Total antioxidant performance: a validated fluorescence assay for the measurement of plasma oxidizability

Anal Biochem. 2006 Jul 15;354(2):290-8. doi: 10.1016/j.ab.2006.04.001. Epub 2006 May 2.

Abstract

The antioxidant capacity of human plasma was determined by following the oxidation kinetics of the lipid-soluble fluorescent marker BODIPY using 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) as the lipophilic radical initiator. The results are expressed as a total antioxidant performance (TAP) value based on the inhibition of BODIPY oxidation, as determined by the appearance of green fluorescence, with respect to a control sample (phosphatidylcholine with or without delipidized human serum). The suitability of the assay was evaluated on the basis of its precision, reproducibility, and specificity. The intra- and interassay coefficients of variation both were less than 5%. The addition of a representative substrate of plasma peroxidation, phosphatidylcholine, up to 750mug/ml did not induce significant changes in the TAP value. Also, BODIPY photooxidation was not observed during the experimental time course (220min). The TAP values of 6 plasma samples from healthy donors were measured and correlated with the main plasma water- and lipid-soluble antioxidants (uric acid and ascorbic acid, alpha-tocopherol, and carotenoids) and lipid profiles. Significant correlations were found between TAP and uric acid (R=0.97, P<0.05) and cholesterol-adjusted alpha-tocopherol (R=0.93, P<0.01). The results confirm that the TAP assay is suitable to measure the antioxidant activity of plasma antioxidants localized in both the lipophilic and hydrophilic compartments.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Validation Study

MeSH terms

  • Adult
  • Aged
  • Antioxidants / metabolism*
  • Azo Compounds
  • Blood Chemical Analysis / methods*
  • Boron Compounds
  • Fluorescence
  • Fluorescent Dyes
  • Humans
  • In Vitro Techniques
  • Middle Aged
  • Nitriles
  • Oxidation-Reduction
  • Photochemistry
  • Plasma / metabolism*
  • Spectrometry, Fluorescence / methods*

Substances

  • 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
  • Antioxidants
  • Azo Compounds
  • Boron Compounds
  • Fluorescent Dyes
  • Nitriles
  • V 70