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Related Experiment Videos

The methanol method for the quantification of ascorbic acid and dehydroascorbic acid in biological samples.

Curd-David Badrakhan1, Frank Petrat, Maren Holzhauser

  • 1Institut für Physiologische Chemie, Universitätsklinikum Essen, Hufelandstrasse 55, 45122 Essen, Germany.

Journal of Biochemical and Biophysical Methods
|March 18, 2004
PubMed
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This study introduces a rapid spectrophotometric method for measuring vitamin C (ascorbic acid) and its oxidized form in biological samples. The new methanol-based assay offers a simpler, single-point determination, outperforming existing test kits.

Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Cell Biology

Background:

  • Ascorbic acid (vitamin C) and its oxidized form, dehydroascorbic acid, play crucial roles in biological systems.
  • Accurate quantification of both forms in biological samples is essential for understanding cellular processes and health status.
  • Existing methods for vitamin C determination can be complex or lack sensitivity.

Purpose of the Study:

  • To develop a fast and simple spectrophotometric method for quantifying both ascorbic acid and dehydroascorbic acid in biological samples.
  • To validate the method's performance against commercially available test kits.
  • To assess intracellular vitamin C levels in response to extracellular ascorbate and oxidative stress.

Main Methods:

  • A novel spectrophotometric assay utilizing the reaction of dehydroascorbic acid with methanol in a phosphate/citrate buffer.

Related Experiment Videos

  • Quantification of ascorbic acid in the presence of ascorbate oxidase.
  • Single endpoint determination for simplified analysis.
  • Application of the method to cell cultures (L-929 cells, RAW 264.7 macrophages) and human blood serum.

    • Main Results:

    • The developed methanol method provides a simple, single endpoint spectrophotometric determination for both vitamin C forms.
    • The assay demonstrated improved performance compared to a commercial vitamin C test kit.
    • Intracellular ascorbic acid levels increased dose-dependently with extracellular ascorbate in L-929 cells and macrophages.
    • Vitamin C concentrations in blood serum ranged from 46 to 97 microM, with good recovery upon supplementation.
    • Intracellular vitamin C in L-929 cells remained largely unoxidized even under high reactive nitrogen species conditions.

    Conclusions:

    • The methanol method is a fast, efficient, and reliable spectrophotometric technique for determining ascorbic acid and dehydroascorbic acid in biological matrices.
    • This assay offers a significant advantage over existing methods due to its simplicity and single endpoint measurement.
    • The method is suitable for studying vitamin C metabolism in cell cultures and its levels in biological fluids like blood serum.