Oxidative stress refers to the imbalance between reactive oxygen species and reductive antioxidant pathways, and is implicated in a range of pathologies including cancer, psychiatric disorders, and ageing. Peroxiredoxins (Prxs) are a superfamily of thiol-based redox proteins with six isoforms in mammals (Prx1-6), which are critical in many biological processes. Prxs react with high sensitivity and specificity towards peroxides, become oxidised, and are recycled by reducing power derived from NADPH. Prxs are important in cells, particularly the mitochondrial isoform Prx3 that protects cells against mitochondrial oxidants produced during respiration. The Prx2 isoform is highly abundant in anuclear red blood cells, where it is critical in neutralising oxidants produced by the continual autoxidation of haemoglobin and the wide range of oxidants encountered in circulation.
Novel biomarkers are needed to better study oxidative stress, as traditional markers are limited due to the inherent instability of reactive oxidants, poor sensitivity, and artefactual oxidation during analysis/storage. Prxs have emerged as promising biomarkers due to the formation of intermolecular disulfides, which can be readily detected due to the change in oligomeric state. Their high abundance, reactivity with hydroperoxides, and specific subcellular localisation may address many of these limitations. Using a combination of non-reducing SDS-PAGE and immunoblotting, we can measure the relative proportion of oxidised Prxs in a sample. This has been applied to human blood samples by measuring the basal oxidation of Prx3 in platelet mitochondria and Prx2 in red blood cells, as well as their rate of reduction following oxidant challenge. These assays were repeatedly applied to a small group to investigate variation within individuals over time, and also applied to a large cohort to show variation between individuals.