Gas Chromatographic Analysis of Malondialdehyde as an Index of Lipid Peroxidation: Validation and Application in Cultured Cells

Abstract

Malondialdehyde (MDA) is very often taken as an index of lipid peroxidation but its measurement by colorimetric methods has been heavily questioned particularly in the case of cultured cells. In the present work, we have thus adapted a gas chromatographic method coupled with nitrogen–phosphorus detection to measure in various cell types (PC₁₂, fibroblasts) the formation of MDA in the cells as well as in the extracellular medium. When measuring MDA formation in cells treated by hydrogen peroxide/ferrous iron, we observed that the addition of increasing concentrations of H₂O₂/Fe²⁺ (0.1–1 mm) led to a linear formation of MDA in the cells and in the culture medium (fibroblasts) or in the culture medium only (PC₁₂ cells). In contrast, cumene hydroperoxyde (CHP) failed to induce MDA formation and release in both cell types. In conclusion, our method using hydrazinobenzothiazole (HBT) derivatisation is simple and reliable and sensitive enough to evaluate MDA production at the cellular level and to investigate the role of lipid peroxidation in cell degeneration.

Keywords:

• Malondialdehyde
• Lipid peroxidation
• Gas chromatography
• Hydrogen peroxide
• Cumene hydroperoxyde
• Fibroblasts
• PC₁₂ cells

Acknowledgments

We are grateful to “le Conseil Régional de Basse‐Normandie” (Caen, France) for its financial support. We wish to thank Dr. A.R. Young for correcting the English.