Detection of activated O₂ species in vitro and in rat lungs by chemiluminescence

This study used chemiluminescence, an 'on-line' photon-counting technique, to detect and characterize activated O₂ species in vitro and in isolated rat lungs. The sensitivity and specificity of enhanced chemiluminescence for superoxide anion and hydrogen peroxide (H₂O₂) was evaluated in vitro. The effect of media conditions (such as O₂ tension, albumin concentration, and sulfhydryl group availability) on luminescence was assessed in vitro. Xanthine-xanthine oxidase (X-XO) primarily produced superoxide anion in vitro. Enhanced chemiluminescence varied directly with the dose of luminescent probe used and the quantity of activated O₂ species administered. The strength of the luminescent signal was also dependent on the concentration of albumin and O₂ in the media. Lucigenin was more sensitive than luminol to the presence of superoxide anion and, unlike luminol, lucigenin did not alter radical production by XO. However, neither luminescent probe was specific for superoxide anion, as both detected H₂O₂ and O₂ in vitro. H₂O₂-induced chemiluminescence was inhibited by catalase but not superoxide dismutase (SOD), while X-XO-induced luminescence was inhibited by SOD but not catalase. SOD-inhibitable chemiluminescence was a sensitive and specific marker for superoxide anion production in vitro. Once the sensitivity-specificity of enhanced chemiluminescence was defined in vitro, this technique was used to explore the mechanism by which exogenous X-XO reduced hypoxic vasoconstriction in isolated rat lungs. The vascular paresis, caused by administration of X-XO to the rat lung, resulted from a brief burst of superoxide anion production rather than a sustained alteration of lung radical levels.