1 Performing Oxygen Radical Absorbance Capacity Assays with Synergy HT ORAC Antioxidant Tests Introduction The antioxidant capabilities of foods, cosmetics, supplements and pharmaceutical agents has become of particular interest. This is the result of the evidence demonstrating the relationship of reactive oxygen/nitrogen species (ROS) with aging and pathogenesis. [1,2]. Living organisms: use of a fuel (ATP) whose production results in the formation of toxic compounds, and requires that a balance be maintained between the oxidants and the antioxidants. ROS (reactive oxygen species) are generally held in check by a combination of antioxidant enzymes, proteins, and antioxidants provided by the diet. A breakdown or reduction in antioxidant capability has been associated with a number of chronic diseases. Conceivably, the ingestion of foods or supplements that contain antioxidant activity could provide some protection towards this affect. Figure 1. Illustration of Antioxidant activity determination expressed as the net area under the curve (AUC). The AUC for standards, samples and blanks are calculated and the blank value subtracted. A KC4 kinetic profile of a blank well and an experimental well has been modified to highlight the Net AUC as described by Huang et al. . All living organisms are exposed to ROS on a continual basis. Besides exogenous sources, ROS are formed in vivo by several different mechanisms. The interaction of ionizing radiation with biological molecules can result in free radical formation. ROS are also the byproduct of normal cellular respiration. Electrons passing through the respiratory chain often leak away and BioTek Instruments, Inc., P.O. Box 998, Highland Park, Winooski, Vermont USA COPYRIGHT 26 TEL: FAX: Outside the USA:
2 directly reduce oxygen molecules to the superoxide anion. In addition, several dedicated enzymes in phagocytic cells (e.g. neutrophils and macrophages) synthesize ROS as part of the normal cellular function. ROS molecules fall into two categories: ROS molecules that contain unpaired electrons (e.g. superoxide anion, O 2 - ; hydroxyl ion, OH -, and the hydroxyl radical, OH) and react directly with biological molecules, and molecules that have the ability to pull electrons from other molecules (H 2 O 2 or HOCl) and can damage biomolecules through direct interaction or initiate a chain reaction where ROS species are transferred from molecule to molecule. Figure 2. Schematic depiction of the ORAC assay. Reactive oxygen species generators are added to parallel reactions that contain equal amounts of a fluorescent probe. Reactions contain either a buffer blank or antioxidant samples and standards. The antioxidant capacity of a sample is the net difference between the area under the curve (AUC) of the sample and that of the blank. Figure adapted from Huang et al. . There are several different methods to measure total antioxidant capacity described in the literature (3-7). The total peroxyl radical trapping parameter assays decribed by Wayner et al.  was widely used in the 198s and early 9s. This method was replaced by more precise methods such as the ferric reducing ability of plasma (FRAP) assay  and the Trolox equivalent antioxidant capacity assay (TEAC) . The TEAC assay is based on the inhibition by antioxidants of the absorbance of the cation of 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). ABTS is also a common substrate for absorbance based ELISA. The use of a fluorescent compound to measure antioxidant capability such as phycoerythrin increased the sensitivity , but suffers due to the inherent instability of the compound over time. The oxygen radical absorbance capacity (ORAC) assay replaces phycoerythrin with fluorescein, which is much more stable over the time frame of the assay . This assay has been automated  and over time converted to a microplate format . Ronald Prior at Brunswick Laboratories and his colleagues have accomplished much of the development of the ORAC assay. The ORAC assay depends on the free radical damage to a fluorescent probe, such as fluorescein, to result in a downward change of fluorescent intensity . The assumption, of course, is that the degree of change is indicative of the amount of radical damage. The presence of antioxidants results in an inhibition in the free radical damage to the fluorescent compound. This inhibition is observed as a preservation of the fluorescent signal. Reactions containing antioxidants and or blanks are run in parallel using equivalent amounts of a ROS
3 generator and fluorescent probe (Figure 1). Because the reaction is driven to completion, one can quantitate the protection by calculating the area under the curve (AUC) from the experimental sample. After subtracting the AUC for the blank, the resultant difference would be the protection conferred by the antioxidant compound (Figure 2). Comparison to a set of known standards allows one to calculate equivalents and compare results from different samples and experiments. Typically Trolox, (6-hydroxy-2,5,7,8-tetrametmethylchroman-2-carboxylic acid) a water soluble vitamin E analog, is used as the calibration standard and ORAC results are expressed as Trolox equivalents (Figure 3). The ORAC assay is unique in that because the assay is driven to completion the AUC calculation combines both the inhibition time as well inhibition percentage of free radical damage by the antioxidant into a single quantity. Standardization of the assay with the use of a common calibrator in conjunction with an assay that can be performed easily on many different compounds, foods, and materials has allowed for an easy comparison of antioxidant capabilities of many different materials and the formation of a database . Materials and Methods Sodium Fluorescein was purchased from Invitrogen. 2,2 -Azobis(2-amidinopropane) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox ), gallic acid, Epigallocatechin gallate (EGCG), epigallocatechin (EGC), and quercetin dihydrate were purchased from Sigma-Aldrich (St. Louis, Mo). Black-sided, special optics clear bottom plates (part # 3615) were obtained from Corning. The ORAC assay was performed essentially as described by Huang et. al . Briefly, AAPH (.414g) was dissolved in 1 ml of 75 mm phosphate buffer (ph 7.4) to a final concentration of 153 mm and made fresh daily. A fluorescein stock solution (4 x 1-3 mm) was made in 75 mm phosphate buffer (ph 7.4) and stored wrapped in foil at 5 C. Immediately prior to use, the stock solution was diluted 1:1 with 75 mm phosphate buffer (ph 7.4). The diluted sodium fluorescein solution was made fresh daily. In regards to the plate usage, the exterior wells were not used for experimental determinations (Figure 3). These wells were filed with 3 µl of water, while the interior wells were used for experimental determinations. To all experimental wells, 15 µl of working sodium fluorescein solution was added. In addition blank wells received 25 µl of 75 mm phosphate buffer (ph 7.4), while standards received 25 µl of Trolox dilution and samples received 25 µl of sample. The plate was then allowed to equilibrate by incubating for a minimum of 3 minutes in the Synergy HT Multi-Detection Microplate Reader (BioTek Instruments, Inc., Winooski, VT) at 37 C. Reactions were initiated by the addition of 25 µl of AAPH solution using the microplate reader s injector for a final reaction volume of 2 µl. The fluorescence was then monitored kinetically with data taken every minute. A Synergy HT Multi-Detection Microplate Reader with injectors was used with a 485 nm, 2 nm bandpass, excitation filter and a 528 nm, 2 nm bandpass, emission filter. The plate reader was controlled by KC4 version 3.4 (revision 1). Reactions were initiated by the addition of 25 µl of AAPH reagent (153 mm) followed by shaking at maximum intensity for 1 seconds. The fluorescence of each well was then measured from the bottom every 6 seconds at a sensitivity setting of 6. ORAC values were calculated as described by Cao and Prior . The AUC and the Net AUC of the standards and samples were determined using KC4 Data Reduction Software (BioTek Instruments, Winooski, VT) using equations 1 and 2 respectively. AUC =.5+(R2/R1)+(R3/R1)+(R4/R1)+.+.5(Rn/R1) (Eq. 1) Where R1 is the fluorescence reading at the initiation of the reaction and Rn is the last measurement. Net AUC = AUC sample AUC blank (Eq. 2.)
4 The standard curve was obtained by plotting the Net AUC of different Trolox concentrations against their concentration. ORAC values of samples were then calculated automatically using the KC4 software to interpolate the sample s Net AUC values against the Trolox standard curve. Figure 3. Plate map of wells typically used. Outer wells (blue-filled) were filled with 3 µl of water to provide large thermal mass. Only the inner 6 wells were used for experimental determinations Results Initial experiments examined the effect of AAPH concentration on the kinetics of fluorescence decay of fluorescein in solution. As demonstrated in Figure 4, the presence of AAPH results in a marked loss of fluorescence of sodium fluorescein over time. While samples that do not contain AAPH ( mm) are relatively stable over a period of 12 minutes, a complete loss of fluorescence was observed with AAPH concentrations greater than 28 mm. In addition the effect observed appeared to be graded on the basis of AAPH concentration. Fluorescence AAPH Kinetics mm mm mm mm mm mm Time (min) Figure 4. AAPH Induced Decay of Fluorescence. Various concentrations of AAPH were incubated with sodium fluorescein and the fluorescence was monitored kinetically for 12 minutes. Data was captured using KC4 data reduction software and exported to and plotted using GraphPad software.
5 The loss in fluorescence can be assessed by measuring the area under the curve (AUC) of the kinetic plot for each concentration. The greater the extent of florescent decay, the smaller the expected AUC value would be. When the AUC for each concentration is plotted against AAPH concentration a linear relationship is observed (Figure 5). A 4-parameter logistic fit of the data was used to describe these data. These data confirmed that in our hands a final concentration of AAPH of 19 mm, as described by Prior and his colleagues, was adequate for ORAC determinations of compounds. A final concentration of 19 mm (25 µl of a 153 mm stock solution in a final assay volume of 2 µl) was used for all subsequent ORAC determinations. Figure 5. AAPH Concentration Curve. Various concentrations of the ROS generator AAPH were incubated with constant amounts of sodium fluorescein in solution. Total area under the curve (AUC) for each well and the subsequent result plotted was against its concentration using KC4 data reduction software. A 4-parameter logistics fit was used to describe the data. The data presented in Figure 6 demonstrate the ability of the Synergy HT reader to perform the ORAC assay and measure antioxidant activity of samples. The kinetic curves of several different concentrations of Trolox standard demonstrates varying amounts of protection of fluorescein against oxidation that results in the loss of fluorescence. The highest concentration tested (1 µm) provided virtually full protection for approximately 2 minutes, before fluorescence intensity began to diminish, while the lowest concentration tested (6.25 µm) provided only slight protection above the buffer only control.
6 Figure 6. Trolox Kinetic Curves. Representative kinetic curves from an ORAC assay of Trolox antioxidant standards ranging from to 1 µm were plotted using GraphPad software. The reactions were initiated by the addition of 25 µl of AAPH (153 mm) solution and the fluorescence monitored every minute using a Synergy HT as previously described. When net AUC are calculated from these kinetic curves and plotted against Trolox Concentration a linear relationship is observed (Figure 7). Using a least means squared linear regression analysis a correlation coefficient (R 2 ) of.9987 was obtained. As a result, the determination Trolox equivalents of unknown samples can be made with confidence. Figure 7. Typical Antioxidant Standard Curve. The net AUC of different Trolox standards are plotted as a function of concentration. The subsequent calibration curve is then used to interpolate the antioxidant capacity of unknown samples. The standard curve can then be interpolated to quantitate unknown samples. The resultant determinations are expressed as Trolox equivalents. Several compounds known to have antioxidant properties were assayed using the ORAC assay as previously described. As depicted in Figure 8, all of these compounds show a significant linear concentration dependent antioxidant activity. The specificity of the assay is demonstrated by the lack of any response from Tris buffer (Figure 8C). Grape juice samples have also been measured for ORAC activity using the Synergy HT. Figure 9 demonstrates the significant antioxidant activity found in grape juice, where 1:1 diluted samples exhibit ORAC activity equivalent to 4 µm Trolox. In addition, these data demonstrate the repeatability of the Synergy HT. In these experiments, 5 separate 1:1 dilutions of two different grape juice samples were assayed for ORAC in triplicate. The mean of each determination is in very close agreement with the other corresponding measurement.
7 A Gallic Acid Dose Response Curves B Quercetin C Tris D Myricetin E EGC F EGCG Figure 8. Antioxidant Dose Response Curves. The ORAC of several different known antioxidant compounds, as well as Tris buffer, were measured as described previously and their Net AUC plotted against their concentration.
8 Sample Repeatabilty 6 Trolox Equivalance (µm) Sample 1 Sample 2 Grape Juice Sample Figure 9. Sample Repeatability. Two different grape juice samples were diluted 1:1 with phosphate buffer and assayed for antioxidant capability using the ORAC method. Five discrete samples were diluted separately and assayed in triplicate. Each data point represents the mean of the three determinations. Discussion The ORAC assay in our hands is very sensitive to slight temperature gradations. Despite extreme efforts, an edge effect was observed when the outside wells were used. Edge effects have been reported in any number of different types of microplate-based assays. Because we used the reader s reagent injectors to initiate the assay, plate lids were not used. In these experiments, the tighter control between reading the wells and the initiation of the assay by the addition of AAPH was of greater value than the throughput. HTS type experiments where sample throughput is of greater importance would be served better by plate covers, which would alleviate much of the edge-effect while allowing for an increase in the number of available wells. We found that filling the outer wells with 3 µl of water resulted in more consistent data from the interior wells. Besides avoiding the use of the outer wells, where most of the discrepancies were found, the filled wells served as a significant heat mass that eliminated any temperature fluctuations. In addition, we found that reading the plate from the bottom resulted in more consistent results. The Synergy HT with injectors is an ideal instrument to perform ORAC assays. The Synergy HT Multi-Detection Microplate Reader is a robotic compatible microplate reader that can measure absorbance, fluorescence, and luminescence. The Synergy HT utilizes a unique dual optics design. The fluorescence optics is capable of measuring all plate formats up to 384- well plates. The top probe adjusts up and down automatically via software accommodating different plate heights and different sized probes are available meet specific applications. When the reader is in conventional fluorescence mode, it uses a tungsten-halogen lamp as a light source and band-pass filters in a filter wheel cartridge to provide wavelength specificity and a PMT for detection. Besides special optics, the Synergy HT has numerous features that enhance the reader s capability. Elevated temperatures are regulated by a 4-Zone microprocessor controlled system that assures superior temperature uniformity up to 5 C. The twin injectors module allows for the controlled addition of reagents to wells while the plate is in the reading chamber. This allows for precise timing between the initiation of reactions and their measurement. With a compact footprint, a robot friendly carrier design and RS-232 connection, the Synergy HT is also compatible with many of the commonly preferred robotics systems. The functionality of the Synergy HT is greatly enhanced by its controlling data reduction
9 software, KC4. In these experiments, KC4 was used to control reader function, capture the raw data to a PC, as well as perform all the necessary Net AUC calculations, plot the Trolox standard curve and interpolate it to determine sample ORAC values. Using true Object Linking and Embedding (OLE) technology, KC4 can interact directly with Microsoft Excel and Microsoft Word programs, providing total control over all report formatting using data objects created by KC4. This allows the user to create custom, publication quality reports of microplate applications. An upgrade to CFR 21 Part 11 compliant software is also available. Abbreviations ORAC Oxygen radical absorbance capacity; ROS, reactive oxygen species; AAPH, 2,2 - azobis(2-amidopropane) dihydrochloride; EGC, epigallocatechin; EGCG, epigallocatechin gallate; AUC, area under the curve; Trolox, 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid; TEAC, Trolox equivalent antioxidant capacity; FRAP, ferric reducing antioxidant power. References 1. Halliwell, B., Aruoma, O. (1991) DNA Damage by Oxygen Derived Species. Its Mechanisms and Measurement in Mammalian Systems. FEBS Lett. 281: Ames, B.N., Shigenaga, M.K., and Hagen, T.M. (1993) Oxidants, Antioxidants and the Degenerative Diseases of Aging. Proc. Natl. Acad. Sci. USA 9: Benzie, IFF, Strain, JJ. (1996) The Ferric Reducing Ability of Plasma (FRAP) as a Measure of Antioxidant Power : The FRAP Assay. Anal. Biochem. 238: Rice-Evans, C. and Miller, NJ. (1994) Total Antioxidant Status in Plasma and Body Fluids. Methods Enzymol. 234: Wayner, DDM, Burton, Gw., Ingold, KU., and Locke, S. (1985) Quantitative Measurement of the Total, Peroxyl Radical-trapping Antioxidant Capacity of Human Blood Plasma by Controlled Peroxidation. FEBS Lett. 187: Glazer, AN., (199) Phycoerythrin Fluorescence-based Assay for Reactive Oxygen Species. Methods Enzymol 186: Cao, GH., Alessio, HM.and Cutler, RG., (1993) Oxygen-radical Absorbance Capacity Assay for Antioxidants. Free Radical Biol. Med. 14: Cao, G., Verdon, C., Wu, A., Wang, H., and Prior, R. (1995) Automated Assay of Oxygen Radical Absorbance Capacity with the COBRAS FARA II. Clin. Chem. 41: Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J., and Prior, R. (22) Highthroughput Assay of Oxygen Radical Absorbance Capacity (ORAC) Using a Multichannel Liquid Handling System Coupled with a Microplate Fluorescence Reader in 96-Well Format. J. Agric. Food Chem. 5: Ou, B., Hampsch-Woodill, and Prior, R. (21) Development and Validation of an Improved Oxygen Radical Absorbance Capacity Assay Using Fluorescein as the Fluorescent Probe. J. Agric Food Chem. 49: Wu, X., Gu, L., Holden, J., Haytowitz, D., Gebhardt, S., Beecher, G., and Prior, R. (24) Development of a Database for Total Antioxidant Capacity in Foods: a Preliminary Study. J. Food Composition and Analysis. 17: Cao, G., and Prior, R. (1999) Measurement of Oxygen Radical Absorbance Capacity in Biological samples. Oxidants and Antioxidants. Methods Enzymol. 299:5-62. Paul Held Ph.D. Senior Scientist & Applications Lab Manager Trolox is a registered trademark if Hottman-LaRoche. Rev. 8/15/5
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