Is the Oxygen Radical Absorbance Capacity (ORAC) Method a Peroxyl-radical Scavenging Assay?

Abstract

The oxygen radical absorbance capacity (ORAC) method employs a water soluble azo-radical initiator, AAPH (2,2’-azobis(2-amidinopropane) dihydrochloride) as a free radical generator, by which the fluorescent probe fluorescein is damaged to result in the loss of fluorescence. Antioxidants can protect the probe from the damage and the degree of protection is quantified. Because AAPH has been used as a lipid-peroxidation reagent, “oxygen radical” in ORAC is generally accepted as peroxyl radicals; however, in the present spin trapping experiments using a newly developed spin trap, CYPMPO, there was no indication of peroxyl-radical formation in AAPH decomposition in aqueous media. These spin trapping studies demonstrated that alkoxyl (RO·) radical adduct was the sole product of AAPH decomposition. In contrast, alkyl-peroxyl (ROO·) radical was spin-trapped during the decomposition of a lipid soluble azo-radical initiator AIBN (azobis(isobutyronitrile)) in non-aqueous media. We speculate that alkyl-peroxyl radicals are short-lived in water, rapidly converted into alkoxyl radicals. Although the possibility that ORAC method monitors peroxyl-radical scavenging activity cannot be completely eliminated, spin trapping evidence strongly indicates that ORAC method is a scavenging capacity assay for alkoxyl radicals.

Cyclodextrin encapsulation of the functional group diminishes an antioxidant's free radical scavenging rates

“…Recent spin-trapping studies revealed that alkoxyl radical (RO • ) but not peroxyl radical (ROO • ) is produced after thermal or photolytic decomposition of AAPH [4,5]. As a result, EPR spectrum shown in the bottom panel of Fig.…”

“…When another precursor, azo-initiator AAPH (2,2 -azobis(2-amidinopropane)dihydrochloride), was employed, UV photolysis produced alkoxyl radical [3][4][5]. Using AAPH, scavenging rates of several water-soluble antioxidants against alkoxyl radical were determined [1].…”

Characterization of inclusion complex of vitamin E compound with 2,6-di-O-methylated β-cyclodextrin as the solubility enhancer and its kinetic determination for radical scavenging ability

“…Recent spin-trapping studies revealed that the AAPH radical produced after thermal or photolytic decomposition of AAPH was not the peroxyl radical (ROOÁ), but the alkoxyl radical (RO • ) [14,15]. Figure 4a shows the EPR spectrum obtained from the UV-irradiated solution of AAPH in the presence of the spin-trap DMPO; its hyperfine splitting constant (hfsc) was obtained with a computer spectrum simulation: An inclusion equilibrium constant was calculated based on the relative concentration of the inclusion complex and free species, which was also determined with spectral simulation.…”

Modified Oxygen Radical Absorbance Capacity Assay that can be Implemented at Low Temperatures: A pilot study

“…AAPH is thermally decomposed by heating the reaction mixture to 37℃, thus generating the oxygen radicals. By electron paramagnetic resonance (EPR) spin trapping studies, it has been shown that the oxygen radical generated by AAPH is an alkoxyl radical (Krainev et al, 1996;Sueishi et al, 2010). Fluorescein (FL) (3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one) is used as a fluorescent probe and the fluorescence intensity (FI) of FL is continuously recorded for 35 min (Ou et al, 2001).…”

“…Interestingly, oxygen radicals can also be generated by photolysis of AAPH, i.e., through illumination with ultraviolet (UV) light (Kohri et al, 2009;Sueishi et al, 2010). The photolysis of AAPH efficiently generates a high quantity of oxygen radicals over a short time by illumination with UV light of adequate intensity, while no heating is required.…”