Insights on Antioxidant Assays for Biological Samples Based on the Reduction of Copper Complexes—The Importance of Analytical Conditions
Abstract
:1. Introduction
2. Results and Discussion
2.1. Complexing Agent Selection
2.2. Reagents Addition Order
2.3. Trolox Equivalent Value
TE | Ascorbic Acid | Trolox | Uric Acid | Glutathione |
---|---|---|---|---|
TE(BCS) | 1.15 ± 0.02 | 1.00 ± 0.01 | 1.90 ± 0.02 | 0.62 ± 0.02 |
TE(NC) | 0.90 ± 0.03 | 1.00 ± 0.01 | 1.96 ± 0.02 | 0.54 ± 0.01 |
TE(BCA) | 1.03 ± 0.03 | 1.00 ± 0.04 | 2.46 ± 0.09 | 0.57 ± 0.02 |
[AA] µM | [AU] µM | [GSH] µM | TE Expected | TE Found Average ± SD | RD% a |
---|---|---|---|---|---|
10 | 10 | 10 | 0.037 | 0.038 ± 0.000 | 3.0 |
10 | 10 | 50 | 0.065 | 0.066 ± 0.000 | 1.7 |
10 | 50 | 10 | 0.111 | 0.112 ± 0.001 | 0.5 |
10 | 50 | 50 | 0.139 | 0.142 ± 0.001 | 1.8 |
50 | 50 | 10 | 0.157 | 0.158 ± 0.004 | 0.9 |
50 | 50 | 50 | 0.185 | 0.188 ± 0.001 | 1.9 |
50 | 10 | 10 | 0.082 | 0.085 ± 0.003 | 2.7 |
50 | 10 | 50 | 0.110 | 0.114 ± 0.001 | 2.9 |
2.4. TE of Biological Antioxidants in Mixtures
2.5. Buffer Solutions
Buffer | Ascorbic Acid | Trolox | Uric Acid | Glutathione |
---|---|---|---|---|
Ammonium acetate, pH 7.0 | 1.148 ± 0.024 | 1.000 ± 0.008 | 1.899 ± 0.013 | 0.624 ± 0.018 |
Tris-glycine urea, pH 7.0 | 0.813 ± 0.040 | 1.000 ± 0.050 | 0.983 ± 0.049 | 0.552 ± 0.028 |
PBS, pH 7.4 | 0.991 ± 0.044 | 1.000 ± 0.050 | 0.766 ± 0.038 | 0.668 ± 0.033 |
2.6. Sample Analysis and Validation
Nominal Conc. (µM) | Ascorbic Acid (µM) | Uric Acid (µM) | Glutathione (µM) | Trolox (µM) |
---|---|---|---|---|
10 | 10.0 ± 0.7 | 10.0 ± 0.6 | 9.9 ± 0.5 | 10.5 ± 0.6 |
20 | 20.3 ± 0.9 | 20.0 ± 0.7 | 21.8 ± 0.7 | 20.2 ± 0.7 |
40 | 39.8 ± 2.0 | 39.6 ± 1.7 | 42.0 ± 0.9 | 40.6 ± 0.7 |
60 | 59.3 ± 2.6 | 60.3 ± 1.9 | 61.0 ± 1.3 | 61.0 ± 1.2 |
100 | 99.9 ± 2.8 | 100 ± 1.5 | 97.9 ± 1.9 | 100.9 ± 1.1 |
Sample | Absorbance | TEAC (µM) | Recovery% | ||
---|---|---|---|---|---|
Added Uric Acid Concentration | Trolox Equivalent (µM) | ||||
0 µM | 25 µM | 0 µM | 44.1 µM | ||
S1 | 0.260 ± 0.002 | 0.524 ± 0.006 | 25.7 ± 0.2 | 66.7 ± 0.8 | 93.0 |
S2 | 0.271 ± 0.004 | 0.518 ± 0.006 | 27.4 ± 0.4 | 66.1 ± 0.7 | 87.9 |
S3 | 0.235 ± 0.005 | 0.493 ± 0.004 | 21.6 ± 0.4 | 62.2 ± 0.5 | 92.0 |
S4 | 0.265 ± 0.003 | 0.511 ± 0.005 | 26.4 ± 0.3 | 65.1 ± 0.6 | 87.7 |
S5 | 0.275 ± 0.004 | 0.520 ± 0.005 | 27.9 ± 0.4 | 66.5 ± 0.7 | 87.6 |
3. Experimental Section
3.1. Materials
3.2. Instrumentation
3.3. Validation and Sample Analysis
4. Conclusions
- An additive response should be provided for the targeted group of antioxidants in the applied concentration range, because as long as the additivity is verified, the nature of the used Cu complexing agent does not seem to influence the results;
- Additive response is provided as long as the oxidant complex is maintained in molar excess considering the electron transfer processes;
- Selection of the buffering system affects more the TE values that the complexing agent used, and this is related to the electron transfer mechanisms of the antioxidants;
- The selection of buffer solution is also important to avoid measurement artefacts due to protein precipitation in biological samples.
Acknowledgments
Author Contributions
Supplementary Files
Supplementary File 1Supplementary Information
Conflicts of Interest
References
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Marques, S.S.; Magalhães, L.M.; Tóth, I.V.; Segundo, M.A. Insights on Antioxidant Assays for Biological Samples Based on the Reduction of Copper Complexes—The Importance of Analytical Conditions. Int. J. Mol. Sci. 2014, 15, 11387-11402. https://doi.org/10.3390/ijms150711387
Marques SS, Magalhães LM, Tóth IV, Segundo MA. Insights on Antioxidant Assays for Biological Samples Based on the Reduction of Copper Complexes—The Importance of Analytical Conditions. International Journal of Molecular Sciences. 2014; 15(7):11387-11402. https://doi.org/10.3390/ijms150711387
Chicago/Turabian StyleMarques, Sara S., Luís M. Magalhães, Ildikó V. Tóth, and Marcela A. Segundo. 2014. "Insights on Antioxidant Assays for Biological Samples Based on the Reduction of Copper Complexes—The Importance of Analytical Conditions" International Journal of Molecular Sciences 15, no. 7: 11387-11402. https://doi.org/10.3390/ijms150711387