ABTS was dissolved in water to a 7 mM concentration.
ABTS radical cation (ABTS•1) was produced by
reacting ABTS stock solution with 2.45 mM potassium
persulfate (final concentration) and allowing the mixture
to stand in the dark at room temperature for 12–16 h
before use (Fig. 1). Because ABTS and potassium persulfate
react stoichiometrically at a ratio of 1:0.5, this
will result in incomplete oxidation of the ABTS. Oxidation
of the ABTS commenced immediately, but the absorbance
was not maximal and stable until more than 6 h
had elapsed. The radical was stable in this form for more
than two days when stored in the dark at room temperature.
For the study of phenolic compounds and food
extracts, the ABTS•1 solution was diluted with ethanol
and for plasma antioxidants with PBS, pH 7.4, to an
absorbance of 0.70 (60.02) at 734 nm and equilibrated at
30°C. Stock solutions of phenolics in ethanol, carotenoids
in dichloromethane and plasma antioxidants in
water were diluted such that, after introduction of a 10-
ml aliquot of each dilution into the assay, they produced
between 20%–80% inhibition of the blank absorbance.
After addition of 1.0 ml of diluted ABTS•1 solution
(A734nm 5 0.700 6 0.020) to 10 ml of antioxidant com-pounds or Trolox standards (final concentration 0–15
mM) in ethanol or PBS the absorbance reading was taken
at 30°C exactly 1 min after initial mixing and up to 6
min. Appropriate solvent blanks were run in each assay.
All determinations were carried out at least three times,
and in triplicate, on each occasion and at each separate
concentration of the standard and samples. The percentage
inhibition of absorbance at 734 nm is calculated and
plotted as a function of concentration of antioxidants and
of Trolox for the standard reference data. The concentration-
response curve for 5 sequentially and separately
prepared stock standards of Trolox is illustrated in Fig. 2.