Detection of superoxide radical in all biological systems by Thin Layer Chromatography

The study presents a new method that detects O₂^•-, via quantification of 2-hydroxyethidium (2-ΟΗ-Ε⁺) as low as ∼30 fmoles by High-Performance Thin Layer Chromatography (HPTLC). The method isolates 2-ΟΗ-Ε⁺ after its extraction by the anionic detergent SDS (at 18-fold higher than its CMC) together with certain organic/inorganic reagents, and its HPTLC-separation from di-ethidium (di-Ε⁺) and ethidium (Ε⁺). Quantification of 2-OH-E⁺ is based on its ex/em maxima at 290/540 nm, and of di-E⁺ and E⁺ at 295/545 nm. The major innovations of the present method are the development of protocols for (i) efficient extraction (by SDS) and (ii) sensitive quantification (by HPTLC) for 2-OH-E⁺ (as well as di-E⁺ and E⁺) from most biological systems (animals, plants, cells, subcellular compartments, fluids). The method extracts 2-ΟΗ-Ε⁺ (by neutralizing the strong binding between its quaternary N⁺ and negatively charged sites on phospholipids, DNA etc) together with free HE, while protects both from biological oxidases, and also extracts/quantifies total proteins (hydrophilic and hydrophobic) for expressing O₂^•- levels per protein quantity. The method also uses SDS (at 80-fold lower than its CMC) to extract/remove/wash 2-ΟΗ-Ε⁺ from cell/organelle exterior membrane sites, for more accurate internal content quantification. The new method is applied on indicative biological systems: (1) artificially stressed (mouse organs and liver mitochondria and nuclei, ±exposed to paraquat, a known O₂^•- generator), and (2) physiologically stressed (cauliflower plant, exposed to light/dark).