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

Abstract

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.

Keywords: Ascorbate; Erythrocytes; Haemoglobin; Hydrogen peroxide; Peroxiredoxin; Thiol oxidation.