exposed to pharmacologic millimolar doses.^11,13 Mechanistic studies in malignant or metabolically stressed cells have demonstrated that high-dose vitamin C can suppress glycolytic flux, alter mitochondrial respiration, and activate cell death pathways, leading to depletion of intracellular ATP, despite transient increases in oxidative metabolism.^11–13

The vitamin C concentrations used in this study were within a lower micromolar range that more closely reflects physiologically achievable oral vitamin C intake compared with pharmacological intravenous dosing. Normal vitamin C plasma levels range from 50–70 µM. Oral vitamin C administration is tightly regulated by intestinal absorption and renal clearance, resulting in plasma concentrations that typically plateau around 0.15–0.22 mM, even at high oral doses.¹⁶ In contrast, intravenous administration can transiently increase plasma ascorbate concentrations to the millimolar range, where vitamin C may behave as a pro-oxidant, redox-active compound capable of generating extracellular hydrogen peroxide and inducing mitochondrial dysfunction in susceptible cells.^16,17 The ATP-enhancing effects observed in our study therefore likely reflect modulation of mitochondrial efficiency and redox balance rather than nonspecific oxidative stress or cytotoxic metabolic disruption.

Our findings support the emerging concept of mitochondrial modulation, in which structurally modified micronutrients are used to fine-tune intracellular energy dynamics. Due to the multiple roles of vitamin C in cell metabolism, further mechanistic studies could define the molecular pathways responsible for the observed ATP enhancement, including mitochondrial membrane potential, oxidative phosphorylation, glycolytic flux, and reactive oxygen species signaling. This may have therapeutic relevance in disorders characterized by mitochondrial dysfunction or energetic insufficiency, including muscle degenerative diseases, neuroinflammatory disorders, chronic fatigue-associated conditions, and metabolic syndromes.^4,5

In summary, the present study demonstrates that select vitamin C derivatives can significantly enhance cellular ATP production across diverse mammalian cell systems under non-cytotoxic conditions. These findings suggest that these specific structural derivatives may possess previously underappreciated properties of increasing bioenergy formation, with potential applications in enhancing normal cellular functions as well as in managing mitochondrial and metabolic disorders.

REFERENCES

Figure 1. Effect of different forms of vitamin C on ATP production in human cells in vitro, evaluated in normal dermal fibroblasts (HNDF), the cardiomyocyte cell line AC16, the hepatoma cell line HepG2, the kidney cell line HEK, and differentiated human skeletal muscle cells (hSkM). Cells were treated with a commercial VCF (formula) and laboratory combined VCF (mix) at concentrations raging between 60–500 µM for 12h. ATP production was evaluated by ELISA assay. *p<0.05, #p<0.001 compared to control; control – 0.02% DMSO, positive control – 60 µM of taurine, negative control – 100% dead cells.

Figure 2. Effect of different forms of vitamin C on ATP production in rodent cells in vitro evaluated in immortalized mouse microglial cells (IMG) and differentiated rat skeletal muscle cells myoblasts (L6). Cells were treated with commercial VCF (formula) and laboratory composed VCF (mix) combinations of vitamin C at concentrations ranging between 60-500 µM for 12h. ATP production was evaluated by ELISA assay. *p<0.05, #p<0.001 compared to control; control – 0.02% DMSO, positive control – 60 µM of taurine, negative control – 100% dead cells.