The ORAC assay is based on the protection of a fluorescent probe (either fluorescein or beta-phycoerythrin) from the oxidation induced by thermal decomposition of AAPH or other radical generators. The antioxidant efficacy of a sample is quantified on the ability to induce a significative delay of the fluorescence decay curve, that is the ability of the antioxidant to shift the curve to the right. Since the measurement of the antioxidant activity is calculated with respect to the maximum fluorescence signal (probe + AAPH without any antioxidant shield) - and, for this reason, may be defined as a relative measurement method - it is necessary to normalize the fluorescence signal using the maximum fluorescence reached as 100%, before calculating AUC.
Thank you for the answer Mr Bertin. From the above you have stated that the the maximum fluorescence signal is equivalent to (probe + AAPH without any antioxidant shield), maybe you mean probe +AAPH + reference antioxidant, or I am mixed up? There is still something else which is rather unclear in the AUC calc for ORAC, Why not use the trapezium rule directly from the curve RFU and time, the trapezium rule does take into consideration the F0 and the decay (if decay occurs) to Fn. When using normalisation and making inter-sample comparison, the fact that F0 is different for different sample, I have found that it becomes difficult to compare and for each of my ORAC assessment the F0 changes with the samples which maybe be due to lag time. I would sincerely appreciate further feedback from you on this assessment. Thanking you in advance, Ouma
Firstly you have to run your analysis with a "blank" solution, that is a fluorescein solution with the only AAPH at 37°; in this way you will achieve a rapid decay curve, with a maximum fluorescence at time zero and a minimum fluorescence (or no fluorescence) at time x. Using this "reference" curve, you may set the fluorescence axis in percentages, normalizing the maximum fluorescence joined with the blank to a 100% relative fluorescence intensity. By now, you can drive your analysis with the sample solution, that is [fluorescein solution + AAPH + sample antioxidant] (e.g. a vegetal extract) and standard solution, that is [fluorescence solution + AAPH + Antioxidant reference] (e.g. Trolox at different solutions), plotting all of yours curves in the same graph, starting from time zero. You should find a shift to the right in the time axis for the curves, proportional to the antioxidant protective effect.
The protective effect of antioxidant is calculated on the difference between the areas of the blank decay curve and that of the sample (for this reason, it is called "net AUC"). Of course, to calculate the areas you can use the trapezium rule, but probably you may automatically integrate the area under the curve directly from the plate reader or the spectrophotometer you use (if they are interfaced to the PC), or manually by entering the fluorescence readings point-by-point in a statistic/graphic-editing program (eg, GraphPad).
In any case, for further details on the construction of the calibration curve and for calculations on the antioxidant activity expressed as Trolox equivalents, I recommend you this article: "Gillespie KM, Chae JM, Ainsworth EA (2007). Rapid measurement of total antioxidant capacity in plants. Nature Protocols 2(4), 867-870", that is, in my opinion, the most exhaustive and comprehensive.
For any other questions or suggestions I am at your disposal...greetings! And best wishes for your experiments
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