Efficient assay for total antioxidant capacity in human plasma using a 96-well microplate

Area of Science:

• Biochemistry
• Clinical Chemistry
• Analytical Chemistry

Background:

• Oxidative stress is implicated in various diseases.
• Accurate measurement of total antioxidant capacity (TAC) is crucial for health assessment.
• Existing TAC assays may lack efficiency or require large sample volumes.

Purpose of the Study:

• To establish a rapid and efficient 96-well microplate assay for determining total antioxidant capacity (TAC) in human plasma.
• To validate the assay's reliability and linearity using Trolox standards.
• To apply the assay for preliminary assessment of TAC in healthy individuals and in response to dietary intervention.

Main Methods:

• Developed a 96-well microplate assay measuring lag time against myoglobin-induced ABTS oxidation.
• Quantified TAC using 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and expressed results as Trolox equivalents.
• Validated assay linearity (R(2) = 0.997) within a 2.5–25 µM Trolox range and assessed intra-day (2.4% CV) and inter-day reproducibility.

Main Results:

• The assay demonstrated excellent linearity and reproducibility for TAC measurement in human plasma.
• Plasma TAC was significantly higher in men (569 ± 41 µM Trolox eq.) compared to women (430 ± 28 µM Trolox eq.).
• Consumption of vegetable juice for one week led to a noticeable increase in plasma TAC in most volunteers.

Conclusions:

• An efficient and reliable 96-well microplate assay for human plasma TAC has been successfully developed.
• The assay provides a valuable tool for clinical research, enabling efficient assessment of antioxidant status.
• Findings suggest potential sex-based differences in TAC and highlight the impact of dietary interventions on antioxidant levels.