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A fluorescence-based method for measuring total plasma antioxidant capability

A Ghiselli1, M Serafini, G Maiani

  • 1National Institute of Nutrition, Rome, Italy.

Free Radical Biology & Medicine
|January 1, 1995
PubMed
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The Total Radical-Trapping Antioxidant Parameter (TRAP) assay in human plasma reveals that ascorbate, alpha-tocopherol, urate, and thiol groups account for up to 70% of antioxidant capacity, suggesting synergistic actions. This new fluorometric method is reliable for fresh plasma samples.

Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Human Physiology

Background:

  • Human plasma possesses significant antioxidant capacity crucial for cellular protection.
  • Accurate measurement of total antioxidant status is vital for understanding oxidative stress and disease.
  • Existing methods for measuring antioxidant parameters may have limitations in speed and reliability.

Purpose of the Study:

  • To develop and validate a new fluorometric assay for measuring the Total Radical-Trapping Antioxidant Parameter (TRAP) in human plasma.
  • To identify the primary contributors to the plasma's total antioxidant capacity.
  • To investigate potential synergistic interactions among plasma antioxidants.

Main Methods:

  • A novel fluorometric assay was employed, monitoring the peroxidation rate induced by 2,2'-diazobis (2-amidinopropane) dihydrochloride (ABAP) via the loss of R-Phycoerythrin (R-PE) fluorescence.

Related Experiment Videos

  • The lag-phase induced by plasma antioxidants was compared to that induced by Trolox, a vitamin E analogue.
  • The contribution of specific antioxidants (ascorbate, alpha-tocopherol, urate, thiol groups) to the overall TRAP was quantified.

    • Main Results:

    • Ascorbate, alpha-tocopherol, urate, and thiol groups collectively explained up to 70% of the measured TRAP in freshly prepared human plasma.
    • A significant synergistic effect was observed between ascorbate and thiol groups, where their combined inactivation resulted in a greater loss of antioxidant capacity than the sum of their individual effects.
    • The new fluorometric assay demonstrated simplicity, reliability, and suitability for rapid analysis of multiple fresh plasma samples.

    Conclusions:

    • The novel fluorometric assay provides a reliable and rapid method for determining TRAP in fresh human plasma.
    • While major antioxidants contribute significantly, other plasma components likely play a role in total antioxidant capacity.
    • Evidence suggests a notable synergistic interaction among plasma antioxidants, particularly between ascorbate and thiol groups, which enhances overall radical-trapping ability.