Assays for Hydrophilic and Lipophilic Antioxidant Capacity (oxygen radical absorbance capacity (ORAC_FL)) of Plasma and Other Biological and Food Samples

TLDR

The study presents methods for applying the ORACFL assay to various biological and food samples. The authors developed a modified ORACFL protocol that extracts hydrophilic and lipophilic antioxidants from plasma using hexane/water/ethanol partitioning and optimizes assay conditions to reduce temperature‑related variability. The method enables measurement of total antioxidant capacity in protein‑free plasma, showing efficient partitioning of lipophilic and hydrophilic fractions and that lipophilic antioxidants account for roughly one‑third of the total capacity, with 37 °C incubation reducing assay variability. Keywords: ORACFL, phenolics, antioxidant, free radical, lipophilic, hydrophilic, blueberry, fruit juices.

Abstract

Methods are described for the extraction and analysis of hydrophilic and lipophilic antioxidants, using modifications of the oxygen radical absorbing capacity (ORACFL) procedure. These methods provide, for the first time, the ability to obtain a measure of "total antioxidant capacity" in the protein free plasma, using the same peroxyl radical generator for both lipophilic and hydrophilic antioxidants. Separation of the lipophilic and hydrophilic antioxidant fractions from plasma was accomplished by extracting with hexane after adding water and ethanol to the plasma (hexane/plasma/ethanol/water, 4:1:2:1, v/v). Lipophilic and hydrophilic antioxidants were efficiently partitioned between hexane and aqueous solvents. Conditions for controlling temperature effects and decreasing assay variability using fluorescein as the fluorescent probe were validated in different laboratories. Incubation (37 °C for at least 30 min) of the buffer to which AAPH was dissolved was critical in decreasing assay variability. Lipophilic antioxidants represented 33.1 ± 1.5 and 38.2 ± 1.9% of the total antioxidant capacity of the protein free plasma in two independent studies of 6 and 10 subjects, respectively. Methods are described for application of the assay techniques to other types of biological and food samples. Keywords: ORACFL; phenolics; antioxidant; free radical; lipophilic; hydrophilic; blueberry; fruit juices