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Solutions to decrease a systematic error related to AAPH addition in the fluorescence-based ORAC assay.

Elena Mellado-Ortega1, Iñigo Zabalgogeazcoa1, Beatriz R Vázquez de Aldana1

  • 1Departamento de Estrés Abiótico, Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Cordel de Merinas 52, 37008 Salamanca, Spain.

Analytical Biochemistry
|December 15, 2016
PubMed
Summary

The Oxygen Radical Absorbance Capacity (ORAC) assay can have errors due to inconsistent initiator AAPH dispensing. We propose two methods to improve the accuracy of total antioxidant capacity (TAC) measurements in biological samples.

Keywords:
96-Well plate readerAAPHORACPeroxyl radicalTotal antioxidant capacityTrolox

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Area of Science:

  • Biochemistry
  • Analytical Chemistry

Background:

  • The Oxygen Radical Absorbance Capacity (ORAC) assay is a common method for measuring total antioxidant capacity (TAC).
  • Current ORAC assays using 96-well plate readers face technical limitations in dispensing the free radical initiator, 2,2'-azobis (2-methyl-propanimidamide) dihydrochloride (AAPH).
  • This leads to a time delay between wells, introducing systematic errors and affecting the statistical significance of TAC determination based on antioxidant solution plate position.

Purpose of the Study:

  • To identify and address the AAPH-dependent error in 96-well plate reader-based ORAC assays.
  • To propose alternative solutions for more accurate total antioxidant capacity (TAC) measurements.

Main Methods:

  • Investigated the impact of delayed AAPH dispensing on ORAC assay results.
  • Developed and tested two novel methods to ensure simultaneous initiation of the antioxidant inhibition reaction across all wells.

Main Results:

  • AAPH dispensing limitations cause statistically significant variations in TAC measurements.
  • The proposed alternative methods effectively mitigate the AAPH-dependent error.
  • Improved accuracy and reliability of ORAC assay results were achieved.

Conclusions:

  • The AAPH-dependent error is a critical limitation in standard 96-well ORAC assays.
  • The developed alternative solutions provide a more robust and accurate method for determining total antioxidant capacity (TAC).
  • These improvements are crucial for reliable antioxidant analysis in biological samples.