Advancing antioxidant testing: Standardisation of a human plasma oxidation assay and its integration with chemical and cellular protocols

Chemical antioxidant methods (CAM) are highly reproducible, but they fail to capture the dynamic nature of biological systems. In contrast, cellular antioxidant assays (CAA) offer a more comprehensive evaluation of antioxidant activity by assessing redox status. However, these assays are time- and resource-intensive. To address these limitations, we standardised and validated a micro-analytical method, the Plasma Oxidation Assay (POA), which utilises human plasma as a probe for Cu²⁺-induced lipoperoxidation to assess the antioxidant activity and capacity of bioactive compounds simultaneously. We analysed various honey samples for their total phenolic content (TPC) and antioxidant capacity using the DPPH free radical scavenging method, ferric-reducing antioxidant power (FRAP), and iron-reducing capacity (IRC). These honey samples were categorised into three groups based on their antioxidant capacity: low, intermediate, and high. These groups were further analysed using the POA, revealing a significant correlation (r > 0.80, p < 0.05) between CAM and the antioxidant capacity using the POA, where samples with lower CAM values also exhibited lower antioxidant capacity and activity using the POA parameters. The POA demonstrated limits of detection and quantification at 0.39 and 1.19 mg of ascorbic acid equivalent/L, respectively, with high repeatability (coefficient of variation: 0.58–7.04 %) and accuracy (recovery: 96.4–112 %). Further cellular-based analysis at the cellular level, using AML12 mouse-derived hepatocytes challenged with oleic acid, revealed that the antioxidant activity masured by the POA correlated withthe mRNA expressions of heme oxygenase (HO-1) (r = 0.769) and thioredoxin reductase (TXNRD) (r = 0.615). In summary, a physiologically relevant method was standardised for assessing both the antioxidant activity and capacity simultaneously, offering new insights into the function and evaluation of food-derived antioxidants.