Q: Can you please explain the difference between the ORAC, HORAC, and TAC assays and why I would select one over the other?
A: All three assays measure antioxidant capacity but are slightly different. The ORAC and HORAC assays are both fluorometric assays that use HAT chemistry to detect oxidation of a fluorescent probe. The main difference between the two assays is the free radicals that are generated that oxidize the probe. The ORAC assay uses a free radical initiator to produce peroxyl radicals, while the HORAC assay uses a hydroxyl radical initiator and fenton reagent to produce hydroxyl radicals (HORAC = hydroxyl radical antioxidant capacity, ORAC = oxygen radical antioxidant capacity). The TAC assay is a colorimetric assay that uses a different chemistry than the ORAC and HORAC, specifically a SET mechanism that is based on copper reduction by antioxidants. The ORAC and HORAC assays are more sensitive than the TAC assay, but they are more technically demanding in both assay protocol and data analysis.
The choice between assays is dependent upon the chemistry that is most applicable to the research being performed (ex: SET vs. HAT), or the free radical generated (ex: hydroxyl vs. peroxyl). Researchers tend to prefer ORAC over HORAC because peroxyl radicals appear to be more relevant than hydroxyl radicals.
Q: Is it possible to test samples that have been stored frozen?
A: Two months at -80ºC generally the longest most antioxidant samples should be stored, but we recommend testing fresh samples wherever possible for the best results.
Q: Is this assay compatible with plant samples?
A: The HORAC Activity Assay is compatible with plant samples. Plant samples can be prepared using the guidelines in the product manual for tissue lysates: Sonicate or homogenize tissue sample on cold PBS and centrifuge at 10,000 x g for 10 minutes at 4ºC. Aliquot and store the supernatant for use in the assay.
With samples that contain many different antioxidants, such as plants or food, we recommend preparing both hydrophilic and lipophilic fractions because not all antioxidants will be soluble in each fraction.
Q: Are urine samples compatible with this assay?
A: Urine can be used with the HORAC Assay. Because the antioxidant capacity is low in urine, it should first be tried undiluted.
Q: Will this detect antioxidants in the cell culture media?
A: The HORAC Assay can be used to determine the antioxidant capacity of a variety of sample types, including cell culture supernatants. A media only control should be used to determine the background interference that results from the media itself.
Q: Does hydrophilic/lipophilic refer to the samples or the antioxidants as described in the product manual?
A: Sample preparation is based on the solubility properties of the sample. With samples that contain many different antioxidants, such as plants or food, we recommend preparing both fractions because not all antioxidants will be soluble in each fraction.
Q: Why is a dilution recommended for serum but not for tissue lysates?
A: The reason for this is due to sample preparation, not antioxidant concentrations. There is only one way to harvest serum, making serum protein concentrations consistent and therefore easy to recommend a dilution that always works well. Tissue lysates, on the other hand, have multiple protocols for preparation that produce variable protein concentrations. Because there is so much variability between lysate protocols, we can’t recommend a dilution that will work in all cases. When working with lysates, we recommend first trying undiluted samples, then adjust dilutions if necessary using the Assay Diluent.
Q: How do I test powdered substances?
A: Sample preparation will depend on the solubility properties of your powder samples. For the hydrophilic fractions, you can dilute your powder samples using the Assay Diluent provided with the kit. For lipophilic fractions, you will dissolve samples in 100% acetone, dilute in 50% acetone, and incubate 1 hour at room temperature with mixing. Then further dilute samples as necessary (see page 5 of the product manual). Both fractions will require testing to determine the optimal dilutions.
Q: My powder sample isn’t dissolving in acetone.
A: We recommend trying a different solvent, such as ethanol, to try to get your powder to dissolve. Other options include methanol, chloroform, or hexane, but you will have to find the right solvent that works for your sample. If the sample is very lipophilic and still doesn’t go into solution right away, it may be necessary to dilute it down and apply heat to get it to dissolve.
Q: How do I analyze my results?
A: Here are the steps for analyzing the results:
1. For each concentration, determine the AUC:
AUC= 1 + (RFU value Time5/RFU value time0) + (RFU value Time10/RFU value time0) + .etc.
This is done for the blank, standard, and samples.
2. Then determine the Net AUC based on the calculated AUCs:
Net AUC = AUC (standard) - AUC (blank)
This will give you one value for each Trolox concentration, which you will then plot as Net AUC vs. Trolox (uM).
3. This graph will then be used to analyze sample concentrations by using the equation of the line of the curve (y=mx+b). To determine the equivalent trolox concentration of the sample, you will solve for y in the equation: (Sample Net AUC-b)/m. To determine TE/L, you will multiple this number by the dilution factor used (ex: 9.68 x 10 dilution = 96.8uM TE = 96.8 uMole TE/L.
Q: Why is the blank reading decreasing spontaneously compared to the standard?
A: In the absence of antioxidants, free radicals generated by AAPH will continuously attack the fluorescent probe and result in a decrease in signal. When antioxidants are present, they will quench the free radicals generated by AAPH, resulting in less free radicals available to attack the fluorescent probe. The fluorescence will drop at a slower rate in samples with antioxidants compared to samples without antioxidants.