The effect of reactive oxygen generation on intact livers was studied. Production of reactive oxygen species in perfused livers isolated from normal and endotoxin-treated rats was measured using chemically enhanced chemiluminescence. The resting state chemiluminescence of the livers increased on endotoxin administration and was maximal about 6 h after treatment. Chemiluminescence from the livers was further stimulated severalfold by inclusion of phorbol myristate acetate in the perfusion medium, reaching maximum intensity 3 h after endotoxin treatment. Oxygen consumption by the endotoxin-treated liver showed a transient increase followed by a significant decrease on phorbol myristate acetate stimulation, which was inhibited by dexamethasone. These results are consistent with the occurrence of a respiratory burst followed by oxygen-radical-species-induced vasoconstriction in the intact perfused liver. The evaluation of reactive oxygen species by resident and accumulated macrophages in the intact liver is made possible by these studies, and related effects on the liver could be conveniently and quantitatively followed using this model.

Repletion with Ca2+ often leads to damage of previously Ca(2+)-depleted hearts (the calcium paradox). The behavior of the liver under similar conditions is not well understood. With a perfused rat liver model, we examined liver cell damage and lipid peroxidation during Ca2+ depletion and repletion and used lucigenin-enhanced chemiluminescence as a measure of oxygen radicals. During 30 minutes of Ca2+ depletion, release of lactate dehydrogenase and thiobarbituric acid-reactive substance did not change significantly. When Ca2+ depletion was extended to 150 minutes, release of lactic acid dehydrogenase and thiobarbituric acid-reactive substance and tissue oxygen radical levels all increased progressively, accompanied by decrease in oxygen uptake. Ca2+ repletion after 30 minutes of Ca2+ depletion caused small increases in release of lactic acid dehydrogenase and thiobarbituric acid-reactive substance but significantly suppressed the changes described, compared with expression in depleted livers without Ca2+ repletion. There were large releases of sinusoidal glutathione and glutathione disulfide at the onset of Ca2+ depletion, which declined within 15 minutes. On Ca2+ repletion, sinusoidal glutathione level decreased to its baseline but glutathione disulfide level did not change significantly. During long-term Ca2+ depletion, sinusoidal glutathione level was significantly higher than baseline but glutathione disulfide level remained low. These results indicate that long-term Ca2+ depletion causes oxidative stress and liver damage. Ca(2+)-dependent release of sinusoidal glutathione appears to result from causes other than oxidative stress. There is no evidence for the calcium paradox in the liver; in fact, reexposure to Ca2+ protects the liver from the injury caused by Ca2+ depletion.

The influx of extracellular Ca2+ has been postulated to be one of the mediators of ischemia-reperfusion injury. A possible link between Ca2+ influx and oxygen radical generation has also been suggested. In the present study, using the isolated perfused rat liver, we evaluated the role of extracellular Ca2+ on oxygen radical generation, liver damage, and lipid peroxidation during 30 min ischemia and 60 min of reperfusion. Oxygen radical generation in the liver was continuously monitored by lucigenin-enhanced chemiluminescence. Liver damage and lipid peroxidation were evaluated by measuring lactate dehydrogenase (LDH) and thiobarbituric acid reactive substances (TBARS) release into the effusate, respectively. In the absence of extracellular Ca2+ (much less than 30 microM) oxygen radical generation from the liver increased gradually over 2 hr and there were concomitant increases in LDH and TBARS release. When livers were made ischemic and then reperfused, oxygen radical generation increased at the onset of reperfusion and then decreased over 30 min of reperfusion. After 30 min of reperfusion, livers reperfused with low Ca2+ buffer showed a linear increase in oxygen radical generation as well as progressive increases in LDH and TBARS release. On the other hand, livers reperfused with Ca2+ containing (1.25 mM) buffer showed no further increase in oxygen radical generation and no evidence of progressive liver damage and lipid peroxidation. These results suggest that Ca2+ overload is not a primary cause of liver ischemia-reperfusion injury and that the presence of extracellular Ca2+ during reperfusion is necessary to maintain normal liver function.

Using lucigenin-enhanced chemiluminescence, isolated rat lungs perfused with physiological salt-Ficoll solution were studied to test whether phorbol myristate acetate (PMA)-induced lung injury was mediated by reactive oxygen species (ROS). PMA (0.03 micrograms ml-1) caused small but significant increases in lung ROS levels and pulmonary arterial perfusion pressure (Ppa) but did not induce lung oedema. PMA (0.15 micrograms ml-1) induced lung oedema with large increases in ROS production and Ppa. Superoxide dismutase (SOD) inhibited the increases in ROS, Ppa, and lung oedema. Catalase and dimethylthiourea inhibited lung oedema but did not attenuate the increases in ROS and Ppa entirely. Indomethacin attenuated lung oedema partially but did not inhibit the increases in ROS and Ppa. These data indicate that PMA-induced lung injury is dependent on PMA concentration and ROS are responsible for such lung injury. Thromboxane plays a minor role for PMA-induced lung injury. The different effects of oxygen radical scavengers suggest that different radical species contribute to the increased pulmonary vascular response and lung injury.

Using the isolated perfused rat liver, we examined the effect of stimulation of mitochondrial respiration by 2,4-dinitrophenol (2,4-DNP) and adrenaline on reactive oxygen species (ROS) production, liver damage and lipid peroxidation. ROS production was monitored by luminol- and lucigenin-enhanced chemiluminescence and oxygen uptake was measured simultaneously. Liver damage and lipid peroxidation were evaluated by measuring hepatic lactate dehydrogenase (LDH) and thiobarbituric acid reacting substances (TBARS) release. Tissue ROS level decreased and oxygen uptake increased soon after 2,4-DNP infusion. On termination of 2,4-DNP infusion, there was a sharp increase in lucigenin-enhanced chemiluminescence, which declined slowly, but luminol-enhanced chemiluminescence did not change prominently. Hepatic LDH and TBARS release increased gradually during 2,4-DNP infusion and were manifested by termination of the infusion. Allopurinol did not affect ROS production and TBARS release, but delayed increases in LDH release after termination of 2,4-DNP infusion. Adrenaline, which stimulates mitochondrial respiration without uncoupling caused similar but smaller ROS changes observed in 2,4-DNP. LDH and TBARS release were not affected significantly by adrenaline infusion. These results indicate that uncoupling of oxidative phosphorylation decreases ROS production and restoration of oxidative phosphorylation enhances ROS production and liver damage. Xanthine oxidase is unlikely to contribute to enhanced ROS production after termination of 2,4-DNP but has some protective effect during uncoupling.

Oxygen radical production in isolated perfused rat hearts during 30 minutes of ischemia followed by reperfusion was monitored by continuous measurement of luninol-enhanced chemiluminescence. The luminol-enhanced chemiluminescence of control hearts increased by 75% during ischemia; reperfusion induced a further (50%) increase in chemicluminescence intensity followed by a slow decline during the course of reperfusion. The inclusion of 5 mmol/L mannitol or of 100 U/mL catalase did not lead to any decrease in the luminol-enhanced chemiluminescence intensities during ischemia-reperfusion. The inclusion of 30 U/mL superoxide dismutase in the perfusion buffer prevented the increases in luminol-enhanced chemiluminescence both during ischemia and reperfusion. Our results suggest that the primary oxygen radical produced during ischemia-reperfusion in our system is the superoxide and that the superoxide produced is accesible to superoxide dismutase in the vascular compartment.

We have applied the Luminol enhanced chemiluminescence technique to the isolated perfused rat liver during ischemia and reperfusion to monitor the production of oxygen radicals in tissue. Livers under perfusion with Luminol-containing buffer were subjected to 30 minutes of global ischemia followed by 60 minutes of reperfusion. Their chemiluminescence was continuously monitored to obtain the time course of oxygen radical production. Transient bursts of oxygen radical production were observed in the livers as indicated by chemiluminescence changes on reperfusion. Superoxide dismutase treatment abolished while catalase treatment enhanced the reperfusion-induced chemiluminescence transient.

We show that the production of Luminol reactive oxygen radicals in the perfused rat heart under ischemia and reperfusion can be monitored continuously by measuring the chemiluminescence of Luminol-perfused hearts. Luminol did not affect the monitored physiological parameters of the hearts. Chemiluminescence increased during ischemia and reperfusion. Superoxide dismutase treatment of the heart before ischemia, but not catalase, abolished these increases.

The reaction of peroxide with cytochrome oxidase generates a peroxide compound having a Soret maximum at 428 nm. X-ray absorption spectroscopy analysis of the local structure of the active site iron shows marked similarity to that of the cytochrome c peroxidase intermediate Compound ES, which contains a short iron to proximal nitrogen distance compared to globins. Reductive titration of the 580 nm band of this compound indicates that the iron is one oxidizing equivalent above the resting oxidized form. These results support the presence of a ferryl iron (Fe(IV) = O) in the peroxide compound similar to that found for the peroxidases.

Elementary steps or geminate states in the reaction of gaseous ligands with transport proteins delineate the trajectory of the ligand and its rebinding to the heme. By use of kinetic studies of the 765-nm optical "conformation" band, three geminate states were identified for temperatures less than approximately 100 K. MbCO, which is accumulated by photolysis between 1.2 and approximately 10 K, was characterized by our previous optical and X-ray absorption studies [Chance, B., Fischetti, R., & Powers, L. (1983) Biochemistry 22, 3820-3829]. Between 10 and approximately 100 K, geminate states that are also identified that have recombination rates of approximately 10(3) s-1 and approximately 10(-5) s-1 (40 K). Thus, it is possible to maintain a steady-state nearly homogeneous population of the slowest recombining geminate state, Mb, by regulated continuous illumination (optical pumping). Both X-ray absorption and resonance Raman studies under similar conditions of optical pumping show that the heme structure around the iron in Mb is similar to that of MbCO. In both geminate states, the iron-proximal histidine distance remains unchanged (+/- 0.02 A) from that of MbCO while the iron to pyrrole nitrogen average distance has not fully relaxed to that of the deoxy state. In MbCO the CO remains close to iron but not bound, and the Fe...CO angle, which is bent in MbCO (127 +/- 4 degrees C), is decreased by approximately 15 degrees [Powers, L., Sessler, J. L., Woolery, G. L., & Chance, B. (1984) Biochemistry 23, 5519-5523]. The CO molecule in Mb, however, has moved approximately 0.7 A further from iron. Computer graphics modeling of the crystal structure of MbCO places the CO in a crevice in the heme pocket that is just large enough for the CO molecule end-on. Above approximately 100 K resonance Raman studies show that this structure relaxes to the deoxy state.

X-ray absorption studies of myoglobin peroxide show that although it is not identical with compound I or II of horseradish peroxidase [Chance, B., Powers, L., Ching, Y., Poulos, T., Yamazaki, I., & Paul, K. G. (1984) Arch. Biochem. Biophys. 235, 596-611], it has some structural features in common with both. As seen in compound I, the Fe-O distance is short, but the iron-pyrrole nitrogen distance is contracted with a longer iron-histidine distance like compound II. The iron has a higher oxidation state than Fe3+, suggesting an oxyferryl ion type species. Comparison of the structures of various peroxidase and myoglobin compounds points out systematic differences that may explain the catalytic activity of the pi cation radical as well as some of the differences between globins and heme enzymes.

The oxygen reaction of the fully reduced respiratory chain in membranes from oxygen-limited Escherichia coli was studied at sub-zero temperatures using EPR spectroscopy. Laser photolysis of CO-liganded cytochrome oxidase d precedes oxidation of at least 2 kinetically separable high-spin cytochromes. At -120 to -100 degrees C, a rhombic signal appears, attributable to cytochrome d, followed at above -100 degrees C, by appearance of a second, axial signal near g = 6, here assigned to cytochrome(s) b, and changes in the redox state of iron-sulphur clusters. The data kinetically resolve the 2 high-spin signals attributed to the oxidase complex and suggest schemes for electron flow to oxygen.

The two state model of hemoglobin has been popular for many years but the description of the structural differences between the two states has been established only for the oxy to deoxy transition.