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Using ESR spin-trapping techniques with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we confirmed the 1:1 stoichiometry for the formation of hydroxyl radicals with Fe2+ in the Fenton reaction under experimental conditions wherein [H2O2] is 90 microM and [Fe2+] is very low, 1 microM or less. The stoichiometry decreased markedly as the Fe2+ concentration was increased. The efficiency of hydroxyl radical generation varied with the nature of the iron chelators used and increased in the order of phosphate alone approximately ADP less than EDTA less than diethylenetriaminepentaacetic acid (DETAPAC). The second order rate constant for the Fenton reaction was measured to be 2.0 x 10(4) M-1 s-1 for phosphate alone, 8.2 x 10(3) M-1 s-1 for ADP, 1.4 x 10(4) M-1 s-1 for EDTA, and 4.1 x 10(2) M-1 s-1 for DETAPAC. Measuring the radicals formed as spins trapped in the presence of ethanol, we estimated the amount of total oxidizing intermediates formed in the Fenton reaction, which we concluded consists of hydroxyl radicals and an iron species. The oxidizing species of iron which might be assigned as ferryl, FeO2+, or Fe(IV) = O was generated effectively in the presence of ADP even at low Fe2+ concentrations. In general, as the Fe2+ concentration was increased, the ferryl species predominated over the hydroxyl radical except for the case of Fe(II)-DETAPAC, which generated only hydroxyl radicals as the oxidizing species. Three possible pathways are proposed for the Fenton reaction, the dominant ones depending very much on the nature of the iron chelator being used.

By measuring the absorbance change due to myoglobin oxygenation in hemoglobin-free isolated perfused rat hearts, we analyzed effects of perfusion pressure and heart rate upon the intracellular oxygen concentration. With Langendorff perfusion, the cardiac tissue was kept normoxic (above 50 microM O2) at aortic pressure above 50 cm H2O, but became hypoxic (8 microM O2) at 30 cm H2O. The increase in cardiac work, expressed as the product of peak systolic pressure and heart rate, increased oxygen consumption at aortic pressure of 50-200 cm H2O. The heart was kept normoxic under these conditions. Lactate release, oxygen consumption, and the oxidation-reduction state of pyridine nucleotide were measured as a function of myoglobin oxygenation under various normoxic and anoxic conditions. Pyridine nucleotide fluorescence and lactate release started to increase as the intracellular oxygen concentration decreased to 6 and 10 microM, respectively. Oxygen consumption was kept constant until the oxygen concentration decreased to 10 microM and slowed down below it. A close relationship between oxygen consumption and lactate release was observed. Infusions of epinephrine and norepinephrine under normoxic perfusion conditions increased cardiac work, oxygen consumption, and lactate release. More than 50% of myoglobin was then deoxygenated even under normoxic perfusion conditions. The increase in lactate release was ascribable to the increase in glycolytic flux caused by hypoxia. The change of pyridine nucleotide fluorescence by epinephrine was also explained by hypoxia in cardiac tissue.

Univalent oxidation-reduction reactions coupled with the oxygen-superoxide system were investigated in the reactions shown in eq 3 and 8, where Q and Q.- stand for p-benzoquinone and p-benzosemiquinone, respectively. From kinetic experiments the following rate constants were obtained at pH 7.0:k3 = 4.5 x 10(4) M-1 sec-1 and k8 = 3 x 10(-2) M-1 sec-1. With known values of k-3 and k-8, and of E0' for the systems Q-Q.- (0.10 V) and Cyt c3+ - Cyt c2+ (0.255 V), the calculated values of E0(O2-O2.-) were found to lie in the range between -0.27 and -0.33 V.