PEDIATRICS Vol. 121 No. 3 March 2008, pp. 555-561 (doi:10.1542/peds.2007-2479)
ARTICLE |
Plasma Biomarkers of Oxidative Stress: Relationship to Lung Disease and Inhaled Nitric Oxide Therapy in Premature Infants
a Department of Pediatrics, University of California, San Francisco, California
b Department of Pediatrics, Children's Mercy Hospitals and Clinics/University of Missouri-Kansas City School of Medicine, Kansas City, Missouri
c Department of Pediatrics and Biostatistics, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania
d Department of Pediatrics, New York Medical College/Maria Fareri Children's Hospital at Westchester Medical Center, Valhalla, New York
e Department of Pharmacology and Toxicology, Rutgers University, New Brunswick, New Jersey
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ABSTRACT |
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 TOP ABSTRACT PATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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OBJECTIVES. Inhaled nitric oxide treatment for ventilated premature infants improves survival without bronchopulmonary dysplasia. However, there has been no information regarding possible effects of this therapy on oxidative stress. We hypothesized that inhaled nitric oxide therapy would not influence concentrations of plasma biomarkers of oxidative stress.
PATIENTS AND METHODS. As part of the Nitric Oxide Chronic Lung Disease Trial, we collected blood samples at specified intervals from a subpopulation of 100 infants of <1250 g birth weight who received inhaled nitric oxide (20 ppm, weaned to 2 ppm) or placebo gas for 24 days. Plasma was assayed for total protein and for 3-nitrotyrosine and carbonylation by using immunoassays.
RESULTS. The demographic characteristics and primary outcome for the infants were representative of the entire group of infants who were in the Nitric Oxide Chronic Lung Disease Trial. For all infants at baseline, before receiving study gas, the concentration of total protein was inversely correlated with the respiratory severity score, and plasma carbonyl was positively correlated with severity score, supporting an association between oxidative stress and severity of lung disease. Infants who survived without bronchopulmonary dysplasia had 30% lower protein carbonylation concentrations at study entry than those who had an adverse outcome. At each of 3 time points (1–10 days) during exposure to study gas, there were no significant differences between control and treated infants for concentrations of plasma protein, 3-nitrotyrosine, and carbonylation.
CONCLUSIONS. Inhaled nitric oxide treatment for premature infants who are at risk for bronchopulmonary dysplasia does not alter plasma biomarkers of oxidative stress, which supports the safety of this therapy.
Key Words: nitric oxide • premature infant • 3-nitrotyrosine • carbonyl • bronchopulmonary dysplasia
Abbreviations: NO—nitric oxide • iNO—inhaled nitric oxide • BPD—bronchopulmonary dysplasia • NO-CLD—Nitric Oxide Chronic Lung Disease • RSS—respiratory severity score • PMA—postmenstrual age • FIO2—fraction of inspired oxygen
Although the benefit and safety of inhaled nitric oxide (iNO) therapy for persistent pulmonary hypertension in term and near-term infants is well established, the use of iNO treatment in premature infants with respiratory failure or chronic lung disease is still under investigation. Controlled clinical trials of iNO in moderately ill, ventilated premature infants have reported benefit in prevention of bronchopulmonary dysplasia (BPD) without apparent adverse effects, particularly in infants between 1000 g and 1250 g birth weight.1–6 In addition, there is evidence of neurologic protection in treated infants.1,5
Premature infants experience oxidative stress as a result of lung disease and exposure to oxygen and ventilator therapy (reviewed in ref 7). Reactive oxygen and nitrogen species increase formation of relatively stable carbonyl adducts of proteins by both direct and indirect oxidation reactions.8 Protein carbonylation can modify protein structure, impair protein function or metabolism, and contribute to disease processes.9 In addition, superoxide anion interacts with endogenous NO to form peroxynitrite, a reactive molecule that modifies proteins (nitration of tyrosine residues), lipids, and DNA and may alter function. Peroxynitrite is increased under conditions of oxidative stress or elevated NO production.10 Alternatively, reactive nitrogen species can be generated during disease states by the oxidation of nitrite, one of the stable products of nitric oxide metabolism, by the action of inflammatory cell peroxidases.11 In some studies, treatment of animals with high levels of oxygen and iNO has adverse pulmonary effects.12–15
Because of the known role of NO in oxidative damage and concern that iNO could potentially increase formation of reactive oxygen and nitrogen species, we prospectively examined plasma biomarkers of oxidative stress as part of the safety assessment in the Nitric Oxide Chronic Lung Disease (NO-CLD) Trial, in which premature infants at high risk for BPD were treated with iNO.4 We collected blood samples from a subpopulation of study infants and determined relationships between the content of total plasma protein, carbonyls and 3-nitrotyrosine and severity of lung disease, and the effects of iNO treatment on concentrations of the biomarkers. In this article we describe associations between the severity of lung disease and both total plasma protein concentrations and carbonylation. We found that exposure to iNO did not affect concentrations of either total protein or the biomarkers.
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PATIENTS AND METHODS |
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Study Population
The NO-CLD Trial was a randomized, blinded, and controlled trial of iNO performed at 21 centers. Consent was obtained for infants of 500 to 1250 g birth weight who still required ventilatory support between 7 and 21 days of age; they were enrolled in the NO-CLD Trial to receive either iNO (INO Therapeutics LLC, Clinton, NJ) or placebo for 24 days. The study gas was administered at 20 ppm for 48 to 96 hours and then decreased to 10 ppm for 7 days, to 5 ppm for 7 days, and then to 2 ppm until the trial was discontinued. Clinical data were collected, including the respiratory severity score (RSS), which is the fraction of inspired oxygen (FIO2) x mean airway pressure, as an index of severity of lung disease. RSS was used rather than the oxygenation index, which requires an arterial line for measurement of PAO2. Details of the NO-CLD Trial protocol, infant characteristics, and outcome have been published previously.4,6
Plasma Samples
Blood samples (
0.5 mL) were collected prospectively from a subpopulation of infants who were enrolled in the NO-CLD Trial for whom consent was obtained for blood and tracheal aspirate collection at 4 centers: Children's Hospital of Philadelphia, Hospital of the University of Pennsylvania, Children's Mercy Hospitals and Clinics, and Maria Fareri Children's Hospital at Westchester Medical Center. There were a total of 100 infants with blood samples that were collected when they entered the study (just before receiving study gas) and at 24 hours, 4 days, and 10 days, respectively, after initiation of study gas. Plasma was obtained by centrifugation and stored at –70°C until it was assayed. Because some samples were not obtained on the predetermined day, analyses of time course data combined samples for 3 to 5 days, and 9 to 11 days, respectively; for some infants, 2 samples were collected within the later time intervals, and the mean value was used for analysis. All analyses were limited to the 100 infants with 1 preentry and at least 1 postentry plasma sample of sufficient amount for assay.
Assays
Carbonyl content was assessed by enzyme-linked immunosorbent assay, with a slight modification of the procedure used by Buss et al.16 In this assay, plasma samples were reacted with dinitrophenylhydrazaine, applied to multiwell plates, and probed with antidinitrophenylhydrazaine antibody and secondary antibody. Dinitrophenylhydrazaine, fully reduced bovine serum albumin (negative control), hypochloorus acid–oxidized bovine serum albumin standards, and plasma quality controls were obtained from Zenith Technology Corp Ltd (Dunedin, New Zealand). Polyclonal antibody raised against a dinitrophenylhydrazaine conjugate of keyhole limpet hemocyanin (rabbit immunoglobulin G fraction) was purchased from Invitrogen (Eugene, OR); antirabbit immunoglobulin G secondary antibody, O-phenylenediamine dihydrochloride reagent, and other biochemicals were from Sigma-Aldrich Co (St Louis, MO). Multiwell plates were from Nunc-Immuno Maxisorp (Nalge Nunc International, Rochester, NY). The sensitivity of the assay was 1 nmol/mL, and intraassay and interassay variations were 1.4 and 8.8%, respectively. Carbonyl results for plasma samples are expressed as pmol/mg protein which was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA).
The 3-nitrotyrosine content was assessed by using a sandwich enzyme-linked immunosorbent assay, with monoclonal and polyclonal antibodies and 3-nitrotyrosine standards provided in the Bioxytech Nitrotyrosine-EIA assay kit (Oxis International Inc, Foster City, CA). Two infant plasma levels with low and high 3-nitrotyrosine content were included in each assay as internal controls. The sensitivity of the assay was 100 pg/mL, and the coefficients of variation were 2.3% and 11.2% for intraassay and interassay, respectively. Results for 3-nitrotyrosine are expressed as pg/mg protein.
All measurements were performed before the treatment code was known. For each assay, all plasma samples from each infant were assayed together in the same experiment.
Statistics
Data were available for plasma parameters on 100 patients with both 1 baseline and at least 1 postentry sample. Reasons for unavailable or censored data included inadequate volume of plasma, gross hemolysis, and collection of a sample outside the window of days for each time point during treatment. Data for both carbonyl and 3-nitrotyrosine were skewed and log-transformed for statistical analysis with presentation of the geometrical mean and range. To test for significant differences between the groups that received iNO and placebo, we used Fisher's exact test for categorical data and Student's t test for continuous variables. The possible influence of infant gender on survival without BPD was tested by using both logistic modeling and generalized estimating equation. The statistical software package SAS version 9.13 (SAS Institute, Inc, Cary, NC) was used for analyses, with .05 as the significance level for all tests.
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RESULTS |
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TOPABSTRACTPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The demographic characteristics of the infants included in this study (n = 100) are shown in Table 1. Birth weights ranged from 502 to 1105 g and were not different between control and iNO-treated groups (mean: 744 and 748 g, respectively). Gestational age ranged from 22.7 to 30.0 weeks, and the mean age was the same for the 2 groups (25.7 weeks). Infants in both groups were entered onto the study between 7 and 21 days with mean values of 15 days of age for both groups. There was a wide range of severity of respiratory illness, as judged by the RSS, at study entry (range: 1.2–12.7), with the same mean value (3.9) for control and iNO-treated groups. Fifty-one percent of treated infants survived without BPD compared with 29% of control infants (P = .02). Two control and 3 treated infants died before 36 weeks' postmenstrual age (PMA). The demographic characteristics and primary outcome results for the subpopulation of infants in this study are representative of the entire group of infants who were in the NO-CLD Trial (n = 582).4
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We first examined the relationship between concentrations of plasma parameters and severity of respiratory illness at study entry. For these analyses, we used the entire population of infants and plasma values from samples that were collected 24 hours or less before beginning administration of study gas. Figure 1 presents regression analyses of total protein, carbonyl/protein as a marker of oxidative stress, and 3-nitrotyrosine/protein as a marker of oxidative/nitrative stress. The concentration of total protein was inversely correlated with RSS (r = –0.25; P = .03; Fig 1A). Plasma carbonyl content, normalized to total plasma protein, was positively correlated with RSS (r = 0.49; P < .001; Fig 1B). A similar positive correlation was observed with analysis of RSS and carbonyl content normalized to volume of plasma (r = 0.31; P = .008; data not shown), which is consistent with an association between carbonyl content and severity of lung disease independent of total protein concentration. Plasma 3-nitrotyrosine was not significantly correlated with either RSS (r = 0.18; P = .11; Fig 1C) or carbonyl concentration (r = 0.17; P = .13; data not shown). By contrast, concentrations of plasma protein, 3-nitrotyrosine, and carbonyl for infants at study entry were not significantly correlated with gestational age (for protein: r = 0.15; P = .16) (for 3-nitrotyrosine: r = 0.13; P = .23) (for carbonyl: r = –0.08; P = .47).
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We further examined the relationship between lung disease and concentrations of total protein and carbonylation in an analysis of infant outcome. Table 1 presents levels of plasma parameters at study entry and outcome, which was defined as survival without BPD at 36 weeks' PMA versus death or BPD. This was the primary outcome in the NO-CLD Trial, with the diagnosis of BPD being determined by a room-air-challenge test for infants with an FIO2 of <0.3. Concentration of total protein at study entry did not predict outcome at 36 weeks' PMA. Infants who survived without BPD had
30% lower carbonyl concentrations at study entry (P = .03) than infants with an adverse outcome. By contrast, concentrations of 3-nitrotyrosine were similar in infants with favorable versus adverse outcomes. Consistent with the observed association between carbonyl concentration and RSS (Fig 1B), infants who survived without BPD had a significantly lower mean RSS at study entry (P = .001). Data for concentrations of plasma parameters during administration of study gas are presented in Table 2. At study entry, levels of protein, carbonyl/protein, and 3-nitrotyrosine/protein were comparable for control and iNO-treated infants. The content of carbonyl decreased during the 10-day study period for both treatment groups; however, there was no change for concentration of 3-nitrotyrosine or total protein. At each of 3 time points during exposure to the study gas, there were no statistically significant differences between control and treated infants for concentrations of the 3 parameters. We also examined, at each time point, the change in concentration from the preentry value for each infant (as both absolute and percent change); when using this approach, there were also no significant differences noted between control and treated groups. In addition, the direction of change in protein, 3-nitrotyrosine and carbonyl after 3 to 4 days of iNO treatment at 20 ppm compared with study-entry values was not related to outcome at 36 weeks' PMA (data not shown).
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DISCUSSION |
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TOPABSTRACTPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As part of the safety assessment of the NO-CLD Trial, this study addressed the role of oxidative and nitrative stress in the evolution of infant lung disease and responses to iNO treatment. By using assays of plasma biomarkers, we found an association of protein carbonyl concentration with severity of lung disease in the second and third weeks of life and with the occurrence of BPD at 36 weeks' PMA. By contrast, the concentration of protein 3-nitrotyrosine at study entry was not correlated with disease severity or outcome. The concentration of total plasma protein was inversely correlated with severity of lung disease but did not predict later outcome. These findings are consistent with occurrence of oxidative stress in premature infants during lung disease and its treatment, and they support the concept that oxidative events contribute to the pathogenesis of BPD. Treatment of infants with iNO did not significantly change either 3-nitrotyrosine or carbonyl content of plasma proteins, which supports the safety of this therapy and indicates that benefits of iNO on pulmonary outcomes do not seem to involve reduced levels of oxidative or nitrative stress.
It is generally accepted that oxidant generation and oxidative injury contribute to diseases of the premature infant, in particular chronic lung disease (reviewed in ref 7). Protein carbonyl derivatives have been examined previously as a biomarker of oxidant stress in infants. In a study with premature infants from birth to 28 days of age, Winterbourn et al17 reported elevated plasma carbonyl concentrations compared with adult plasma concentrations. However, carbonyl levels did not correlate with days on supplemental oxygen or requirement for oxygen at 36 weeks' PMA. In our study, with the same assay, levels of carbonyl were comparable with those described by Winterbourn et al,17 and in addition, levels were related to severity of lung disease at both study entry and 36 weeks' PMA. This association of carbonyl content with disease severity was independent of gestational age and birth weight, factors that influence the occurrence and severity of infant lung disease. Our findings are in agreement with other earlier studies that noted an association between protein oxidation18 and other oxidative biomarkers (malondialdehyde, 3-nitrotyrosine, free iron) with chronic lung disease.19–21
The factors involved in oxidative stress in the premature infant are certainly numerous, complex, and interactive and likely include immature lung structure, deficient antioxidant defenses, free iron content, influx of plasma proteins into air spaces, supplemental oxygen, ischemia-reperfusion injury, and NO levels. In an animal model of lung injury with hyperoxia and surfactant deficiency, for example, lung function (oxygenation and surfactant activity) and an oxidative stress biomarker (malondialdehyde) were adversely affected by treatment with iNO (100 ppm) and iron-containing transferrin; these effects reflect, in part, inhibitory effects of oxidatively modified plasma proteins on alveolar surfactant.15,22 Oxidative modification of epithelial proteins also likely occurs with unidentified effects on lung function, growth, and repair. Our findings regarding carbonyl concentration, along with previous studies with other biomarkers,18–21 support the biological association between oxidative stress and pathogenesis of infant chronic lung disease. Therapeutic approaches to improve antioxidant defenses (such as superoxide dismutase23) may have added benefit in conjunction with iNO therapy.
Nitration of proteins in both plasma and tissues is mediated primarily by reactive nitrogen species including peroxynitrite,24 which is formed by a reaction between superoxide and NO. Thus, both oxidative stress, which is increased in the presence of supplemental O2, and NO of either endogenous or exogenous source can contribute to formation of reactive nitrogen species and the subsequent oxidation of lipids and DNA and nitration of tyrosine residues in proteins. Alternatively, excess exogenous NO could scavenge peroxynitrite to form nitrogen dioxide and nitrite, thereby decreasing 3-nitrotyrosine formation. In the NO-CLD Trial we were concerned that iNO therapy administered to infants who received supplemental oxygen might enhance formation of reactive nitrogen species with potentially adverse effects on the lung or other tissues (eg, brain). To address this possibility, we determined the plasma concentration of 3-nitrotyrosine, which has been used as a biomarker of peroxynitrite formation and oxidative stress in both humans and animals.10,25–27 We found no differences in 3-nitrotyrosine concentrations between control and iNO-treated infants during administration of 20 and 10 ppm, respectively, of iNO, which is similar to the observation in premature baboons at 5 ppm iNO.28 This finding further supports the safety of iNO treatment under conditions of the NO-CLD Trial but may not necessarily apply to use of iNO in extremely ill newborn infants receiving a high FIO2 level. In addition, it is possible that NO treatment could increase, or decrease, oxidation of proteins in the lung, which would not be detected in the analysis of plasma biomarkers.
Previously, we reported that 3 days of iNO treatment for infants with established, severe BPD did not significantly change levels of plasma 3-nitrotyrosine; however, the outcome was improved for those infants who had a decrease in 3-nitrotyrosine levels after exposure to iNO.29 In the current study, 3-nitrotyrosine concentrations were also unaffected by iNO therapy; however, the direction of change from baseline value for individual infants was not associated with outcome at 36 weeks' PMA. The current patient population was less ill and received iNO in the second and third weeks compared with >4 weeks in the previous study. Also, treated infants in the NO-CLD Trial had no immediate improvement in oxygenation after starting iNO therapy, in contrast to improvement in many infants in the earlier study, which likely reflected better ventilation:perfusion match and, perhaps, reduced pulmonary hypertension. The difference in lung pathology between the 2 groups of iNO-treated infants may account for the different observed responses of 3-nitrotyrosine to iNO therapy, and differences in absolute concentrations of 3-nitrotyrosine between studies likely reflect the different assays and antibodies.
Earlier, we reported that infants who developed BPD, compared with infants without BPD, had elevated plasma 3-nitrotyrosine levels between 1 and 28 days of age.20 By contrast, in this study, 3-nitrotyrosine concentration at study entry (mean: 16 days) did not predict outcome at 36 weeks' PMA. This different result may reflect the higher-risk status of infants in the NO-CLD Trial, all of whom were intubated for ventilatory support at study entry. To our knowledge, there have been no other studies of this topic in infants.
A relationship between plasma protein concentration and lung disease in premature infants was first described by Bland,30 who found lower cord plasma protein levels in infants who developed respiratory distress syndrome. More recently, Moison et al31 described lower plasma total protein and albumin at days 1 through10 for infants with respiratory distress syndrome and an oxygen requirement at 28 days; protein concentration was influenced by both duration and level of oxygen therapy. Our finding of an inverse correlation of plasma protein levels with an index of severity of lung disease supports the previous observations. Because blood samples were not available before study entry, we cannot determine whether plasma protein was lower from birth for the sicker infants or developed during the first 2 weeks of life. It is generally felt that lower plasma protein levels result from increased alveolar permeability and influx of plasma protein into alveoli, and that the amount of pulmonary edema is related to severity of lung disease. In addition, plasma protein levels may be lower, and lung disease more severe, in infants who are less developmentally mature for their gestational age. This possibility is consistent with the observed association of respiratory distress syndrome with low protein levels in cord blood.30
There are limitations to this study. Blood samples were collected over a period of 4 years and stored for prolonged periods at –70°C, which possibly affected concentrations of carbonyl or 3-nitrotyrosine (instability) or plasma protein (lyophylization), thus increasing variation in the data. However, both biomarkers are reported to be stable with storage,17,32 and only a few protein values were unusually high; these values were omitted from analyses of protein concentration. The 3-nitrotyrosine assay uses a monoclonal antibody for capture of nitrated proteins; thus, the antibody may not recognize all 3-nitrotyrosine residues because of a restricted epitope spectrum. At present, there is no apparent gold-standard assay for 3-nitrotyrosine. The absolute levels of 3-nitrotyrosine in this study are lower than those that we reported previously, which likely reflects different antibodies and assay conditions. The absolute 3-nitrotyrosine concentrations may be underestimated in both control and iNO-treated infants, and it is possible that iNO affected nitration of specific proteins that were not detected by the antibody used. Postnatal dexamethasone treatment reduces carbonyls and increases plasma protein levels.17,31 The use of dexamethasone at any time was similar in control and treated groups (30.6% and 35.3%, respectively) and, thus, should not substantially impact our results. Our findings of the apparent safety of iNO treatment related to oxidative and nitrative stress biomarkers may apply only to infants treated under conditions of the NO-CLD Trial: 7 to 21 days of age at study entry, treatment with 20 ppm iNO, and generally moderate disease severity. Finally, the study was not sufficiently powered to detect relatively small differences between groups that might have physiologic significance.
We recently reported other laboratory results related to the safety of iNO in the NO-CLD Trial. Treatment did not affect concentrations of various tracheal aspirate inflammatory biomarkers nor the level of 8-isoprostane, a marker of membrane oxidative injury.33 In other studies of tracheal aspirates, we found that iNO therapy did not alter surfactant recovery or protein composition and may have transiently improved surfactant function. The current findings for plasma oxidative/nitrative biomarkers complement the other data and support the benefit/risk profile for iNO use to prevent BPD. Collectively, these laboratory data also suggest that benefit from iNO treatment does not involve reduced inflammation or oxidative stress; accordingly, the findings are consistent with clinical benefit secondary to other mechanisms such as improved alveolarization and airway structure as observed in animal studies.34–36 Other therapies such as antioxidants, late surfactant doses, and caffeine and improved ventilatory strategies may provide additional benefit for infants who receive iNO treatment.
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ACKNOWLEDGMENTS |
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This research was supported by National Institutes of Health grants U01 HL62514, P50 HL56401, P30 HD26979, and General Clinical Research Centers Program grants MO1 RR00240, M01 RR00084, M01 RR00425, M01 RR001271, M01 RR00064, and MO1 RR00080.
We thank the nurses, respiratory therapists, and physicians at participating hospitals; NO-CLD Trial coordinators S. Wadlinger and C. Coburn; the families and infants who participated in this study; Y. Ning, T. McDevitt, C. Castor, and M. Norberg for technical assistance; H. Ischiropoulos for discussions; and INO Therapeutics, Inc, for providing study equipment and gas.
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FOOTNOTES |
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Accepted Oct 9, 2007.
Address correspondence to Philip L. Ballard, MD, PhD, University of California, 3333 California St, Suite 150, San Francisco, CA 94118. E-mail: ballardp{at}peds.ucsf.edu
Financial Disclosure: Drs R. A. Ballard and P.L. Ballard received research grant support and advisor fees from INO Therapeutics, and Drs Golombek and Parton received speaker's fees from INO Therapeutics; the other authors have indicated they have no financial relationships relevant to this article to disclose.
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[Abstract/Free Full Text] - Balasubramaniam V, Maxey AM, Morgan DB, Markham NE, Abman SH. Inhaled NO restores lung structure in eNOS-deficient mice recovering from neonatal hypoxia.
Am J Physiol Lung Cell Mol Physiol.2006; 291
(1):L119
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[Abstract/Free Full Text]
PEDIATRICS (ISSN 1098-4275). ©2008 by the American Academy of Pediatrics
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