An improved method for determination of L-ascorbic acid and L-dehydroascorbic acid in blood plasma

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Accurate quantification of L-ascorbic acid (vitamin C) and its oxidized form, L-dehydroascorbic acid, is crucial for understanding cellular redox status and nutritional health.
• Existing methods may not reliably distinguish between the reduced and oxidized forms in biological samples like blood plasma.

Purpose of the Study:

• To develop and validate an improved method for the separate determination of L-ascorbic acid and L-dehydroascorbic acid in human blood plasma.
• To optimize the reduction conditions of L-dehydroascorbic acid using dithiothreitol.

Main Methods:

• Studied various conditions for reducing L-dehydroascorbic acid with dithiothreitol.
• Determined L-ascorbic acid content using the alpha,alpha'-dipyridyl method (A).
• Reduced L-dehydroascorbic acid to L-ascorbic acid with dithiothreitol, quenched excess reagent with N-ethylmaleimide, and measured total ascorbic acids (B) via the same method. Calculated L-dehydroascorbic acid as (B-A).

Main Results:

• Established optimal conditions for dithiothreitol-mediated reduction of L-dehydroascorbic acid at room temperature (10 min, pH 6.5-8.0).
• Successfully implemented a method allowing for the distinct quantification of both L-ascorbic acid and L-dehydroascorbic acid in plasma.
• Demonstrated that the difference between total ascorbic acid (B) and reduced ascorbic acid (A) accurately reflects the dehydroascorbic acid concentration.

Conclusions:

• The developed method provides a reliable and improved approach for the separate analysis of L-ascorbic acid and L-dehydroascorbic acid in blood plasma.
• This technique enhances the ability to assess vitamin C status and oxidative stress markers in clinical and research settings.