AAPH

This method is also used to measure ex vivo low-density lipoprotein (LDL) oxidation. LDL is isolated fresh from blood samples, oxidation is initiated by Cu(II) or AAPH, and peroxidation of the lipid components is followed at 234 nm for conjugated dienes (Prior and others 2005). In this specific case the procedure can be used to assess the interaction of certain antioxidant compounds, such as vitamin E, carotenoids, and retinyl stearate, exerting a protective effect on LDL (Esterbauer and others 1989). Hence, Viana and others (1996) studied the in vitro antioxidative effects of an extract rich in flavonoids. Similarly, Pearson and others (1999) assessed the ability of compounds in apple juices and extracts from fresh apple to protect LDL. Wang and Goodman (1999) examined the antioxidant properties of 26 common dietary phenolic agents in an ex vivo LDL oxidation model. Salleh and others (2002) screened 12 edible plant extracts rich in polyphenols for their potential to inhibit oxidation of LDL in vitro. Gongalves and others (2004) observed that phenolic extracts from cherry inhibited LDL oxidation in vitro in a dose-dependent manner. Yildirin and others (2007) demonstrated that grapes inhibited oxidation of human LDL at a level comparable to wine. Coinu and others (2007) studied the antioxidant properties of extracts obtained from artichoke leaves and outer bracts measured on human oxidized LDL. Milde and others (2007) showed that many phenolics, as well as carotenoids, enhance resistance to LDL oxidation. 

This assay measures the ability of antioxidant components in test materials to inhibit the decline in (3-phycoerythrin ((3-PE) fluorescence that is induced by 2,2 -azobis(2-amidinopropane) dihydrochloride (AAPH) as peroxyl radical generator (ORACroo.X H202-Cu2+ as hydroxyl radical generator (ORACho.X and Cu2+ as a transition metal oxidant (ORACcu)-... 

DCFH-DA assay was first developed by Valkonen and Kuusi (1997) as an alternative to measure the total peroxyl radical-trapping antioxidant potential of plasma. This assay uses AAPH to generate peroxyl radicals and DCFH-DA as the oxidizable substrate for the generated radicals. The oxidation of DCFH-DA by peroxyl radicals converts DCFH-DA to dichlorofluorescein. DCF is highly fluorescent (excitation 480 nm, emission 526 nm) and also shows absorbance at 504 nm. Therefore, the produced DCF can be monitored either fluorometrically or spectrophotometrically. 

This method was first reported by Winston and others (1998) and it is based on the oxidation of alpha-kclo-y-mcthiolbutyric acid (KMBA) to ethylene by peroxyl radicals produced from AAPH. The ethylene formation, which is partially inhibited... 

These assays measure the level of protection provided to the naturally occurring carotenoid derivative crocin from bleaching by the radical generator AAPH. The assay was originally suggested by Bors and others (1984) and modified by Tubaro and others (1998), who used it to show that plasma antioxidant capacity is deeply influenced by the consumption of wine. The addition of a sample containing chain-breaking antioxidants results in the decrease in the rate of crocin decay. The sample is monitored for 10 min at 443 nm. 

More generally, one-electron oxidation of protein-bound phenols to form reactive ary-loxyl radicals is a possible pro-oxidant mechanism since these radicals can propagate H-atom or electron transfers within the protein. In addition to phenol protein covalent coupling, these phenol-mediated oxidative damages to proteins could be detrimental to their function as enzymes, receptors, and membrane transporters. For instance, investigations by capillary electrophoresis have shown that quercetin in concentrations lower than 25 pM potentiates HSA degradation by AAPH-derived peroxyl radicals. 

The ORAC method was first proposed by Cao and co-workers in 1993. Like in the TRAP method, they used a fluorescent indicator. Determination of antioxidant activity by this method is based on measurement of decreasing fluorescence of the indicator caused by the radicals generated in the system. The reaction mixture in their proposal consisted of a fluorescent indicator p-phycoerythrin (p-PE), 2,2 -azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator and the analysed sample [42]. Attributing the low purity of p-phycoerythrin (approx. 30%) to the low reproducibility of fluorescence and the occurrence of different forms of phycoerythrin, Ou and co-workers [43] modified the method by replacing the indicator with fluorescein (3, 6 -dihydroxyspiro[isobenzofuran-l[3H],9 [9H]-xanthen]-3-one). 

The reaction of antioxidants in a sample with the radicals generated by AAPH and fluorescein is conducted in a phosphate buffer at pH 7.4 and at the temperature of 37°C. As the reaction progresses, antioxidants in the analysed sample react with the radicals. With an excess of radicals, the ability of antioxidants to reduce them becomes exhausted and radicals react with fluorescein, oxidising it to a non-fluorescing form. Observation of the fluorescence of the reaction mixture is conducted at the excitation wavelength of 485 nm and emission wavelength of 525 nm. Measurements are conducted every 60-90 seconds until the resulting curve reaches a plateau. 

MIP films, applied to a QCM transducer, have been employed for chiral recognition of the R- and 5-propranolol enantiomers [107]. MIP films were prepared for that purpose by surface grafted photo-radical polymerization. First, a monolayer of 11-mercaptoundecanoic acid was self-assembled on a gold electrode of the quartz resonator. Then, a 2,2 -azobis(2-amidinopropane) hydrochloride initiator (AAPH), was attached to this monolayer. Subsequently, this surface-modified resonator was immersed in an ACN solution containing the MAA functional monomer, enantiomer template and trimethylolpropane trimethacrylate (TRIM) cross-linker. Next, the solution was irradiated with UV light for photopolymerization. The resulting MIP-coated resonator was used for enantioselective determination of the propranolol enantiomers under the batch [107] conditions and the FIA [107] conditions with an aqueous-ACN mixed solvent solution as the carrier. The MIP-QCM chemosensor was enantioselective to 5-propranolol at concentrations exceeding 0.38 mM [107]. 

Joshi G, Perluigi M, Sultana R, Agrippino R, Calabrese V, Butterfield DA. 2006. In vivo protection of synaptosomes by ferulic acid ethyl ester (FAEE) from oxidative stress mediated by 2,2-azobis(2-amidino-propane)dihydrochloride (AAPH) or Fe(2 +)/ H(2jO(2) Insight into mechanisms of neuroprotection and relevance to oxidative stress-related neurodegenerative disorders. Neurochem Int 48 318-327. 

Total radical trapping parameter (TRAP) assay is widely used in investigations and has various modifications [45-48]. This method presumes antioxidants capability to react with peroxyl radical 2.2-azobis (2-amidinopropane) dihydrochloride (AAPH). TRAP modifications differ in methods of registering analytical signal. Most often the final stage of analysis include peroxyl radical AAPH reaction with luminescent (luminol), fluorescent (dichlorofluorescein-diace-tate, DCFH-DA) or other optically active substrate. Trolox is often used as a standard. 

Fig. 3. Thermal homolysis of 2,2/-azobis(2-amidopropane) (ABAP) in the presence of oxygen leads to formation of peroxyl and alkoxyl radicals. This compound is usually available as mono- or dihy-drochlorode and then is abbreviated also as AAPH.

Ximenes VF, Pessoa AS, Padovan CZ et al (2009) Oxidation of melatonin by AAPH-derived peroxyl radicals Evidence of a pro-oxidant effect of melatonin. Biochim Biophys Acta 1790 787-792... 

---