Comparison Of two Analytical Methods Used for the Measurement of Total Antioxidant Status

Background: Antioxidants play an important role in maintenance of human health and prevention of disease. Effective supplementation of antioxidants requires laboratory monitoring of antioxidant status. An understanding of the methods used to determine the TAS helps in better interpretation of values obtained using a particular method and also to select a suitable method. Material and Methods: Forty subjects including 25 healthy volunteers and 15 patients diagnosed with rheumatoid arthritis were studied. All samples were analysed for TAS using Ferric reducing ability of plasma (FRAP) method and Trolox equivalent antioxidant capacity (TEAC) assay. Results: Mean TAS values obtained by TEAC method were higher than those obtained by FRAP method (p<0.0001); no difference was observed when TEAC values were corrected for proteins and FRAP values were corrected for uric acid (p=0.420). No correlation was found between TEAC and FRAP methods (p=0.102). However, when TEAC was corrected for proteins, positive correlation was observed with FRAP (p=0.044). There was agreement between the two methods when TEAC values were corrected for proteins. Conclusion: Although the reaction conditions differ, similar compounds react in both the assays and thus TEAC and FRAP assays are comparable. However, the two methods differ with respect to –SH groups and uric acid contributions. This contributes to the higher TAS values obtained by TEAC assay. Thus, in conditions with altered protein or uric acid levels, the two methods may not be used interchangeably. The TEAC assay is to be corrected for protein for comparison of reports of the two assays. DOI : 10.14302/issn.2471-2140.jaa-14-617 Corresponding author: Dr P.V.L.N. SrinivasaRao, , Professor and Head,, Department of Biochemistry, Sri Venkateswara Institute of Medical Sciences, , Tirupati 517507. India, Email: seenupvln@yahoo.com Running Title: Comparison of methods for serum TAS


Introduction
Cells, tissues and body fluids have developed effective antioxidant defence systems that help to counteract the oxidative challenge posed by potentially harmful free radicals which are generated continuously during cellular metabolism.To match with the diversity of the prooxidants, the antioxidant defence mechanism comprises various components which include enzymatic and nonenzymatic antioxidants (1).Superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase are important enzymatic antioxidants whereas albumin, ascorbic acid, glutathione, uric acid, tocopherol, carotenoids comprise major non-enzymatic antioxidants (2).These antioxidant molecules protect the body from free radical induced damage by preventing formation of free radicals, scavenging them or by promoting their decomposition (3).An increase in oxidants and/or a decrease in antioxidants can shift the balance towards a state of oxidative stress that has been implicated in over 100 disorders (4).In this context, the role of antioxidants in the maintenance of human health and prevention of diseases has attracted attention as some of them can be supplemented.Therapeutic interventions have to aim at either decreasing the exposure of patient to reactive metabolites or supplementing with antioxidants, to counter the oxidative stress.Antioxidant supplementation, to be effective, needs laboratory monitoring of antioxidant status, which further helps in evaluating the effect of treatment on plasma redox status (5) and the ability of an individual to withstand the oxidative stress.Thus, the main hindrance for antioxidant therapy is the need to monitor several parameters.
Measuring individual parameters and making necessary specific supplementations is a tedious process that may not be a clinically viable option.So the aim would be to consider methods that measure the combined antioxidant status of the individual rather than individual parameters.
Measured TAS of a sample depends on the method used for its estimation.The methods differ with respect to the components measured.Moreover, various components of TAS are affected in different clinical situations implicated in causing oxidative stress such as uric acid in chronic kidney disease.Hence, there is a need for the identification of suitable method for the measurement of TAS.Comparison of different analytical methods for TAS measurement constitutes an important factor to select a convenient method and also to understand and interpret the results obtained using a particular method for TAS estimation as well as its suitability to monitor antioxidant supplementation.In this background, we evaluated two commonly used methods, the FRAP and TEAC assays for estimation of TAS with respect to the major components measured, clinical utility and comparative interpretation of results.

Results:
The intra assay coefficient of variation by FRAP and TEAC assays was 0.8% and 2.4% respectively.The inter assay coefficient of variation obtained by FRAP and TEAC assays was 3.5% and 4.5% respectively.Mean TAS values obtained by TEAC method were found to be significantly higher than those obtained by FRAP method (p<0.0001).However, when TAS values by TEAC assay were corrected for total proteins and TAS values by FRAP method were corrected for uric acid, no significant difference was observed (p=0.420)(Table 1).The range of values for TEAC and FRAP with and without correction for total protein and uric acid were shown in Figure 1.This shows that the antioxidant activity measured by these parameters is almost the same except for total protein and uric acid (Figure 1D).

Discussion:
The present study compared FRAP assay with TEAC assay for assessment of serum antioxidant capacity.The mean total antioxidant status (TAS) of the samples determined by TEAC method was found to be significantly higher than that obtained by FRAP method (p < 0.0001).Serum contains different antioxidant compounds and the TAS of a sample depends upon the method employed for its measurement as the methods differ in their ability to measure different components of the serum that contribute to the total antioxidant When we further evaluated for the agreement between FRAP and TEAC assays using Bland Altman plot and ICC, we could not observe good agreement (Figure 2-A) between the two methods.This is expected and can be explained based on the fact that the parameters measured by the two methods are different.However, TAS values by TEAC method corrected for total proteins showed better agreement with TAS values by FRAP method (Figure 2-B).This indicates that the main difference between the two assays is measurement of proteins and the antioxidant status measured by both assays is comparable.Moreover, although the reaction conditions differ, since the reduction potential of Fe 3+ -TPTZ and ABTS .*are comparable, similar compounds react in both the assays (17).Hence, we have included these two assays for comparison.However, in conditions where uric acid, one of the main components measured by FRAP method, is altered, measurement of TAS using TEAC method may be beneficial; likewise, in conditions affecting total protein levels, FRAP method may be useful.

Conclusion
The mean TAS values obtained by FRAP method were lower than those obtained by TEAC method as the FRAP method does not measure the -SH containing antioxidants, mainly contributed by proteins.Moreover, although the reaction conditions differ, since the reduction potential of Fe 3+ -TPTZ and ABTS .*are comparable, similar compounds react in both the assays (17).The FRAP assay is simple and inexpensive but does not measure -SH containing antioxidants.The TEAC assay measures the antioxidative effects of -SH containing antioxidants also.Thus, although both FRAP and TEAC methods are comparable and can be used for measuring the anti oxidant capacity, the two methods may not be used interchangeably when there are alterations in protein and uric acid levels.If reports from these two methods have to be matched for any reason, TEAC corrected for total proteins can be used to compare with FRAP values.
Several methods have been developed for the measurement of TAS along with their automation.The most widely used methods are based on colorimetric, fluorescence and chemiluminiscence assays (9-11).The fluorescence and chemiluminiscence methods require sophisticated techniques and are not commonly employed in most routine biochemistry laboratories (12).
No correlation was observed between TAS values by TEAC and FRAP methods (r=0.262,p=0.102).However, TAS values by TEAC method corrected for total proteins showed significant positive correlation with FRAP (r=0.320,p=0.044).Significant positive correlation was also observed between TAS values obtained by TEAC and Total proteins (r=0.327, p=0.039), and both FRAP and TEAC showed significant positive correlation with UA (r=0.485,p=0.001 and r=0.335, p=0.034 respectively) Table2.TAS values by TEAC showed poor agreement with FRAP as assessed using Bland Altman plot and ICC (Figure2-A, ICC=0.1975);also, TAS values by TEAC corrected for total proteins showed poor agreement with TAS values by FRAP corrected for uric acid (Figure 2-C, ICC= -0.0591).However, when TAS values by TEAC were corrected for total proteins, the agreement with FRAP improved (Figure 2-B, ICC=0.4450).

Figure1.
Figure1.Dot plots of the parameters studied