[논문]Validation of Analytical Methods for Plasma Total Antioxidant Capacity by Comparing with Urinary 8-Isoprostane Level

Lee, Sang Gil; Wang, Taoran; Vance, Terrence M.; Hurbert, Patrice; Kim, Dae-Ok; Koo, Sung I.; Chun, Ock K.

doi:10.4014/jmb.1604.04053

[국내논문] Validation of Analytical Methods for Plasma Total Antioxidant Capacity by Comparing with Urinary 8-Isoprostane Level 원문보기

Journal of microbiology and biotechnology, v.27 no.2, 2017년, pp.388 - 394

Lee, Sang Gil (Department of Nutritional Sciences, University of Connecticut) , Wang, Taoran (Department of Nutritional Sciences, University of Connecticut) , Vance, Terrence M. (Department of Nutritional Sciences, University of Connecticut) , Hurbert, Patrice (Department of Nutritional Sciences, University of Connecticut) , Kim, Dae-Ok (Department of Food Science and Biotechnology, Kyung Hee University) , Koo, Sung I. (Department of Nutritional Sciences, University of Connecticut) , Chun, Ock K. (Department of Nutritional Sciences, University of Connecticut)

Abstract ▼ AI-Helper

Although several analytical methods for measuring total antioxidant capacity (TAC) have been applied to biological samples, there were often dissimilar results due to the different principles of methods applied. Thus, this study aimed to validate four conventional analytical methods for measuring plasma TAC, including the ABTS assay, DPPH assay, FRAP assay, and ORAC assay, by comparing with urinary 8-isoprostane concentration. In addition, TAC results were compared with antioxidant enzyme activities including superoxide dismutase (SOD) and glutathione peroxidase in erythrocyte, and catalase in plasma. Plasma TAC measure by ABTS assay was strongly correlated with the result by FRAP assay. Plasma TAC by FRAP and ORAC assays were negatively correlated with erythrocyte SOD activity. The agreement among the four TAC assay methods and 8-isoprostane was determined using 95% prediction limits of linear regression, expressed as the mean of 8-isoprostane ${\pm}95%$ prediction limits. The ABTS method better agreed with 8-isoprostane than the other methods, demonstrating narrow prediction of limits. Furthermore, only plasma TAC determined by the ABTS assay was inversely correlated with urinary 8-isoprostane (r = -0.35, p < 0.05). In summary, the ABTS assay would be an appropriate method to measure overall plasma antioxidant capacity and predict the body's antioxidant status.

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문제 정의

The aim of this study was to evaluate the compatibility of the four conventional TAC assay methods (i.e., ABTS, DPPH, FRAP, and ORAC), using human plasma. This study also tested the validity of the assays in predicting the body’s oxidative stress status by comparing the results with urinary 8-isoprostane levels.

제안 방법

Blood samples after overnight fasting and urine samples collected during the overnight period, from 45 subjects who participated in an intervention study on the effects of a chokeberry supplement in former smokers, were used to measure TAC by the ABTS, DPPH, FRAP, and ORAC assays. Plasma samples were isolated from baseline blood samples in the study by centrifugation for 15 min at 2,000 ×g.
SAS was used to evaluate the compatibility between plasma TAC measured by each assay and 8-isoprostane. In order to validate the analytical methods for plasma TAC, the agreement of the four TAC assay methods with urinary 8-isoprostane level was determined by comparing the mean and 95% prediction limits for 8-isoprostane from linear regression; the narrower the prediction limits, the better agreement with the urinary 8-isoprostane level [18]. The 95% prediction limits were calculated as #, where MSE is the mean squared error from linear regression [18, 19].
One study determined the correlation between plasma ORAC values with a 70 min reaction time and trolox equivalent antioxidant capacity (TEAC) results at different time points measured by ABTS assay. In this study, a sufficient reaction time in the ABTS assay (30 min) led to a significant correlation with the ORAC assay. However, TEAC values with less than 5 min reaction did not show significant correlation with ORAC values [26].
This study also tested the validity of the assays in predicting the body’s oxidative stress status by comparing the results with urinary 8-isoprostane levels.
Plasma samples were isolated from baseline blood samples in the study by centrifugation for 15 min at 2,000 ×g. Urine samples corresponding to plasma samples were used to investigate the agreement between TAC by each of the four methods with urinary 8-isoprostane concentrations. After the plasma was collected, erythrocytes were mixed with 4 volumes of cold water and centrifuged at 10,000 ×g for 15 min to prepare erythrocyte lysate for antioxidant enzyme measurement.

대상 데이터

ABTS, 2 2’-azobis(2-amidinopropane) dihydrochloride (AAPH), methanol, DPPH, 2,4,6-tri[2-pyridyl]-s-triazine (TPTZ), ferric chloride, sodium acetate, glacial acetic acid, sodium chloride, potassium chloride, sodium phosphate dibasic, potassium phosphate monobasic, sodium phosphate monobasic, fluorescein, Trolox, and L-ascorbic acid were purchased from Sigma-Aldrich (USA).

데이터처리

SAS was used to evaluate the compatibility between plasma TAC measured by each assay and 8-isoprostane. In order to validate the analytical methods for plasma TAC, the agreement of the four TAC assay methods with urinary 8-isoprostane level was determined by comparing the mean and 95% prediction limits for 8-isoprostane from linear regression; the narrower the prediction limits, the better agreement with the urinary 8-isoprostane level [18].

이론/모형

The 95% prediction limits were calculated as #, where MSE is the mean squared error from linear regression [18, 19]. Correlations between the TAC assay methods, antioxidant enzyme activities, and urinary 8-isoprostane level were analyzed using the Pearson Correlation method.

성능/효과

The prediction interval of the mean of measured TAC via the ABTS, DPPH, FRAP, and ORAC assays corresponding to the 8-isoprostane level were 66.8 ± 70.2 ng/ mmol creatinine for ABTS, 66.8 ± 72.4 ng/ mmol creatinine for DPPH, 66.8 ± 74.8 ng/ mmol creatinine for FRAP, and 66.8 ± 73.3 ng/ mmol creatinine for ORAC assays, respectively.

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