Evaluation of hydroxyl radicals production using 13CO2 gas chromatography-isotope ratio mass spectrometry.
A gas chromatography-isotope ratio mass spectrometry (GC-IRMS) technique for detecting the production of hydroxyl radicals is described. The decarboxylation of [7-13COOH]benzoic acid in the presence of a hydroxyl radicals source (a mixture of porphyrin and hydrogen peroxide) was followed by direct measurement of the 13CO2/12CO2 isotopic ratio. The production of hydroxyl radicals by the mixtures of porphyrin-hydrogen peroxide was proved by comparative study with electron spin 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 the inhibition of 13CO2 production from [7-13COOH]benzoic acid. In contrast, high concentrations of the antioxidant ascorbate strongly increased [7-13COOH]benzoic acid decarboxylation. Finally, the use of anaerobic conditions showed that decarboxylation was independent of the presence of oxygen. The absence of the effect of superoxide dismutase could exclude a possible effect of the superoxide ion. This nonradioactive technique offers many advantages compared to the well-established method for detecting hydroxyl radicals based on the decarboxylation of [7-14COOH]benzoic acid. It is rapid and easy to perform as a simple tube test and is highly reliable for detecting hydroxyl radicals. This method provides an on-line analysis of carbon dioxide compared to the radiochemical method. In addition, 13CO2-enrichment measurements led to easy kinetic studies with high sensitivity and semiquantitative determinations.
Unité de Pathologie Moléculaire, Hôpital Edouard Herriot, Lyon, France.
R
R
Lamrini
J M
JM
Crouzet
A
A
Francina
R
R
Guilluy
J P
JP
Steghens
J L
JL
Brazier
D001565:Benzoates / Q000737:chemistry; D019817:Benzoic Acid; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid; D003653:Decarboxylation; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005069:Evaluation Studies as Topic; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006861:Hydrogen Peroxide / Q000737:chemistry; D017665:Hydroxyl Radical; D006900:Hydroxylation; D008353:Mannitol / Q000737:chemistry; D000432:Methanol / Q000737:chemistry; D011166:Porphyrins / Q000737:chemistry; D015203:Reproducibility of Results
D001565:Benzoates; D002247:Carbon Isotopes; D011166:Porphyrins; D017665:Hydroxyl Radical; D008353:Mannitol; D019817:Benzoic Acid; D006861:Hydrogen Peroxide; D000432:Methanol; D004121:Dimethyl Sulfoxide