James D. Crapo

[41] Preparation and assay of superioxide dismutases

Publisher Summary This chapter discusses the preparation and assay of superoxide dismutase (SOD). SODs are found in all oxygen-utilizing organisms and constitute a defense against oxygen toxicity. SODs were first isolated from erythrocytes as a copper protein of unknown function. Thus, some SODs contain copper and zinc, others contain manganese, and still others contain iron. Assay techniques for each of these enzymes are similar, but distinct isolation procedures are used in their purification. Most mammalian tissues contain both a cuprozinc and a mangano superoxide dismutase. SODs are unique among enzymes in that their substrate is an unstable free radical. This complicates the measurement of their catalytic activity. Convenient assays of SODs have necessarily been of the indirect type. Such assays consist of two components: a superoxide generator and a superoxide detector. The control reaction rate can be completely inhibited by large amounts of SOD if a xanthine oxidase of high quality is being used. Partially degraded xanthine oxidase can, to a small extent, reduce cytochrome c by a nonsuperoxide mediated mechanism.

Genetic Epidemiology of COPD (COPDGene) Study Design

ABSTRACT Background: COPDGene is a multicenter observational study designed to identify genetic factors associated with COPD. It will also characterize chest CT phenotypes in COPD subjects, including assessment of emphysema, gas trapping, and airway wall thickening. Finally, subtypes of COPD based on these phenotypes will be used in a comprehensive genome-wide study to identify COPD susceptibility genes. Methods/Results: COPDGene will enroll 10,000 smokers with and without COPD across the GOLD stages. Both Non-Hispanic white and African-American subjects are included in the cohort. Inspiratory and expiratory chest CT scans will be obtained on all participants. In addition to the cross-sectional enrollment process, these subjects will be followed regularly for longitudinal studies. A genome-wide association study (GWAS) will be done on an initial group of 4000 subjects to identify genetic variants associated with case-control status and several quantitative phenotypes related to COPD. The initial findings will be verified in an additional 2000 COPD cases and 2000 smoking control subjects, and further validation association studies will be carried out. Conclusions: COPDGene will provide important new information about genetic factors in COPD, and will characterize the disease process using high resolution CT scans. Understanding genetic factors and CT phenotypes that define COPD will potentially permit earlier diagnosis of this disease and may lead to the development of treatments to modify progression.

Requirement for superoxide in excitotoxic cell death.

We tested the pathogenic role of O2-) radicals in excitotoxic injury. Inactivation of the TCA cycle enzyme, aconitase, was used as a marker of intracellular O2- levels, and a porphyrin SOD mimetic was used to scavenge O2-. The selective, reversible, and SOD-sensitive inactivation of aconitase by known O2- generators was used to validate aconitase activity as a marker of O2- generation. Treatment of rat cortical cultures with NMDA, KA, or the intracellular O2- generator PQ2+ produced a selective and reversible inactivation of aconitase, which closely correlated with subsequent cell death produced by these agents. The SOD mimetic, but not its less active congener, attenuated both aconitase inactivation and cell death produced by NMDA, KA, and PQ2+. These results provide direct evidence implicating O2(-) generation in the pathway to excitotoxic injury.

The effect of hyperoxia on superoxide production by lung submitochondrial particles

Hyperoxia increases CN−-resistant respiration in rat lung mitochondria, rat lung submitochondrial particles, and porcine lung submitochondrial particles. Cyanide-resistant respiration increases from 0 at normal lung tissue oxygen tensions to 1.2 ± 0.06 nmol O2 · min−1 · mg protein−1 (X ± SD) in rat lung mitochondria and 2.7 ± 0.2 nmol O2 · min−1 · mg protein−1 in rat lung submitochondrial particles, when assayed at an oxygen concentration of 85%. Superoxide production by submitochondrial particles washed free of superoxide dismutase was directly measured by quantitating superoxide dismutase-inhibitable epinephrine oxidation or cytochrome c reduction, and compared to parallel measurements of CN− resistant respiration. Succinate or NADH was used as substrate and rotenone, antimycin, or CN− were used as respiratory chain inhibitors. At least two components of the respiratory chain produced O2−, the ubiquinone-cytochrome b region and the NADH dehydrogenase complex. Superoxide generation by the NADH dehydrogenase is 1.5-fold greater than superoxide production by the ubiquinone-cytochrome b segment. Oxidation of NADH by porcine lung mitochondria, treated with either rotenone, antimycin, or CN−, parallels rates of O2− formation and increases directly with oxygen tension, providing another indirect measurement of superoxide production by mitochondrial membranes. These findings support the hypothesis that oxygen toxicity is in part a consequence of increased rates of intracellular O2− and H2O2 production.

Hyperoxia increases H2O2 release by lung mitochondria and microsomes.

Hyperoxia increased H2O2 release by lung mitochondria. A 10-fold increase in the rate of H2O2 production was observed when oxygen concentration increased from 21 to 100%. The rate of H2O2 release increased linearly from 0 to 60% oxygen. Above 60% oxygen, the rate of H2O2 release increased dramatically up to 0.19 ± 0.03 nmol · min−1 · mg protein−1 at 100% oxygen. The same effect was observed when mitochondria were supplemented with malate + malonate + glutamanate in the presence of rotenone or when endogenous substrates were oxidized by rotenone-supplemented particles. The three antioxidant enzymes (catalase, glutathione peroxidase, and superoxide dismutase) were present in the mitochondrial fraction obtained from porcine lungs. The effect of oxygen concentration on extramitochondrial H2O2 release may be the consequence of a sufficient increase in the rate of intramitochondrial H2O2 production to overwhelm H2O2 scavenging by intramitochondrial peroxidases. Hydrogen peroxide formation by lung microsomes supplemented with NADPH increased linearly with oxygen concentration from 0.91 ± 0.16 at 21% to 1.6 ± 0.3 nmol · min−1 · mg protein−1 at 100% oxygen. When supplemented with NADH, microsomes produced H2O2 at rates that were about half of those with NADPH, at all oxygen concentrations. From these results, it was estimated that mitochondria can produce 2.9 nmol H2O2/g tissue while microsomes produce 19.7 nmol H2O2/g tissue, when exposed to 100% oxygen. This suggests that the enhanced release of H2O2 by hyperoxic mitochondria and microsomes may be responsible, in part, for the cellular damage observed in lungs of animals breathing oxygen concentrations of 60% or greater.

Variants in FAM13A are associated with chronic obstructive pulmonary disease

We performed a genome-wide association study for chronic obstructive pulmonary disease (COPD) in three population cohorts, including 2,940 cases and 1,380 controls who were current or former smokers with normal lung function. We identified a new susceptibility locus at 4q22.1 in FAM13A and replicated this association in one case-control group (n = 1,006) and two family-based cohorts (n = 3,808) (rs7671167, combined P = 1.2 × 10−11, combined odds ratio in case-control studies 0.76, 95% confidence interval 0.69–0.83).

The clinical features of the overlap between COPD and asthma

BackgroundThe coexistence of COPD and asthma is widely recognized but has not been well described. This study characterizes clinical features, spirometry, and chest CT scans of smoking subjects with both COPD and asthma.MethodsWe performed a cross-sectional study comparing subjects with COPD and asthma to subjects with COPD alone in the COPDGene Study.Results119 (13%) of 915 subjects with COPD reported a history of physician-diagnosed asthma. These subjects were younger (61.3 vs 64.7 years old, p = 0.0001) with lower lifetime smoking intensity (43.7 vs 55.1 pack years, p = 0.0001). More African-Americans reported a history of asthma (33.6% vs 15.6%, p < 0.0001). Subjects with COPD and asthma demonstrated worse disease-related quality of life, were more likely to have had a severe COPD exacerbation in the past year, and were more likely to experience frequent exacerbations (OR 3.55 [2.19, 5.75], p < 0.0001). Subjects with COPD and asthma demonstrated greater gas-trapping on chest CT. There were no differences in spirometry or CT measurements of emphysema or airway wall thickness.ConclusionSubjects with COPD and asthma represent a relevant clinical population, with worse health-related quality of life. They experience more frequent and severe respiratory exacerbations despite younger age and reduced lifetime smoking history.Trial registrationClinicalTrials.gov: NCT00608764

Extracellular superoxide dismutase in vessels and airways of humans and baboons

Extracellular superoxide dismutase (EC SOD) is generally the least abundant SOD isozyme in tissues, while the intracellular Cu,Zn SOD is usually the most abundant isozyme. The biological significance of EC SOD is unknown. Immunolocalization studies show that EC SOD is in the connective tissue surrounding smooth muscle in vessels and airways within the lung. Endothelium derived relaxing factor, thought to be a nitric oxide (NO) species, is a primary mediator of vascular relaxation. During NO.s diffusion between the endothelium and smooth muscle, extracellular superoxide would be the most efficient scavenger of NO(.). High levels of extracellular superoxide dismutase in vessels could, therefore, be essential to enable NO. to modulate vascular tone. To evaluate the hypothesis that vessel walls are functionally rich in extracellular superoxide scavenging capacity, this study quantitates the EC SOD levels in pulmonary and systemic vessels and in airways. Both pulmonary and systemic arteries in humans and baboons were found to contain high activities of EC SOD. The level of EC SOD in all human and baboon arteries examined is greater than or equal to the level of intracellular Cu,Zn SOD, and EC SOD accounted for over 70% of the total SOD activity in some vessels examined. Immunolocalization of EC SOD in human and baboon vessels show similar distributions of this enzyme in pulmonary and systemic vessels. EC SOD is located beneath the endothelium, surrounding smooth muscle cells, and throughout the adventitia of vessels. The high level of EC SOD in vessels, and its localization between endothelial and smooth muscle cells, suggest that regulation of superoxide may be particularly important in this region, possibly in regulating vascular tone.

The potential role of peroxynitrite in the vascular contractile and cellular energetic failure in endotoxic shock

Peroxynitrite is a toxic oxidant species produced from nitric oxide (NO) and superoxide. We have recently observed that the cell‐permeable superoxide dismutase mimetic Mn(III)tetrakis(4‐benzoic acid) porphyrin (MnTBAP) inhibits the suppression of mitochondrial respiration elicited by authentic peroxynitrite in vitro. Here we have investigated the relative potency of MnTBAP and a range of related compounds in terms of inhibition of peroxynitrite‐induced oxidation and cytotoxicity. In addition, we tested the effects of MnTBAP on the vascular and the cellular energetic failure in rodent models of endotoxic shock. We observed a dose‐related inhibition of the peroxynitrite‐induced oxidation of dihydrorhodamine 123 to rhodamine by MnTBAP, ZnTBAP and FeTBAP, but not by MnTMPyP [(5,10,15,20‐tetrakis(N‐methyl‐4′‐pirydyl)porphinato)‐manganese (III)]. In addition, MnTBAP, ZnTBAP and FeTBAP, but not MnTMPyP prevented the suppression of mitochondrial respiration by authentic peroxynitrite in cultured J774 macrophages. In rat cultured aortic smooth muscle cells, MnTBAP protected against the suppression of mitochondrial respiration in response to authentic peroxynitrite, immunostimulation and nitric oxide (NO) donor compounds. MnTBAP slightly reduced the amount of nitrite/nitrate produced in response to immunostimulation in these cells. Administration of MnTBAP, 15 mg kg−1 i.v., before the administration of endotoxin (15 mg kg−1, i.v.) to rats ameliorated the development of vascular hyporeactivity and the development of endothelial dysfunction in the thoracic aorta ex vivo. MnTBAP also prevented the endotoxin‐induced decrease in mitochondrial respiration, the development of DNA single strand breaks, and the depletion of intracellular NAD+ in peritoneal macrophages ex vivo. MnTBAP did not inhibit the expression by endotoxin of the inducible NO synthase in lung samples. MnTBAP did not alter survival rate in mice challenged with high dose endotoxin. Our findings, taken together with previous data demonstrating protective effects of NO synthase inhibitors against the endotoxin‐induced contractile and energetic failure in the models of shock used in the current study, and with the known ability of peroxynitrite to cause cellular energy depletion, suggest a role for peroxynitrite in the pathogenesis of cellular energetic failure and contractile dysfunction in endotoxin shock.

Hyperoxia increases oxygen radical production in rat lung homogenates

Abstract Lung damage during hyperoxia has been postulated to be due to increased rates of local organ oxygen radical production. Lung homogenate respiration was inhibited with cyanide, and residual respiration was used as an indicator of electron diversion to O 2 − and H 2 O 2 . Cyanide-resistant respiration in lung homogenates, supplemented with 1 m m NADH, increased linearly with oxygen tension, and accounted for 7% of total respiration in air and for 17% of total respiration when homogenates were incubated in 80% oxygen. Exposure of rats to 85% oxygen for 7 days induces tolerance to the lethal effects of 100% oxygen. Rats which previously breathed 85% oxygen for 7 days had a greater CN − -resistant respiration than control rats. This implies that adaptation to hyperoxia does not include decreased lung tissue oxygen radical production as indicated by CN − -resistant respiration. One possible explanation for the increased CN − -resistant respiration in oxygen tolerant rat lungs is that they contain increased cell mass. Lung homogenates of rats exposed to 85% oxygen for 7 days also had 2.5 times greater thiobarbituric acid positive material than controls, indicating that increased lung lipid peroxidation occurs as a consequence of hyperoxia. Incubation of normal rat lung homogenates under hyperoxic conditions also acutely increased lipid peroxidation, which could be inhibited by both superoxide dismutase and catalase. This confirms that hyperoxia enhances cellular production of O 2 − and H 2 O 2 and implies an essential role for both O 2 − and H 2 O 2 in hyperoxic lung damage.