In vivo quantitation of peroxides in the vitreous humor by fluorophotometry.

PURPOSE To detect intravitreal peroxides in vivo by a new fluorophotometric method with a hydrogen peroxide (H2O2)-sensitive fluorescent dye. METHODS The authors used a 2',7'-dichlorofluorescin (DCFH) assay to measure oxidative status in the rabbit vitreous. In the presence of H2O2 and lipid hydroperoxides, nonfluorescent DCFH in the vitreous is oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF; excitation, 495 nm; emission, 520 nm) that is detectable by fluorophotometry. Reactions of DCFH with various concentrations of H2O2 were investigated in vitro and in vivo. An inhibitory effect of catalase also was monitored. Vitreous fluorophotometry with DCFH was performed immediately and at 3, 7, and 28 days after constant light exposure to the retina (1800 lux, 24 hours) as an oxidative stress. RESULTS In vitro study revealed that H2O2 oxidized DCFH to DCF in a dose-dependent manner, ranging from 0.1 to 100 mmol/1 in concentration. Catalase inhibited DCF production. Vitreous fluorophotometry demonstrated that H2O2 oxidized DCFH to DCF in vivo in a dose-dependent manner, ranging from 0.06 to 60 mmol/1 in concentration. DCF production in the vitreous significantly increased immediately (P = 0.03) and at 3 days (P = 0.01) and 7 days (P = 0.01) after light exposure, and it returned to the pretreatment level by day 28. CONCLUSIONS The results suggest that this fluorophotometric method quantitatively can detect intravitreal peroxides in vivo. This method will be helpful to study the oxidative status in some experimental pathologic conditions.