DETECTION OF H2O2 RELEASE FROM VASCULAR ENDOTHELIAL-CELLS

Endothelial cells are both significant sources and targets of reactive oxygen species, including O2.-, H2O2, .OH, .NO, and ONOO-, which play important roles in vascular homeostatic mechanisms and pathogenic processes. To better quantify cell oxidant metabolism processes, a fluorescence technique has been developed to measure H2O2 release from bovine aortic endothelial cells. Incubation of H2O2 with horseradish peroxidase (HRP) results in HRP-mediated oxidation of p-hydroxyphenylacetic acid (PHPA) to the fluorescent PHPA dimer, 2,2-dihydroxy-biphenyl-5,5' diacetate [(PHPA)2]. The HRP-mediated dimerization of 5 mM PHPA with concentrations of H2O2 up to 2.5 mM resulted in a linear increase in fluorescence (R = .995, n = 8). Maximal fluorescence occurred at 2.9 mM H2O2, with greater H2O2 concentrations yielding products with altered spectrophotometric characteristics and decreased fluorescent yield. The fluorescence of (PHPA)2 Was pH sensitive and increased 500-fold from pH 7 to 9. Fluorescence versus pH profiles were adjusted to a Henderson-Hasselbalch fitting, with a 50% maximal emission at pH = 8.1 +/- 0.2. The apparent pK(a) of fluorescence emission correlated well with a weak range of buffering capacity of (PHPA)2, which had a pK(a) = 8.0 +/- 0.1. With cells maintained in Hank's balanced salt solution (HBSS), the pH can increase to 7.90 during 3 h, with the increased pH due to the loss of HCO3- from HBSS. After adjustment for pH changes, a linear cellular H2O2 release of 217 pmol H2O2.min-1.mg protein-1 was observed. When bovine aortic endothelial cells (BAEC) were incubated with HBSS and PHPA alone, 50% less fluorescence was observed than when HRP was added. The HRP-independent production of (PHPA)2 May be due to cellular peroxidase activity or other cell-mediated oxidations. The H2O2-dependent HRP-mediated oxidation of PHPA is a sensitive and specific assay for quantifying cell H2O2 release if careful considerations are made for possible pH changes and overoxidation of PHPA.