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Technical Briefs |
Departments of 1 Obstetrics & Gynaecology and 3 Anaesthesia & Intensive Care, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong2 Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, University Eye Centre, Hong Kong Eye Hospital, Kowloon, Hong Kong
aaddress correspondence to this author at: 1st Floor, Block E, Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong; fax 852-2636-0008, e-mail ccwang{at}cuhk.edu.hk
Because of difficulty in measuring each antioxidant component separately and interactions among antioxidants, methods have been developed to assess the total antioxidant capacity of serum or plasma. The 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)-equivalent antioxidant capacity (TEAC) assay (1), the oxygen radical absorbance capacity (ORAC) assay (2), and the ferric reducing ability of plasma (FRAP) assay (3) are commonly used and have been extensively evaluated. Although comparable mean results have been obtained with the TEAC and ORAC assays (4)(5), no correlation has been found between ORAC and TEAC values or between FRAP and TEAC values (6). This lack of correlation between TEAC and other assays is likely attributable to underestimation of overall antioxidant capacity. Underestimation may be related to the effects of dilution (7) and to premature measurement of inhibition percentage at a fixed time of 3 min (6). In fact, both the ORAC and TEAC assays are inhibition methods: a sample is added to a free- radical-generating system, and the inhibition of the free radical action is measured. This inhibition is related to the antioxidant capacity of the sample. In addition, both assay methods measure antioxidants in serum or plasma proteins, including albumin (6). In this study we investigated the performance of the TEAC assay, modified the procedure, and then reevaluated the TEAC for comparison with the ORAC assay.
The TEAC assay, commercialized by Randox Laboratories Ltd., is based on the suppression of the absorbance of radical cations of 2,2'-azinobis(3-ethylbenzothiazoline 6-sulfonate) (ABTS) by antioxidants in the test sample when ABTS incubates with a peroxidase (metmyoglobin) and H2O2 (1). If the inhibition time is fixed at 3 min, as stated in the manufacturer’s instructions, the added antioxidants quench ABTS radicals in a nonlinear dose–response fashion (6). To optimize the incubation period for the complete inhibition of ABTS radical formation in the system, we extended the reaction up to 40 min and monitored the absorbance changes at 3-min intervals at 600 nm (Fig. 1A
). A reaction mixture containing 20 µL of H2O2 (100 µmol/L), 100 µL of metmyoglobin (6.1 µmol/L), ABTS (610 µmol/L), and 4 µL of Trolox (concentration range, 0–1.65 µmol/L) was incubated and subjected to spectrophotometry (µQuant; Bio-Tek Instruments) at 37 °C. We found that the formation of ABTS radicals increased in proportion to the incubation period until a plateau was achieved after 30 min. The higher the concentration of Trolox used in the reaction mixture, the more the absorbance of ABTS radicals was suppressed. There was a lag time, reported as the time needed for the rate of production of chromogen ABTS radicals to stabilize (8). If 1.65 µmol/L Trolox was incubated in the mixture, no ABTS radicals were detectable until after 15 min. The detection limit was 0.825 µmol/L at 9 min, 0.413 µmol/L at 6 min, and 0.206 µmol/L at 3 min. The dose–response curve of standard Trolox confirmed that the reaction is nonlinear if the incubation time is fixed at 3 min. The nonlinear dose–response relationship persisted even if the incubation period was extended to 21 min; the relationship became linear only when incubation was extended beyond 24 min (R2 >99.5%).
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To test the accuracy of the TEAC assay, we repeatedly measured the TEAC value of control serum from Randox (1.36 mmol/L; lot no. 144NX) without dilution for different incubation periods. When the end-point measurement was fixed at 3 min, the variation in TEAC values was >0.004 mmol/L (SD, 0.063 mmol/L; CV = 3.7%) for five repeated within-batch assays. The lowest variance (<0.001 mmol/L) and CV (<1.5%) were observed when the reaction mixture was incubated >24 min. The best performance was established when 30 min was used as the end-point of the TEAC assay: the assay was linear (R2 = 0.9952; y = –0.2788x + 0.1469 mmol/L; variance <0.0001 mmol/L; SD, 0.015 mmol/L; CV = 1.1%). This performance was maintained even when the control serum was diluted up to fourfold (from 1.36 to 0.17 mmol/L).
The ORAC assay is based largely on the work reported by Glazer (9) and modified by Cao and Prior(10), in which the decrease in fluorescence of B- or R-phycoerythrin (PE) is measured in the presence of 2,2'-azobis(2-amidinopropane) dihydrochloride, and the lag phase or rate constant for PE fluorescence decay is used to determine the antioxidant capacity of the added sample. The ORAC method is the only method that takes free radical action to completion and uses the area under the curve for quantification; it thus combines both the percentage of inhibition and the length of inhibition of free radical formation by antioxidants into a single quantity (11). We considered 30 min of inhibition as an equivalent end-point equilibrium in our modified TEAC protocol.
We compared plasma total antioxidant capacity as measured by the TEAC and ORACs assay in the same plasma samples from adult rats administered decaffeinated green tea extract (SUNPHENON DCF-1®; Taiyo Kagaku Co. Ltd.; Fig. 1B
). The Animal Research Ethics Committee of the University approved the experiments. A single dose of SUNPHENON (reconstituted to give a dose of 53.7 mg/kg (–)-epigallocatehin-3-gallate) was given to acclimatized and fasted adult female rats via a sterile gastric tube. Blood samples were collected at 0, 0.5, 1, 2, 3, 5, and 8 h after administration. Total plasma ORAC was measured as described previously (10): 10 µL of 2,2'-azobis(2-amidinopropane) dihydrochloride (160 mmol/L; Wako) was added to a reaction mixture containing 300 µL of B-PE (3.8 mg/L; Sigma) and 20 µL of Trolox (concentration range, 0–100 µmol/L; Sigma) or whole plasma (1:200 dilution) and incubated in a dual-scanning spectrofluorometer (GEMINI SpectraMAX; Molecular Devices) at 37 °C. The PE fluorescence decay was monitored at 2-min intervals for 70 min (excitation, 546 nm; emission, 565 nm). ORAC values were calculated by use of the areas under the curves with SOFTmax PRO (Molecular Devices). TEAC values at 30 min were significantly correlated with ORAC values over 70 min (Pearson correlation, r = 0.453; P <0.01). The correlation was not significant if TEAC values obtained after 3 min of incubation were used (Pearson correlation, r = 0.371; P = 0.133). The TEAC values at 30 min in our experiments were 98% of the ORAC values (6.0–10.5 mmol Trolox-equivalents/L for TEAC; 7.0–9.5 mmol Trolox equivalents/L for ORAC) compared with TEAC values of only 26–50% of the ORAC values at 3 min (4)(5). Both the TEAC and ORAC assays revealed a significant 35–40% increase in total plasma antioxidant capacity (P <0.05, ANOVA) 1 h after SUNPHENON administration in rats. There was no significant difference between estimates of change in total plasma antioxidant capacity as measured by the TEAC or ORAC method.
Despite modification of the TEAC assay to improve agreement with the ORAC assay, the correlation of the two assays was not high (Fig. 1B
). The correlation was higher at higher TEAC and ORAC values (8.0 mmol Trolox-equivalents/L for both). A poor correlation may be expected because different free radical sources are used in the two methods. The TEAC assay uses exogenous ABTS radicals, whereas the ORAC assay uses peroxyl radicals. Because peroxyl radicals are the most common radicals found in the human body, ORAC measurements should be more biologically relevant. On the other hand, different antioxidants respond differently in different measurement methods. For example, uric acid in serum contributes 19% in TEAC but only 7% in ORAC assays, whereas 
-tocopherol, ascorbic acid, and bilirubin contribute more in the TEAC assay than in the ORAC assay (6). Meanwhile, it is still possible that further optimization could improve the analytical performance of both methods.
In summary, this report demonstrates that the length of the inhibition time for the TEAC assay must be taken into account when determining the total antioxidant capacity of plasma. Under our conditions, we found that 30 min of inhibition was required for complete suppression of ABTS radical formation in the TEAC assay. We validated the results from the TEAC assay at 30 min and confirmed that values were similar and correlated with the results obtained by the ORAC assay over 70 min.
Acknowledgments
This study was partially supported by the Research Grants Council of the Hong Kong Special Administrative Region (Grants CUHK4077/01M, CUHK4081/01M, and CUHK4060/02M). We thank Taiyo Kagaku Co. Ltd. (Tokyo, Japan) for the generous gift of SUNPHENON DCF-1 green tea extract tablets.
References
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, 30 min (R2 = 0.9952); 
, 27 min (R2 = 0.9958);
, 24 min (R2 = 0.9955); x, 21 min (R2 = 0.9932); 
, 18 min (R2 = 0.9838); •, 15 min (R2 = 0.9560); +, 12 min (R2 = 0.8775); 
, 9 min (R2 = 0.7798);
, 6 min (R2 = 0.6569);
, 3 min (R2 = 0.4977). (B), results of the TEAC reaction at 30 min are significantly correlated with the results of ORAC incubation over 70 min. Solid line, Pearson correlation (y = 0.9876x + 0.5877 mmol Trolox-equivalents/L; r2 = 0.2053; P = 0.009); dotted line, Lowess regression. (Inset), changes in rat plasma total antioxidant capacity (n = 5 x 7 time points) after consumption of SUNPHENON DCF-1 green tea extract. Error bars, SE.