Published ahead of print on January 6, 2006, doi:10.1164/rccm.200509-1387OC
© 2006 American Thoracic Society doi: 10.1164/rccm.200509-1387OC
Genetically Increased Antioxidative Protection and Decreased Chronic Obstructive Pulmonary DiseaseDepartment of Clinical Biochemistry, Herlev University Hospital, Herlev; Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital; The Copenhagen City Heart Study, Bispebjerg University Hospital, Copenhagen; Department of Respiratory Medicine, Hvidovre University Hospital, Hvidovre, Denmark; and Department of Clinical Chemistry, Umeå University Hospital, Umeå, Sweden Correspondence and requests for reprints should be addressed to Børge G. Nordestgaard, M.D., D.M.Sc., Department of Clinical Biochemistry, Herlev University Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark. E-mail: brno{at}herlevhosp.kbhamt.dk
Rationale: Increased oxidative stress is involved in chronic obstructive pulmonary disease (COPD); however, plasma and bronchial lining fluid contains the antioxidant extracellular superoxide dismutase. Approximately 2% of white individuals carry the R213G polymorphism in the gene encoding extracellular superoxide dismutase, which increases plasma extracellular superoxide dismutase 10-fold and presumably also renders bronchial lining fluid high in extracellular superoxide dismutase. Objective: We tested the hypothesis that R213G reduces the risk of COPD. Methods: We studied cross-sectionally and prospectively (during 24 yr) 9,258 individuals from the Danish general population genotyped for R213G. Measurements: We determined plasma extracellular superoxide dismutase concentration, pulmonary function and COPD diagnosed by means of spirometry or through national hospitalization and death registers. Main Results: In the general population, 97.5% were noncarriers, 2.4% were heterozygotes, and 0.02% were homozygotes. Among R213G noncarriers, extracellular superoxide dismutase plasma concentration was 148 ± 52 and 142 ± 43 ng/ml (mean ± SD) in individuals with and without COPD (Student's t test, p = 0.02). Among heterozygotes, corresponding concentrations were 1,665 ± 498 ng/ml and 1,256 ± 379 (p < 0.001). The adjusted odds ratio for spirometrically diagnosed COPD in heterozygotes versus noncarriers was 0.5 (95% confidence interval: 0.3–0.9). After stratification, the equivalent adjusted odds ratio was 1.5 (0.3–6.6) among nonsmokers and 0.4 (0.2–0.8) among smokers (p value for interaction = 0.10). The adjusted hazard ratio for COPD hospitalization or death during follow-up in heterozygotes versus noncarriers was 0.3 (0.1–0.8). Conclusions: Extracellular superoxide dismutase R213G heterozygosity protects against development of COPD in the Danish general population. This was observed in smokers, but not in nonsmokers.
Key Words: chronic obstructive pulmonary disease • epidemiology • genetics • smoking Oxidative stress is implicated in the pathogenesis of several lung diseases, most notably those with an inflammatory element such as chronic obstructive pulmonary disease (COPD) (1–12). Major oxidants are the reactive oxygen species (e.g., superoxide) and the most important extracellular scavenger of superoxide is extracellular superoxide dismutase (EC-SOD) (13). Interestingly, EC-SOD is expressed at particularly high concentrations in the lungs, where it has been immunohistochemically demonstrated in bronchial lining fluid, in epithelium of airways, and in pulmonary blood vessel walls (14, 15). This pattern of expression may reflect the importance of EC-SOD as an enzymatic defense in the lungs against oxidation by molecular oxygen and against oxidative stress from inhaled air pollutants such as cigarette smoke (3). We studied a polymorphism causing substitution of arginine for glycine at position 213 in the positively charged heparin-binding domain of EC-SOD. Heterozygous and homozygous carriers of EC-SOD R213G found in, respectively, 2.4 and 0.02% of the population have an approximately 10-fold and 40-fold plasma concentration of functional EC-SOD compared with noncarriers (16). It has been speculated that this increase is due to accelerated release of EC-SOD from heparan sulfate and collagen in the interstitial matrix where it is normally anchored (17–20). Because the mutation is associated with high EC-SOD plasma concentrations, the bronchial lining fluid may also be enriched in EC-SOD and could therefore offer enhanced protection against inhaled oxidants. We tested the hypothesis that EC-SOD R213G heterozygosity is associated with decreased risk of COPD. For this purpose, we genotyped 9,258 individuals from the Danish general population, and studied them cross-sectionally for COPD prevalence diagnosed by means of spirometry, as well as during 24-yr follow-up for incident hospitalization or death from COPD. Because we found only two homozygotes, this hypothesis could not be tested for EC-SOD R213G homozygotes.
Participants The Copenhagen City Heart Study is a prospective cardiopulmonary study of the Danish general population initiated in 1976–1978 with follow-up examinations in 1981–1983 and 1991–1994 (21–25). Informed consent was obtained from all participants, and the study was approved by the ethics committee for the City of Copenhagen and Frederiksberg (#100.2039/91) as well as by Herlev University Hospital. The Danish ethics committee system does not require that we ask participants for consent to each specific DNA analysis.
Study Design
Prospective studies.
EC-SOD Plasma Concentration In addition to this subsample, we measured EC-SOD on the 136 participants with COPD who had the lowest FEV1/FVC ratio. EC-SOD was determined in duplicate using ELISA, as previously described (27).
Spirometry Instruments were calibrated daily against a 1-L syringe. Each spirometry was performed in triplicate and results accepted only if variation between two of these were less than 5%. The highest measurements of FEV1 and FVC were used. FEV1 and FVC were used as percent of predicted values. Algorithms for the calculation of FEV1% predicted and FVC% predicted were made using multiple regression with age and height as covariates on a subsample of never-smokers for men and women separately.
COPD Even though smoking is the major determinant of COPD, it is well established that not all COPD can be attributed to smoking. In both the cross-sectional and the prospective study, we therefore chose to diagnose COPD in all participants who met the stated criteria irrespective of smoking history. In the Copenhagen City Heart Study using the COPD diagnostic criteria of the cross-sectional study, 6% of COPD patients (GOLD stage II or higher) claimed to be never-smokers.
DNA Analyses
Statistical Analyses
Among participants from the Danish general population, 97.5% (n = 9,031), 2.4% (n = 225), and 0.02% (n = 2) were EC-SOD R213G noncarriers, heterozygotes, or homozygotes, respectively. This genotype distribution did not differ from that predicted by the Hardy-Weinberg equilibrium (  2 test, p = 0.65). EC-SOD R213G genotype was not associated with any of the basic characteristics examined (Table 1). Plasma EC-SOD concentration was 142 ± 45 ng/ml (mean ± SD) in noncarriers, 1278 ± 394 in heterozygotes (p < 0.001 vs. noncarriers), and 4,147 ± 377 in homozygotes (p < 0.001 vs. noncarriers) (Table 1) (16). Apart from FEV1% predicted, which did not differ between patients with COPD GOLD stage I and controls, and FVC% predicted, which was higher in COPD GOLD stage I patients compared with controls, FEV1% predicted, FVC% predicted, and FEV1/FVC were lower in COPD patients compared with controls irrespective of GOLD stage (Table 2).
Cross-sectional Study Plasma EC-SOD concentration. Among EC-SOD R213G noncarriers, plasma EC-SOD concentrations were 148 ± 52 (mean ± SD) and 142 ± 43 ng/ml in those with and without COPD (Student's t test, p = 0.02; Figure 1). Corresponding plasma concentrations among heterozygotes were 1,665 ± 498 and 1,256 ± 379 ng/ml (p < 0.001). This difference remained significant in noncarriers after adjustment for sex, smoking, age, exposure to occupational dust, and ischemic heart disease (adjusted EC-SOD values 142 ± 17 vs. 137 ± 19 ng/ml, analysis of covariance, p = 0.02), but was attenuated in heterozygotes (1,609 ± 212 vs. 1,217 ± 180 ng/ml, p = 0.08). A similar pattern of higher plasma EC-SOD concentrations in COPD patients compared with controls was also observed after stratification into smokers and nonsmokers (Figure 1).
Pulmonary function. Among smokers and nonsmokers combined or among nonsmokers alone, pulmonary function did not differ by EC-SOD R213G genotype (Figure 2). Among smokers, FEV1/FVC was higher for EC-SOD R213G heterozygotes than for noncarriers, a difference largely explained by a higher FEV1 in heterozygotes.
COPD. Among participants with (n = 978) and without (n = 7,604) COPD, 1.5% (n = 15) and 2.6% (n = 196) were EC-SOD R213G heterozygotes ( 2 test, p = 0.05). This corresponds to an unadjusted odds ratio of 0.6 (95% confidence interval, 0.3–1.0; Table 3). Adjusting for gender, age, smoking, and exposure to occupational dust produced an odds ratio for COPD in heterozygotes versus noncarriers of 0.5 (0.3–0.9). Equivalent adjusted odds ratios were 1.5 (0.3–6.6) among nonsmokers and 0.4 (0.2–0.8) among smokers (test for interaction: p = 0.10). None of the two EC-SOD R21G homozygotes had spirometrically defined COPD.
Prospective Study The cohort was followed for 186.877 person-years (median follow-up: 23.9 yr), during which 496 participants were hospitalized with or died from COPD (incidence rate 27 events/10.000 person-years [95% confidence interval: 24–29]). At the age of 90 yr, the cumulative incidence of COPD hospitalization or death was 7% in heterozygotes versus 17% in noncarriers (log-rank: p = 0.08; Figure 3). The unadjusted hazard ratio for COPD in heterozygotes versus non-carriers was 0.5 (0.2–1.1; Table 3). Adjustment for age at entry, gender, smoking, exposure to occupational dust, and calendar date produced an equivalent hazard ratio of 0.3 (0.1–0.8). None of the two EC-SOD R213G homozygotes were hospitalized with or died of COPD.
Cigarette smoking is the major cause of COPD, yet only a minority of smokers develop clinically relevant COPD (11). This suggests varying lung susceptibility to the deleterious effects of cigarette smoke among individuals in the general population. Genetic polymorphisms are likely explanations for such differences (4, 11). However, despite the fact that COPD is a leading cause of death in developed countries and has been extensively studied, the only well-established genetic risk factor for COPD is  l-antitrypsin deficiency. A novel candidate gene for COPD is EC-SOD, which is interesting for several reasons: (1) Compared with other tissues in the human body, lung parenchyma has an exceptionally high concentration of EC-SOD (11); (2) animal studies have demonstrated a reduced inflammatory and fibrotic response to bleomycin and lipopolysaccharides in transgenic mice overexpressing human EC-SOD (30, 31); (3) plasma EC-SOD concentration has been reported to be higher in COPD patients than in healthy controls (32), which was confirmed in the present study stratified for R213G; (4) EC-SOD or other SOD isoenzymes have been implicated in other lung diseases such as asthma, interstitial lung disease, respiratory distress syndrome, bronchopulmonary dysplasia, and lung malignancy, yet little is known about EC-SOD and COPD (3); and (5) cigarette smoke contains vast amounts of reactive oxygen species (e.g., superoxide anions), the major extracellular scavenger of which is EC-SOD (13). These highly reactive substances are capable of inducing pulmonary tissue damage characteristic of COPD (2, 12) Additionally, smoking increases the number of leucocytes in the airways and their superoxide generation (33, 34). More than 90% of EC-SOD is located in the extravascular space bound to heparan sulfate proteoglycans in the glycocalyx of endothelial cell surfaces and in connective tissue matrix (35–37). The EC-SOD R213G polymorphism affects a cluster of positively charged amino acids that are responsible for binding to the extracellular matrix (17, 20). Therefore, carriers of this polymorphism have an enzyme with reduced affinity for negatively charged glucosaminoglycans and type 1 collagen, presumably leading to accelerated loss of EC-SOD from the extracellular matrix into surrounding fluids (17, 18). Such redistribution can explain the 10-fold and 40-fold increased plasma concentration observed in heterozygous and homozygous carriers of EC-SOD R213G (16). We report an association between COPD and EC-SOD R213G, which is known to profoundly affect EC-SOD plasma concentration. Although this is an interesting finding, the results would be much more persuasive if there were evidence that the polymorphism affected EC-SOD levels in the lung. Such evidence has yet to be published. A higher EC-SOD concentration in the bronchial lining fluid could be beneficial in counteracting deleterious effects of cigarette smoke and could explain the association between reduced risk of COPD and EC-SOD R213G heterozygosity observed in our study. Alternatively, the high plasma concentration of EC-SOD in heterozygotes could exert a protective effect directly in lung parenchyma. Such a mechanism seems equally likely, considering the small anatomic distance between plasma and inhaled oxidative stressors in the alveoli. Our study did not demonstrate differences in pulmonary function measurements by EC-SOD R213G genotype apart from a higher FEV1/FVC ratio in smoking heterozygotes compared with smoking noncarriers. This may at first glance seem contradictory to the finding of a decreased COPD prevalence among heterozygotes compared with noncarriers because COPD in the cross-sectional study is diagnosed spirometrically. However, R213G heterozygosity may not influence lung function in the average person, but could still exert a profound influence in those exposed to excessive oxidative stress in the lung, those most prone to develop COPD. In addition, data from the prospective study, which investigates COPD diagnosed in a completely different manner (hospitalization or death), confirm our finding of reduced COPD in R213G heterozygotes. Maybe the major clinical effect of the EC-SOD R213G polymorphism is an ability to reduce exacerbations in COPD and therefore lower the rate of hospitalization. Interestingly, Bowler and colleagues found that lipopolysaccharide-induced neutrophilic lung inflammation was exaggerated in EC-SOD–deficient mice and diminished in mice that overexpressed EC-SOD. Alternatively, EC-SOD R213G heterozygotes are protected from respiratory muscle dysfunction induced by oxidative stress (38). An interesting observation in the cross-sectional part of our study was the fact that the reduced risk of COPD in R213G heterozygotes versus noncarriers observed overall appeared to be due to an association among smokers. We believe that this finding makes biological sense considering the pronounced oxidative stress exerted by inhaled cigarette smoke. However, there was no statistical evidence to support an interaction between the EC-SOD R213G polymorphism and smoking; therefore, we cannot statistically exclude that R213G heterozygosity may also protect against COPD in nonsmokers. Unfortunately, we were not able to investigate the apparent interaction between smoking and genotype on COPD in the prospective study because of insufficient statistical power.
Limitations We chose to exclude participants claiming to have asthma from this study. The diagnosis of asthma in epidemiologic studies may be problematic and the differential diagnosis between asthma and COPD is even more difficult than in the clinical setting. The use of a self-reported diagnosis of asthma invariably leads to some degree of misclassification, because some subjects with mild asthma remain undiagnosed, whereas some subjects with COPD are likely to have reported asthma and are thus excluded from the group with COPD. However, previous studies have shown that using the question "Do you have asthma?" is a reasonable approach (40, 41). Importantly, we believe that misclassification of end-point is of limited importance in our study for two reasons: (1) Misclassification of end-point is unrelated to EC-SOD R213G genotype and will invariably drive risk estimates toward unity (i.e., underestimates a true association). Therefore, end-point misclassification cannot account for the reduced COPD risk in EC-SOD R213G carriers observed. (2) The use of two independent and different ways of diagnosing COPD (COPD diagnosed using spirometry or COPD hospitalization/death) serves to validate results. We did not apply the American Thoracic Society criteria on spirometric testing but used the criteria described in this article. We acknowledge that our approach is less accurate, but on the other hand, more subjects are able to comply with our criteria, making exclusion of the subjects because of an inability to perform a correct spirometry a less common event. We have previously compared our field spirometric data with the Danish reference values produced in a laboratory and found no significant differences (24). We observed higher EC-SOD levels in plasma of COPD patients than among healthy individuals irrespective of genotype, which at first seemed surprising. This is because R213G heterozygosity with 10-fold the plasma levels of noncarriers were protected against COPD, whereas COPD patients had slightly higher plasma EC-SOD levels than controls irrespective of genotype status. One possible explanation for such apparently conflicting data could be that extremely high EC-SOD levels protect against COPD, but that COPD in itself causes a small increase in EC-SOD levels in plasma. It should be stressed that the EC-SOD measurements reported in this study were performed for other purposes than for investigating EC-SOD in relation to COPD (16). Although EC-SOD levels were not the primary focus of this article, we nevertheless chose to include this data. EC-SOD comparisons in this study between COPD patients and controls could be influenced by differences other than COPD status (such as sex, age, smoking habits, exposure to occupational dust, or ischemic heart disease). However, adjusting for these covariates did not alter our conclusions. Our conclusion of higher EC-SOD in patients with COPD compared with healthy control subjects is further supported by data from Folz and coworkers (32), who reported a 30% higher plasma EC-SOD concentration in 155 patients with a variety of chronic lung diseases versus 18 control subjects. The synthesis of EC-SOD is regulated by a variety of growth factors, vasoactive factors, and inflammatory cytokines but not by oxidant stress (42–44); however, the specific factors causing higher plasma levels in COPD cases are at present difficult to deduce. We measured levels of EC-SOD in plasma and not in lung lining fluid, and levels in plasma may not reflect concentrations in lung lining fluid. COPD damages most of all the lungs and thus the levels of EC-SOD, which are important are the lung levels. Because this is a study of a sample of the general population, we do not have approval from the Danish ethics committee to perform bronchoalveolar lavage on these participants. However, we hope that other research groups can examine whether EC-SOD levels are indeed elevated in bronchoalveolar lavage in heterozygotes for R213G. A possible source of bias comes from the fact that DNA samples were not obtained until 1991–1994. However, because EC-SOD R213G genotype is not acquired but remains constant throughout life, genotype precedes the development of disease. Also, results from the prospective part of this study are supported by results obtained in the traditional cross-sectional part of the study. Misclassification of EC-SOD genotype is another possible limitation. However, all carriers of the EC-SOD R213G polymorphism had their genotype verified using DNA sequencing. Furthermore, because EC-SOD plasma concentration is 10 times higher in heterozygotes and 40 times higher in homozygotes than in noncarriers, measurements of EC-SOD plasma concentration serves as an additional check to verify genotype. No inconsistencies were observed between the restriction enzyme based assay, the sequencing assay or the plasma EC-SOD measurements. The Copenhagen City Heart Study is an ethnically very homogenous study population, which comprises almost exclusively individuals of Danish decent. Thus 99% of the participants were whites. Obviously, studying COPD by EC-SOD R213G genotype stratified by ethnicity in our study was not possible because of insufficient statistical power in other ethnic groups than whites. Including ethnicity in the multifactorial logistic regression model or the multifactorial Cox proportional hazards model did not change the risk estimate for COPD in EC-SOD R213G heterozygotes in the present study. Despite this, our findings cannot automatically be extended to other ethnic groups, particularly as frequency of heterozygotes may differ between different ethnic groups (Table 4). It is interesting that the R213G heterozygous state seems to be more prevalent among Asians and Eskimos than among whites, opening the possibility that at a population level EC-SOD R213G heterozygosity may have an even larger impact on COPD protection in Asians and Eskimos than among whites.
In conclusion, we find that COPD is associated with a higher plasma concentration of EC-SOD irrespective of EC-SOD R213G genotype. In addition, heterozygous carriers of the EC-SOD R213G polymorphism in the Danish general population have an approximate 40–70% reduction in the risk of COPD compared with noncarriers.
The authors thank biotechnicians Nina Dahl and Karin Hjertkvist for their skillful technical assistance.
Supported by the Danish Heart Foundation, The Danish Medical Research Council, Chief Physician Johan Boserup and Lise Boserup's Fund, Lykfeldt's Fund, Dagmar Marshall's Fund, Wedelborg's Fund, Lily Benthine Lund's Fund, The Beckett Fund, and P. Carl Petersen's Fund. The sponsors of the study are public or nonprofit organizations that support science in general. They had no role in gathering, analyzing, or interpreting the data and had no right to approve or disapprove of the submitted paper. This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200509-1387OC on January 6, 2006 Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form September 7, 2005; accepted in final form January 4, 2006
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ABSTRACT
METHODS
80 %predicted; GOLD stage III: FEV1/FVC < 70% and 30% predicted < FEV1 
Gly extracellular superoxide dismutase (EC-SOD) in plasma is caused by a reduction of both heparin and collagen affinities. Biochem J 2005;15:427–432.
