Aging Aging

Assessing Antioxidant Status 135Fig. 1. Measuring concept of FRASC. This figure shows the absorbance changeowing to Fe III reduction by antioxidants in the sample. Calculation of FRAP value is bytaking the 0–4-min change in absorbance at 593 nm for test sample (closed circles, 1)and relating it to the 0–4-min absorbance change for the Fe II standard (closed triangles,2), with a reagent blank correction (open triangles, 3) for both. Calculation of ascorbicacid results is by subtracting the 0–1-min absorbance reading of the ascorbate oxidasetreatedtest sample (open circles) from the matching water-treated sample (closed circles,4); this signal is then related to that given by a standard solution of Fe II (closed triangles)(or ascorbic acid, closed squares, 5) of appropriate concentration. (Reproducedwith permission from Benzie, I. F. F. and Strain, J. J. (1997) Redox Report 3, 233–238.)mL of glacial acetic acid (BDH Laboratory Supplies, England), and make up to afinal volume of 1 L with distilled and deionized water. This solution can be storedat room temperature for up to 1 mo.2. 0.01 M TPTZ (2,4,6 tripyridyl-s-triazine, Fluka Chemicals, Switzerland) in0.04 M HCl (BDH). This solution can be stored at room temperature for up to2 wk (see Note 1).3. 0.02 M FeCl 3 . 6H 2 O (BDH). This solution can be stored at room temperature forup to 2 wk.4. 4000 U/L of ascorbate oxidase (EC 1.10.3.3) (Sigma Chemical, St. Louis, MO,USA) in distilled water. Aliquots of this solution should be stored at –70°C andthawed when required.5. To prepare working FRASC reagent, mix 20.0 mL of 0.3 M acetate buffer, pH3.6, 2.0 mL of 0.01 M TPTZ solution in 0.04 M HCl; and 2.0 mL of 0.02 MFeCl 3·6H 2 O solution just before use.