EVALUATION OF HYDROXYL RADICALS PRODUCTION USING (CO2)-C-13, GAS-CHROMATOGRAPHY ISOTOPE RATIO MASS-SPECTROMETRY

A gas chromatography-isotope ratio mass spectrometry (GC-IRMS) techniq ue for detecting the production of hydroxyl radicals is described. The decarboxylation of [7-(COOH)-C-13]benzoic acid in the presence of a h ydroxyl radicals source (a mixture of porphyrin and hydrogen peroxide) was followed by direct measurement of the (13)C0(2)/(CO2)-C-12 isotop ic ratio. The production of hydroxyl radicals by the mixtures of porph yrin-hydrogen peroxide was proved by comparative study with electron s pin resonance spectrometry and high-performance liquid chromatography analysis of hydroxylation products of benzoic acid. The water-soluble radical scavengers methanol, mannitol, and dimethyl sulfoxide led to t he inhibition of(13)CO(2) production from [7-(COOH)-C-13]benzoic acid. In contrast, high concentrations of the antioxidant ascorbate strongl y increased [7-(COOH)-C-13]benzoic acid decarboxylation. Finally, the use of anaerobic conditions showed that decarboxylation was independen t of the presence of oxygen. The absence of the effect of superoxide d ismutase could exclude a possible effect of the superoxide ion. This n onradioactive technique offers many advantages compared to the well-es tablished method for detecting hydroxyl radicals based on the decarbox ylation of [7-(COOH)-C-14]benzoic acid. It is rapid and easy to perfor m as a simple tube test and is highly reliable for detecting hydroxyl radicals. This method provides an on-line analysis of carbon dioxide c ompared to the radiochemical method. In addition, (CO2)-C-13,-enrichme nt measurements led to easy kinetic studies with high sensitivity and semiquantitative determinations. (c) 1994 Academic Press, Inc.