The use of fluorescent probes to assess oxidative processes in isolated-perfused rat heart tissue

The formation of reactive oxygen species (ROS) in intact heart tissue has been assessed by direct ESR measurements, and indirectly by the formation of characteristic tissue products and the protective effects of various antioxidants. The development of lipid soluble esters of compounds which can be trapped intracellularly after hydrolysis, and which fluoresce after oxidation, has provided a new tool to investigate ROS in vitro. The utility of 2',7'-dichlorofluorescin diacetate (DCFDA) in isolated-perfused rat heart tissue was investigated in the present study. DCFDA and its deacetylated form were incubated with various levels of hydrogen peroxide or t-butylhydroperoxide (tBOOH). Conversion of the diacetate form to a fluorescent product required 4-5 h with hydrogen peroxide and up to 24 h with tBOOH. In contrast, the deacetylated form fluoresced at 80% of maximum levels 1 h after the addition of 100 mM tBOOH. DCFDA was loaded into heart tissue by infusing for 10 min at a final concentration of 10 microM in Krebs-Henseleit bicarbonate buffer. After a 10 min washout period, analysis of freeze-clamped heart tissue revealed that the trapped material was readily converted to a fluorescent product by tBOOH, indicating hydrolysis had occurred. Fluorescence of material trapped in heart tissue was approximately 24% of the maximum achieved after oxidation with 100 mM tBOOH. This value decreased to 18 and 13% when the loading and washout periods were from 0 to 20 or 10 to 30 min of hypoxia, respectively. Similar results were obtained with the less readily oxidized dicarboxy derivative of DCFDA. Infusion of 500 microM tBOOH increased the oxidation of DCFDA in heart tissue from 24 to 31%.(ABSTRACT TRUNCATED AT 250 WORDS)