Peroxiredoxin 2 oxidation reveals hydrogen peroxide generation within erythrocytes during high-dose vitamin C administration.

Intravenous infusion of high dose (>10 g) vitamin C (IVC) is a common alternative cancer therapy. IVC results in millimolar levels of circulating ascorbate, which is proposed to generate cytotoxic quantities of H2O2 in the presence of transition metal ions. In this study we report on the in vitro and in vivo effects of millimolar ascorbate on erythrocytes. Addition of ascorbate to whole blood increased erythrocyte intracellular ascorbate approximately 35-fold. Within 10 min of ascorbate addition, we detected increased oxidation of erythrocyte peroxiredoxin 2 (Prx2), a major thiol antioxidant protein and a sensitive marker of H2O2 production. Up to 50% of Prx2 was present in the oxidised form after 60 min. The presence of extracellular catalase, removal of plasma or the addition of a metal chelator did not prevent ascorbate-induced Prx2 oxidation, suggesting that the H2O2 responsible for Prx2 oxidation was generated within the erythrocyte. Ascorbate is known to increase the rate of haemoglobin autoxidation and H2O2 production. Through spectral monitoring of oxidised haemoglobin we estimated a generation rate of 15 µM H2O2/min inside erythrocytes. We also investigated changes in erythrocyte ascorbate concentration and Prx2 oxidation following IVC infusion in a cohort of patients with cancer. Plasma ascorbate levels ranged from 7.8 to 35 mM immediately post infusion, while erythrocyte ascorbate levels reached 1.5-3.4 mM 4 h after beginning infusion. Transient oxidation of erythrocyte Prx2 was observed. We conclude that erythrocytes accumulate ascorbate during IVC infusion, providing a significant reservoir of ascorbate, and this ascorbate increases H2O2 generation within the cells. The consequence of increased erythrocyte Prx2 oxidation warrants further investigation.

Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells.

Purpose: Protein levels and activity of the hypoxia-inducible transcription factors HIF-1 and HIF-2 are controlled by hydroxylation of the regulatory alpha chains. Proline hydroxylases (PHDs) target the protein for degradation via the von-Hippel-Lindau (VHL)-ubiquitin-ligase complex, and asparagine hydroxylation by Factor Inhibiting HIF (FIH) leads to transcriptional inactivation. In cell-free systems, these enzymes require ascorbate as a cofactor, and this is also inferred to be an intracellular requirement for effective hydroxylation. However, how intracellular concentrations of ascorbate affect hydroxylase activity is unknown. In this study, we investigated the modulation of the regulatory hydroxylases in cancer cells by intracellular ascorbate. Materials and methods: To facilitate this investigation, we used clear cell renal carcinoma cell lines that were VHL-proficient (Caki-1), with a normal hypoxic response, or VHL-defective (Caki-2 and 786-0), with uncontrolled accumulation of HIF-α chains. We monitored the effect of intracellular ascorbate on the hypoxia-induced accumulation of HIF-1α, HIF-2α and the expression of downstream HIF targets BNIP3, cyclin D1 and GLUT1. Changes in hydroxylation of the HIF-1α protein in response to ascorbate were also investigated in 786-0 cells gene-modified to express full-length HIF-1α (786-HIF1). Results: In VHL-proficient cells, hypoxia induced accumulation of HIF-1α and BNIP3 which was dampened in mild hypoxia by elevated intracellular ascorbate. Increased HIF-2α accumulation occurred only under severe hypoxia and this was not modified by ascorbate availability. In VHL-defective cells, ascorbate supplementation induced additional accumulation of HIF under hypoxic conditions and HIF pathway proteins were unchanged by oxygen supply. In 786-HIF1 cells, levels of hydroxylated HIF-1α were elevated in response to increasing intracellular ascorbate levels. Conclusion: Our data provide evidence that the hypoxic pathway can be modulated by increasing HIF hydroxylase activity via intracellular ascorbate availability. In VHL-defective cells, accumulation of HIF-alpha proteins is independent of hydroxylation and is unaffected by intracellular ascorbate levels.

Ascorbate availability affects tumor implantation-take rate and increases tumor rejection in Gulo-/- mice.

In solid tumors, HIF1 upregulates the expression of hundreds of genes involved in cell survival, tumor growth, and adaptation to the hypoxic microenvironment. HIF1 stabilization and activity are suppressed by prolyl and asparagine hydroxylases, which require oxygen as a substrate and ascorbate as a cofactor. This has led us to hypothesize that intracellular ascorbate availability could modify the hypoxic HIF1 response and influence tumor growth. In this study, we investigated the effect of variable intracellular ascorbate levels on HIF1 induction in cancer cells in vitro, and on tumor-take rate and growth in the Gulo-/- mouse. These mice depend on dietary ascorbate, and were supplemented with 3,300 mg/L, 330 mg/L, or 33 mg/L ascorbate in their drinking water, resulting in saturating, medium, or low plasma and tissue ascorbate levels, respectively. In Lewis lung carcinoma cells (LL/2) in culture, optimal ascorbate supplementation reduced HIF1 accumulation under physiological but not pathological hypoxia. LL/2, B16-F10 melanoma, or CMT-93 colorectal cancer cells were implanted subcutaneously into Gulo-/- mice at a range of cell inocula. Establishment of B16-F10 tumors in mice supplemented with 3,300 mg/L ascorbate required an increased number of cancer cells to initiate tumor growth compared with the number of cells required in mice on suboptimal ascorbate intake. Elevated ascorbate intake was also associated with decreased tumor ascorbate levels and a reduction in HIF1α expression and transcriptional activity. Following initial growth, all CMT-93 tumors regressed spontaneously, but mice supplemented with 33 mg/L ascorbate had lower plasma ascorbate levels and grew larger tumors than optimally supplemented mice. The data from this study indicate that improved ascorbate intake is consistent with increased intracellular ascorbate levels, reduced HIF1 activity and reduced tumor initiation and growth, and this may be advantageous in the management of cancer.

Human skeletal muscle ascorbate is highly responsive to changes in vitamin C intake and plasma concentrations.

BACKGROUND: Vitamin C (ascorbate) is likely to be essential for skeletal muscle structure and function via its role as an enzyme cofactor for collagen and carnitine biosynthesis. Vitamin C may also protect these metabolically active cells from oxidative stress. OBJECTIVE: We investigated the bioavailability of vitamin C to human skeletal muscle in relation to dietary intake and plasma concentrations and compared this relation with ascorbate uptake by leukocytes. DESIGN: Thirty-six nonsmoking men were randomly assigned to receive 6 wk of 0.5 or 2 kiwifruit/d, an outstanding dietary source of vitamin C. Fasting blood samples were drawn weekly, and 24-h urine and leukocyte samples were collected before intervention, after intervention, and after washout. Needle biopsies of skeletal muscle (vastus lateralis) were carried out before and after intervention. RESULTS: Baseline vastus lateralis ascorbate concentrations were ~16 nmol/g tissue. After intervention with 0.5 or 2 kiwifruit/d, these concentrations increased ~3.5-fold to 53 and 61 nmol/g, respectively. There was no significant difference between the responses of the 2 groups. Mononuclear cell and neutrophil ascorbate concentrations increased only ~1.5- and ~2-fold, respectively. Muscle ascorbate concentrations were highly correlated (P < 0.001) with dietary intake (R = 0.61) and plasma concentrations (R = 0.75) in the range from 5 to 80 µmol/L. CONCLUSIONS: Human skeletal muscle is highly responsive to vitamin C intake and plasma concentrations and exhibits a greater relative uptake of ascorbate than leukocytes. Thus, muscle appears to comprise a relatively labile pool of ascorbate and is likely to be prone to ascorbate depletion with inadequate dietary intake. This trial was registered at the Australian New Zealand Clinical Trials Registry (www.anzctr.org.au) as ACTRN12611000162910.