Human plasma dynamically quenches the fluorescein at the initial point of oxygen radical absorption capacity (ORAC) assay

Abstract

ObjectiveOxygen radical absorbance capacity (ORAC) assay measures the quenching of fluorescent probe by peroxyl radicals. Antioxidants present in biological systems block the quenching of fluorescence probe. We experienced the dynamic quenching of fluorescein, the fluorescence probe used in ORAC assay by the human plasma while plasma ORAC assay was optimized. Therefore, for the first time, we report the quenching of fluorescein by human plasma at the initial point of ORAC assay.ResultsAqueous whole and non-protein fractions of plasma were used in the analysis. Since the both fractions showed a similar pattern of quenching at the initial stage, quenched percentage of fluorescein was calculated and added to each sample in subsequent analysis. Addition of extra 20% fluorescein allowed plasma samples to quench the required amount of fluorescein and follow the normal decay curves afterwards. Further, change of fluorescein quenching (ΔF/F0) disclosed a dose dependent linear relationship with plasma (R2 = 0.8). It can be speculated that dynamic quenching exhibited by human plasma biomolecule/s at the initial stage would be of non-protein aqueous phase molecule/s. We suggest initiating further studies to detect, identify and quantify the fluorescein quenching biomolecules present in human plasma for further improvements in plasma ORAC assay.

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Proteome Analysis and In Vitro Antiviral, Anticancer and Antioxidant Capacities of the Aqueous Extracts of Lentinula edodes and Pleurotus ostreatus Edible Mushrooms

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Screening the Chemical Composition and Bioactivity of Cerasus serrulata Flower Extracts Using Two Extraction Methods

“…To determine the oxygen radical absorption capacity of the extracts, azo compounds were used as free radical sources, fluorescein sodium was used as an indicator, and water-soluble Vitamin E (Trolox) was used as a reference substance for antioxygen analysis (Gunawardena et al, 2019). Oxygen free radicals oxidize the fluorescent probes via hydrogen transfer, quenching the fluorescent probes overtime (Kameya et al, 2014).…”