Assay dilution factors confound measures of total antioxidant capacity in polyphenol-rich juices

Abstract

The extent to which sample dilution factor (DF) affects total antioxidant capacity (TAC) values is poorly understood. Thus, we examined the impact of DF on the ORAC, FRAP, DPPH, and total phenols (TP) assays using pomegranate juice (PJ), grape juice (GJ), selected flavonoids, ascorbic acid, and ellagic acid. For ORAC, GJ was comparable to PJ at DF 750, but at DF 2000, the ORAC value of GJ was 40% more than PJ. Increasing DF increased GJ and PJ, DPPH, TP, and FRAP values 11% and 14%, respectively. Increased test concentrations of quercetin and catechin resulted in 51% and 126% greater ORAC values, but decreased naringenin by 68%. Flavonoids, but not ellagic acid or ascorbic acid, may contribute to the dilution effect on the variation of final TAC values. Thus, reporting TAC or TP using a single DF may introduce uncertainty about the confidence of TAC assay values, especially when comparing different juices. These results underscore the importance of using compatible test standards for reporting TAC values.

Practical application: Total antioxidant capacity (TAC) values such as the ORAC assay are increasingly used for comparison of polyphenol-rich foods and beverages. Choice of standards and test concentrations, even within the linear range of standards, may introduce variation probably due to synergy/antagonism between antioxidant and thereby, confound final TAC values. Thus, test concentration or dilution factors of samples should be considered in the design of TAC assays and interpretation of their results.

Figure 1

A. Linear regression of ORAC area under the curve verse concentration for EA and Trolox. B. Transformation of EA to TE results in a constant value.

Figure 2

GAE of flavonoid standards assessed by the TP assay.

Figure 3

GAE or TE values of polyphenol-rich juices are constants values over a range of dilution factors in the (A) TP, (B) DPPH, (C) FRAP, and (D) ORAC assays. PJ, pomegranate juice; PNJ, pomegranate nectarine juice; GJ, grape juice.

Figure 4

The dilution factors employed in the assays had a significant effect on final TAC and TP values, (A) TP and (B) ORAC. Values between bars are the % difference of pomegranate juice. PJ, pomegranate juice; PNJ, pomegranate nectarine juice; GJ, grape juice. P-values for 2-tailed t-test comparisons for TP are P = 0.0003 for 20/20 DF; P < 0.0001 for 120/20 DF; P < 0.0001 for 20/120 DF; P = 0.0051 for 120/120 DF; and for ORAC are P = 0.5467 for 750/750 DF; P = 0.0016 for 2000/750 DF; P = 0.0799 for 750/2000 DF; P = 0.0446 2000/2000 DF.

Figure 5

Potential strategies to report TAC as TE in the ORAC assay for fruit juices. EC: effective concentration, defined as the assay response (in TE) of a sample at a predetermined standard concentration, for example, 20 μmol/L Trolox. EC_n is the TAC of a test sample, which is the multiplication product of a predefined Trolox concentration (n) and the calculated dilution factor. The calculated dilution factor is obtained using a regression plot of raw responses of at least three diluted samples falling in the range of the standard curve and corresponding dilution factors when the response of the diluted sample at the calculated dilution factor is equal to the target response of a predefined Trolox concentration. DF, Dilution Factor; PJ, pomegranate juice; PNJ, pomegranate nectarine juice; GJ, grape juice.