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.

Positive Association of Ascorbate and Inverse Association of Urate with Cognitive Function in People with Parkinson's Disease.

Oxidative stress is thought to contribute to the aetiology of neurological disorders such as Parkinson's disease. Ascorbate (vitamin C) is a potent antioxidant and is associated with neurological and cognitive function. In this study we assessed the ascorbate status of a cohort of people with Parkinson's disease (n = 215), aged 50-90 years, compared with a cohort of age matched healthy controls (n = 48). The study sample's cognitive status ranged from normal to mild cognitive impairment and dementia. There was no difference between the Parkinson's disease and healthy control groups with respect to mean ascorbate status, however, a higher proportion of participants with Parkinson's disease had hypovitaminosis C (i.e., <23 µmol/L) compared with healthy controls (20% vs. 8%, respectively). Within the Parkinson's disease group, Montreal Cognitive Assessment (MoCA) scores correlated positively with ascorbate concentrations, with higher ascorbate status associated with better cognitive function (r = 0.14, p = 0.045). Participants with hypovitaminosis C had significantly lower MoCA scores relative to participants with ascorbate concentrations >23 µmol/L (p = 0.014). Ascorbate concentrations were significantly lower in the cognitively impaired subgroup compared with the normal cognition subgroup in the Parkinson's disease cohort (p = 0.03). In contrast, urate showed an inverse correlation with cognitive function (r = -0.19, p = 0.007), with higher urate concentrations observed in the cognitively impaired subgroup compared with the normal cognition subgroup (p = 0.015). There was an inverse association between ascorbate status and urate concentrations (r = -0.15, p = 0.017). Plasma protein carbonyls, a measure of systemic oxidative stress, were not significantly different between the Parkinson's disease cohort and healthy controls, and there was no association with cognitive function (r = 0.09, p = 0.19) or with ascorbate status (r = -0.05, p = 0.45). Overall, our study showed ascorbate status was positively associated with cognitive function in Parkinson's disease, suggesting that longitudinal studies investigating the temporal sequence of cognitive decline and ascorbate status are warranted.

Multiple binding modes of isothiocyanates that inhibit macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has roles in the innate immune response, and also contributes to inflammatory disease. While the biological properties of MIF are closely linked to protein-protein interactions, MIF also has tautomerase activity. Inhibition of this activity interferes with the interaction of MIF with protein partners e.g. the CD74 receptor, and tautomerase inhibitors show promise in disease models including multiple sclerosis and colitis. Isothiocyanates inhibit MIF tautomerase activity via covalent modification of the N-terminal proline. We systematically explored variants of benzyl and phenethyl isothiocyanates, to define determinants of inhibition. In particular, substitution with hydroxyl, chloro, fluoro and trifluoro moieties at the para and meta positions were evaluated. In assays on treated cells and recombinant protein, the IC50 varied from 250 nM to >100 µM. X-ray crystal structures of selected complexes revealed that two binding modes are accessed by some compounds, perhaps owing to strain in short linkers between the isothiocyanate and aromatic ring. The variety of binding modes confirms the existence of two subsites for inhibitors and establishes a platform for the development of potent inhibitors of MIF that only need to target one of these subsites.