Protein–Incorporated Serum Total Antioxidant Capacity Measurement by a Modified CUPRAC (CUPRIC Reducing Antioxidant Capacity) Method

Abstract

Due to inadequacies in analytical methodology of total antioxidant capacity (TAC) measurement, proteins are initially separated from the human serum matrix by precipitation and are left unmeasured, thereby causing an important “antioxidant gap.” The aim of this work is to measure the TAC of serum with the modified CUPric Reducing Antioxidant Capacity (CUPRAC) method, and to identify the contribution of serum proteins, especially thiol-containing proteins, to TAC. CUPRAC results were statistically compared to those found by reference methods, namely ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), FRAP (ferric reducing antioxidant power), and Ellman thiols assay. The curves of absorbance vs thiol concentration, as well as of absorbance vs diluted serum (whole serum, trichloroacetic acid (TCA)−precipitated and redissolved serum protein solution, and TCA supernatant fractions) volume, of three distinct serum samples showed excellent linearity and low intercept values only with the modified CUPRAC method. The proposed method will help characterize the “antioxidant gap” of serum TAC originating from protein components which should not be neglected in future antioxidant measurements.

Keywords:

• Ellman method
• Human serum antioxidants
• Modified CUPRAC method
• Protein thiols
• Total antioxidant capacity

Acknowledgments

One of the authors (Nilay Kara) would like to thank Istanbul University Research Fund for the support given to her M.Sc. thesis Project T - 6475.

Notes

*Because serum protein solution and supernatant caused turbidity during the experiments, FRAP method could be applied only to whole serum.

**Ellman method of thiol quantitation basically does not respond to antioxidants other than thiols.

***(N.D): Not detected.

¹The modified CUPRAC dilution curve of 1:5 (one to five volumes) diluted whole serum (WS), serum protein solution (SPS), supernatant (S); (V_final = 2.0 mL; N = 5).

²The ABTS dilution curve of 1:5 diluted whole serum (WS), serum protein solution (SPS), supernatant (S); (V_final = 2.0 mL; N = 5).

³The FRAP dilution curve of 1:1 diluted whole serum (WS); (V_final = 3.4 mL; N = 5).

TAC = x¯ ± (t_.95 s/); N = 5 (x¯: mean, s: standard deviation).

*Because serum protein solution and supernate caused turbidity during the experiments.

FRAP method could only be applied to whole serum.