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Detection of Intracellular Superoxide using HPLC!

Proof for Oxy-Ethidium formation

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HPLC chromatograms of dihydroethidium, ethidium, and oxy-ethidium formed by exposure of dihydroethidium by various oxidants. (A) HPLC tracing of ethidium (1 然); (B) HPLC trace of authentic dihydroethidium (25 然); (C) Elution profile of the reaction of 2 mg KO2 to 25 然 dihydroethidium in 0.9% NaCl solution; (D) Same as C except using 4 mg KO2; (E) Incubation of dihydroethidium (25 然) in Krebs-HEPES buffer (20 mM, pH 7.4) with xanthine oxidase (5mU/ml) and xanthine (0.5 mM); (F) Same as D but in the presence of 100 U/ml of SOD; (G) Incubation of dihydroethidium (25 然) with H2O2 (10 mM); (H) Incubation of dihydroethidium (25 然) with peroxynitrite (10 mM). HPLC traces E, F, G, and H were obtained 30 min after incubation with the respective agents. Evaluation of original HPLC traces was performed after subtraction of water or methanol background spectra using 32-Karat HPLC software from Beckman-Coulter.

In Mouse Aorta

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A) Effect of long-term angiotensin II treatment (0.7 mg/kg/day x 14 days) on formation of oxy-ethidium formation without (hatched bars) and with pre-incubation of aorta segments with (squared bar) PEG-SOD (1 hour, at 37蚓, 100 U/ml). Angiotensin II was infused subcutaneously via an osmotic minipump. Data are mean SEM (n = 4). * P < 0.05 vs. control, ** P < 0.05 vs. angiotensin II; B). Oxy-ethidium formation in DOCA-salt mice aorta segments was performed in vessels treated without (hatched bar) and with (squared bar) pretreatment with L-NAME (100 然). Data are mean SEM (n = 4). * P < 0.05 vs. control, ** P < 0.05 vs. DOCA; C) Comparison of O2- production measured by SOD-inhibited cytochrome c reduction (hatched bars, n = 13-4-14) and oxy-ethidium formation from dihydroethidium (open bars, n = 4-6) in vessels from mice with either angiotensin II-induced or DOCA-salt hypertension. Five 2 mm vessel segments were incubated for 15 minutes in a Krebs-HEPES buffer containing dihydroethidium (50 mM) at 37oC. Data are mean SEM. * P < 0.01 vs. control.

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