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

Harshi Gunawardena,Renuka Silva,Pathmasiri Ranasinghe

doi:10.1186/s13104-019-4845-4

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.

Highlights

The oxygen radical absorbance capacity (ORAC) assay is a widely accepted method of measuring the antioxidant capacity of different biological samples
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
The fluorescence quenching is used in the ORAC assay, this is the first time we report the dynamic quenching of fluorescence given by fluorescein in plasma analysis

Summary

Introduction

The oxygen radical absorbance capacity (ORAC) assay is a widely accepted method of measuring the antioxidant capacity of different biological samples. It is based upon the early work of Ghiselli et al [1] and Glazer [2], and developed further by Cao et al [3]. We found that the human plasma samples initially tend to quench the fluorescence exerted by fluorescein when we tried to optimize the plasma ORAC assay. After the dynamic quenching of fluorescein at initial point, plasma samples followed normal fluorescence decay curves. Fluorescein quenching of human plasma was appeared to be quite significant.

Results

Discussion

Conclusion