Inhaled NO and Markers of Oxidant Injury in Infants with Respiratory Failure

Original Article Inhaled NO and Markers of Oxidant Injury in Infants with Respiratory Failure

Krisa P. Van Meurs, MD INTRODUCTION Toby L. Cohen, MD Term infants experience substantial morbidity and mortality from Guang Yang, PhD respiratory failure.1,2 This condition can result from meconium Marco Somaschini, MD aspiration or infection complicated by persistent pulmonary 3 Pavani Kuruma, MD hypertension of the newborn among other causes. Recent clinical Phyllis A. Dennery, MD trials have demonstrated that the endogenous vasodilator nitric oxide (NO) may be a useful adjunct in the treatment of term infants with respiratory failure and decreases the need for extracorporeal membrane oxygenation.4–6 Despite the known BACKGROUND: benefits of iNO, there are also theoretical considerations as to the Inhaled nitric oxide (iNO) is an effective adjunct in the treatment of effects of NO on oxidative stress since NO can combine with infants with respiratory failure. Although there are clear benefits to this molecular oxygen to form nitrogen dioxide at a rate dependent on therapy, potential toxicity could result from reactive nitrosylated species. the concentration of oxygen and the square of the concentration of NO.7,8 Additionally, NO can react with superoxide radicals to form a OBJECTIVE: To evaluate whether iNO therapy is associated with increased serum secondary stable cytotoxic species, peroxynitrite, which eventually markers of oxidative stress. decomposes to NO and the hydroxyl radical, a highly toxic and reactive molecule.9 In animals, the combination of oxygen with DESIGN/METHOD: NO resulted in increased toxicity in a ventilated piglet model.9 In Multiple markers were prospectively evaluated in the serum of term contrast, NO significantly reduced oxygen toxicity at low doses (5 to infants with severe respiratory failure treated with iNO for 1 to 72 hours. 10 parts per million (p.p.m.)) in a rat model10 and did not worsen These were compared to those of patients exposed to greater than 80% oxidative stress in a premature lamb model.11 In human studies, oxygen for more than 6 hours and room air controls. increased nitrotyrosine residues were observed in the serum of RESULTS: premature infants receiving iNO who developed chronic lung After 24 hours of exposure, the iNO-treated infants had increased serum disease (CLD).12 lipid hydroperoxides (LPO), protein carbonyls and nitrotyrosine residues It has been suggested that NO in the presence of superoxide can as well as increased serum total glutathione (GSH) content. The increase inactivate surfactant protein A.13 This may account in part for the in LPO peaked at 24 hours and correlated with the cumulative dose of NO-mediated lung injury. The purpose of this study was to evaluate iNO whereas other markers did not. The presence of chronic lung disease the effects of iNO on markers of oxidative stress. Serum samples (CLD) did not correlate with serum markers of oxidative injury. were obtained from full term infants with severe respiratory distress CONCLUSIONS: treated with iNO. These were compared to values in air exposed In term infants with respiratory failure, prolonged iNO exposure is controls as well as infants treated with oxygen alone. The serum associated with a transient increase in markers of oxidative stress, but this samples were assayed for markers of oxidative injury. The finding does not appear to predict the development of CLD. incidence of CLD, defined as the need for oxygen at 30 days, was Journal of Perinatology (2005) 25, 463–469. doi:10.1038/sj.jp.7211327 also recorded. Published online 12 May 2005 MATERIALS AND METHODS Patient Selection Serum samples were obtained from three groups of infants after

Department of Pediatrics (K.P.V.M., P.K.), Stanford University School of Medicine, Palo Alto, CA, parental consent. All of the infants were greater than 36 weeks USA; Divisione di Patologia Neonatale (M.S.), Ospedale Bolognini di Seriate, Bergamo, Italy; gestation, less than 7 days of age and had serum bilirubin levels of Division of Neonatology (G.Y., P.A.D.), The Children’s Hospital of Philadelphia, Philadelphia, less than 10 mg/dl. The ‘‘room air’’ cohort consisted of infants in Bergamo, PA, USA; and Department of Pediatrics (P.A.D.), University of Pennsylvania School of Medicine, Philadelphia, PA, USA. Aurora Bay Care Medical Center, Green Bay, USA (T.L.C). room air with no prior oxygen treatment. The ‘‘oxygen-exposed’’

Address correspondence and reprint requests to Krisa P. Van Meurs, MD, Department of cohort were infants with respiratory failure in > 80% oxygen for Pediatrics, Stanford University School of Medicine, 750 Welch Rd #315, Palo Alto, CA 94304, USA. more than 6 hours. The ‘‘iNO’’ cohort were infants who had

received iNO and had serum samples drawn after approximately compound can then be quantitated colorimetrically at 675 nm. The 1, 24 and 48 hours of iNO therapy. linear range of detection is between 2 and 200 nmol/ml. Above this level, values are unreliable. Normal human serum has values of 0 iNO Therapy to 1.3 nmol/ml. All infants receiving iNO were participating in the open label phase of the Neonatal Inhaled Nitric Oxide Study.5 In this trial, therapy Detection of GSH. Total serum GSH was measured using the with 20 p.p.m. iNO was initiated when eligibility criteria were met. Glutathione Assay kit Cat #703002 (Cayman Chemical Company, These included reaching an oxygenation index of greater than 25 Ann Arbor, MI). GSH reacts with 5, 50-dithiobis-2-nitrobenzoic acid, on two measurements obtained at least 15 minutes apart. A full Ellman’s reagent to produce a yellow colored product, 5-thio-2- response was defined as an increase in blood oxygen of more than nitrobenzoic acid. Measurement of the absorbance of thio-2- 20 millimeters of mercury (mm Hg); a partial response was defined nitrobenzoic acid at 405 nm accurately measures GSH. as an increase in blood oxygen of 10 to 20 mmHg and no response was defined as an increase of less than 10 mmHg of blood oxygen 30 minutes after iNO exposure. When the infant had less than a Nitrotyrosine residues. Nitration of tyrosine residues in complete response, iNO was then administered at 80 p.p.m. and an proteins occurring in the presence of NO was detected by Western arterial blood gas was again measured after 30 minutes. Infants blotting. Serum samples were electrophoresed on a 12% with full responses continued to receive 80 p.p.m. iNO; infants with polyacrylamide gel and transferred to polyvinyl difluoridine partial responses received the lowest concentration of iNO that membrane blots. The samples were incubated overnight with rabbit produced at least a partial response. If there was no response to polyclonal IgG anti-nitrotyrosine antibodies (Upstate either 20 or 80 p.p.m., iNO was discontinued. After 12 hours of iNO Biotechnology, Lake Placid, NY) at a 1:1000 dilution in a 1% therapy, the iNO dose was decreased by 50% every 12 hours until a solution of bovine serum albumin in phosphate-buffered saline dose of 5 p.p.m. was reached and then was weaned by 1 p.p.m. with 0.05% Tween-20. Blots were washed in 0.05% Tween-20 and every 12 hours. incubated for 1 h at 251C with a 1:5000 dilution of horse radish Primary grade NO was supplied at 800 p.p.m. with nitrogen peroxidase-conjugated goat anti-rabbit IgG. Antigen antibody (Ohmeda, Liberty Corner, NJ). The gas was certified to contain less complexes were visualized with the horse radish peroxidase than 5 p.p.m. of nitrogen dioxide. NO was titrated into the chemiluminescence system according to the manufacturer’s ventilator circuit and the gas mixture was continuously sampled at instructions (Bio-Rad). Quantification was performed by 50 cm distal to the injection site on the inspiratory limb of the densitometry (Molecular Analyst image analysis software, Bio-Rad, ventilator circuit just prior to the endotracheal tube using either a Hercules, CA) and values were normalized to air exposed controls chemiluminescence analyzer (ECOPhysics model CLD 700 AL, on each blot. Durten, Switzerland) or an electrochemical sensor (Pulmonox II, Pulmonox, Tolfield, Alta, Canada) to measure both NO and Protein carbonyls. Protein oxidation was measured by detecting nitrogen dioxide. oxidatively generated carbonyl groups using the OxyBlot Kit For each patient, the cumulative number of p.p.m. hours of iNO (Oncor, Gaithersburg, MD). Briefly, 20 mg protein aliquots, mixed therapy as well as the hours in greater than 80% oxygen were with 1 volume of 12% sodium dodecyl sulfate, were incubated with calculated at the time each serum sample was drawn. two volumes of 1 Â 2,4-dinitrophenylhydrazine for 15 minutes at room temperature. The samples were then neutralized by addition Assays of 1.5 volumes of neutralization solution (Oncor) and All serum samples were assayed for lipid hydroperoxides (LPO), electrophoresed on a 12% polyacrylamide gel. After transfer to glutathione (GSH), nitrotyrosine residues, protein carbonyls and polyvinyl difluoridine membranes, blots were briefly washed in total antioxidant status (TAS) as described below. blocking buffer (1% bovine serum albumin in phosphate-buffered saline–0.05% Tween) for 1 hour and then incubated for 1 hour at Measurement of LPO. Serum LPO was measured using the 251C with rabbit anti-dinitrophenylhydrazine IgG diluted 1:150 in Determiner LPO-CC kit (Kamiya Biomedical Company, Thousand blocking buffer. Blots were washed in blocking buffer and Oaks, CA) for the determination of lipid hydroperoxides in the incubated for 1 h at 251C with a 1:300 dilution of HRP-conjugated serum. This assay is based on the reaction of LPO with the goat anti-rabbit IgG. The antigen–antibody signal was visualized methylene blue derivative (10-N-methylcarbamoyl-3, 7- by chemiluminescence using the horseradish peroxidase dimethylamiNO-10 H-phenothiazine). The LPO determiner kit chemiluminescence system according to the manufacturer’s allows for the detection of lipid hydroperoxides. In the presence of instructions (Bio-Rad, Hercules, CA). The bands showing the most hemoglobin, LPO are reduced to lipid alcohols that react with a consistent signal in controls and samples were used for comparison chromogen after oxidative cleavage to form methylene blue.14 This between samples. Quantification was performed by densitometry.

464 Journal of Perinatology 2005; 25:463–469 iNO and Respiratory Failure Van Meurs et al.

Evaluation of TAS. Serum TAS was measured using the Total values were not statistically different for the groups and did not Antioxidant Status Kit (Randox Laboratories, Antrim, UK). In this correlate with the incidence of CLD. In addition, the incidence of assay, 2,2-AziNO-di-[3-ethylbenzthizoline sulfonate is incubated CLD also did not correlate with the cumulative dose of iNO. Infants with peroxidase (metmyoglobin) and H202 yielding a blue colored with CLD had a mean of 2.2±2.6 p.p.m.-hours/100 exposure to compound, which is quantitated colorimetrically at 600 nm. Serum iNO vs 3.6±4.0 p.p.m.-hours/100 in infants without CLD antioxidants cause suppression of this reaction in proportion to (P ¼ 0.41). their concentration. Serum LPO Interestingly, exposure to oxygen alone was not associated with RESULTS increased LPO compared to room air (Figure 1). In contrast, serum A total of 108 samples were drawn from 81 patients entered in the LPO was significantly increased after 24 hours of iNO. The mean study. All patients were of similar birth weight, gender and LPO values for the infants receiving greater than 24 hours of iNO chronological age at the time of study entry. The iNO-exposed was 4.5 times greater than that of the oxygen group (Figure 1). infants were at a more advanced gestational age (1 week) when After 48 hours of iNO, serum LPO values were no longer compared to the room air control infants (Table 1). All patients statistically different from those of air and oxygen-exposed controls, were at least 36 weeks gestational age and no more than 7 days suggesting a resolution of oxidative stress. Nonetheless, the 2 old. Since bilirubin pigment can interfere with the cumulative iNO dose was correlated to serum LPO (R ¼ 0.355; spectrophotometer measurements, it was important to determine p ¼ 0.046) (Figure 2). However, one patient was a significant bilirubin values. All values were less than 10 mg/dl upon study outlier due to low LPO values despite high cumulative exposure to entry. In the room air control group, patient diagnoses included iNO at 24 and 48 hours and accounted for two data points. 2 suspected sepsis, encephalocele, esophageal atresia, Exclusion of this single patient resulted in an increased R value of myelomeningocele, infant of a diabetic mother, transient 0.52, p<0.0001. tachypnea of the newborn, dehydration and polycythemia with the majority of infants (67%) admitted for suspected sepsis. Diagnoses Serum GSH in the oxygen-treated group included meconium aspiration Serum GSH levels were no different in the air and hyperoxia- syndrome, sepsis/pneumonia, perinatal depression, persistent exposed group. In the iNO group, no change in serum GSH was pulmonary hypertension of the newborn, congenital diaphragmatic noted after 1 h(NO-1) or 24 hours (NO-24). After 48 hours (NO- hernia, respiratory distress syndrome and blood aspiration. In the 48), serum GSH levels were significantly higher than all other iNO group, diagnoses included meconium aspiration syndrome, groups (Figure 3). sepsis/pneumonia, persistent pulmonary hypertension of the newborn, congenital diaphragmatic hernia and respiratory distress syndrome. The majority of the oxygen and iNO-exposed patients were diagnosed with meconium aspiration syndrome or sepsis/ pneumonia (56 and 66%, respectively). Patients were exposed to >80% oxygen for 38±50.4 hours (oxygen group), 42.6±45.5 hours (NO-24 group) and 61.3±55.8 hours (NO-48 group). The incidence of CLD was 15% for the oxygen group and 40% for the iNO group. The oxygen exposure

Table 1 Patient Demographics

Room air group Oxygen group iNO group

Number of patients 30 34 17 Birth weight (kg) 3.3±0.5 3.4±0.6 3.4±0.7 Figure 1. Serum LPO in infants treated with iNO. Values represent the Gestational age (weeks) 38.4±1.7 38.9±1.9 39.4±1.6* mean±se of samples from each group. Room air: controls not exposed Chronological age (days) 2.2±1.6 2.0±1.6 2.4±1.7 to oxygen (n ¼ 27); oxygen: patients exposed to >80% oxygen Gender (male/female) 16/14 19/15 11/6 (n ¼ 33); NO-1: patients treated with iNO for 1 hour (n ¼ 13); NO-24: patients treated with iNO for 24 hours or greater (n ¼ 14); NO-48: Values are mean±SD. patients treated with iNO for 48 hours or greater (n ¼ 7). *p<0.05 vs *p<0.05 vs room air. air controls; wp<0.05 vs all others.

Journal of Perinatology 2005; 25:463–469 465 Van Meurs et al. iNO and Respiratory Failure

Figure 2. Regression plot of serum LPO values with cumulative dose of iNO. Dose of iNO was calculated for each patient as the: [sum of (hours Â ppm NO) for each ppm dose]/100. Individual LPO values are represented on the scattergram in relation to the iNO cumulative dose at the time of sampling.

Figure 4. (a) A representative Western analysis of serum protein carbonyl detection in iNO-treated infants. Lane U: unoxidized protein molecular weight standard mixture treated with dinitrophenylhydrazine as described (negative control); lane Ox: protein molecular weight standard mixture oxidized overnight with 100 mM ferric chloride and 25 mM ascorbic acid to induce oxidation then subjected to dinitrophenylhydrazine incubation as described (positive control); other lanes represent patients in the room air (room air), oxygen (oxygen), 1 hour iNO (NO-1), 24 hours iNO (NO-24) and 48 hours iNO (NO-48) groups. (b) Densitometric evaluation of serum protein carbonyl levels in iNO-treated infants. Figure 3. Total serum GSH in infants treated with iNO. Values Values represent the mean±SE of samples from each group. Room represent the mean±SE of samples from each group. Room air: air: controls not exposed to oxygen (n ¼ 7); oxygen: patients exposed controls not exposed to oxygen (n ¼ 25); oxygen: patients exposed to to >80% oxygen (n ¼ 7); NO-1: patients treated with iNO for >80% oxygen (n ¼ 33); NO-1: patients treated with iNO for 1 hour 1 hour (n ¼ 7); NO-24: patients treated with iNO for 24 hours (n ¼ 13); NO-24: patients treated with iNO for 24 hours or greater or greater (n ¼ 7); NO-48: patients treated with iNO for 48 hours or (n ¼ 14); NO-48: patients treated with iNO for 48 hours or greater greater (n ¼ 7). *p<0.05 vs room air, oxygen and NO-1. (n ¼ 7). *p<0.05 vs all other groups.

Serum TAS signiﬁcantly different than those obtained from infants exposed to Serum total antioxidant status was no different between groups oxygen. (room air: 0.75±0.38 mmol/g protein; oxygen: 0.81±0.59 mmol/g protein; NO-1: 0.88±0.8; NO-24: 0.78±0.71 mmol/g protein and Correlation of Serum Markers with Chronic Lung NO-48: 0.85±0.89 mmol/g protein, values are mean±SE). Disease Serum LPO, GSH and TAS values did not correlate with the Visualization of Serum Protein Carbonyl and presence of CLD. Serum LPO were 4.06±6.9 nmol/g protein in Nitrotyrosine Residues infants with CLD and 10.9±20.6 nmol/protein in infants without Serum protein carbonyls increased in infants exposed to iNO for 24 CLD, p ¼ 0.24. Serum GSH levels were 68.6±35.9 nmol/g protein hours compared to infants exposed to room air, oxygen and 1 hour in infants with CLD and 92.2±94.1 nmol/g protein in infants of iNO (Figure 4). Increased serum nitrotyrosine residues were without CLD, p ¼ 0.39. Serum TAS were 0.94±0.29 mmol/g observed in all iNO-exposed infants as compared to air-exposed protein in infants with CLD and 0.82±0.25 mmol/g protein in controls (Figure 5). At 24 hours, only the iNO samples were infants without CLD, p ¼ 0.11.

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Most of the iNO-treated infants were also receiving high oxygen concentrations, therefore the observed changes in LPO may have been simply attributed to prolonged exposure to high oxygen. However, the oxygen-treated infants had relatively low serum LPO. Furthermore, the duration of high oxygen exposure did not differ between the oxygen and iNO groups at the time of iNO sampling. Therefore, we could not prove a cumulative effect of iNO and oxygen on markers of oxidative stress. Several groups have shown that iNO has anti-inflammatory properties, which could ameliorate lung injury.11,17 This could explain the lack of sustained oxidative stress in the serum of patients with prolonged iNO exposure (greater than 24 hours). We did not specifically assess markers of inflammation to verify this. The lack of difference in LPO between the hyperoxia and air control groups may result from the relatively heterogeneous nature of the room air control group. This group included many patients with suspected sepsis and therefore possible inflammation. Oxidative stress can result from inflammation.18,19 This could Figure 5. (a) Representative Western analysis of serum nitrotyrosine result in increased LPO in the controls. Furthermore, infants of residue detection in iNO-treated infants. The lanes represent patients in diabetic mothers included in the group could have had increased the room air, oxygen, 1 hour iNO (NO-1), 24 hours iNO (NO-24) and lipid peroxidation with prenatal hyperglycemia.20,21 Ideally, the 48 hours iNO (NO-48) groups. (b) Densitometric evaluation of serum nitrotyrosine levels in iNO-treated infants. Values represent the control group should have consisted of only healthy babies. mean±SE of samples from each group. M: protein markers; room However, obtaining blood samples on such a population would air: controls not exposed to oxygen (n ¼ 7); Oxygen: patients raise ethical concerns. exposed to >80% oxygen (n ¼ 7); NO-1: patients treated with iNO To further corroborate the findings of increased LPO, serum for 1 hour (n ¼ 7); NO-24: patients treated with iNO for 24 hours or GSH was evaluated. Glutathione is an important thiol, which greater (n ¼ 7); NO-48: patients treated with iNO for 48 hours or protects against oxidative injury.22 The initial response to oxidative greater (n ¼ 7). *p<0.05 vs room air controls. wp<0.05 vs oxygen stress involves increased total GSH content due to induction of exposed. glutathione synthetic enzymes followed by later depletion.23 Serum GSH levels were high after 48 hours of iNO, suggesting increased glutathione synthesis with mild oxidative stress. The TAS reflects serum antioxidant capacity. Increased TAS DISCUSSION suggests induction of antioxidant defenses.24 An inverse correlation NO improves oxygenation and decreases the need for ECMO use in between this marker and the development of CLD has been term and near-term in neonates with respiratory failure.4–6 documented.25 In the present study, no differences in serum TAS Although acute benefits are observed, potential deleterious effects were observed between groups suggesting that no significant could result when iNO-treated infants are also exposed to high change in total antioxidant capacity occurred despite an increased concentrations of inspired oxygen because NO can interact with GSH. superoxide radicals to form toxic radicals.15 We measured markers Serum protein carbonyls are another useful marker of oxidative of oxidative injury in the serum of patients treated with iNO and stress in ventilated infants. The presence of these modified proteins observed increased serum markers of oxidative stress with longer in tracheal aspirates in the first days of life correlated with adverse exposures to iNO. We chose to look at lipid peroxidation as a outcome and the development of chronic lung disease,26,27 but marker of injury because this is a well-known index of oxidative serum protein carbonyls did not.28 Infants exposed to hyperoxia did injury.16 Oxidative stress affects the phospholipase pathway not demonstrate increased protein carbonyls but exposure to iNO resulting in oxidation of polyunsaturated fatty acids including for greater than 24 hours was associated with significant increases arachidonic acid. This is detected with the lipid hydroperoxide in serum protein carbonyls further suggesting increased oxidative assay.14 Serum LPO was significantly increased in patients stress. Serum nitrotyrosine residues were also increased in infants receiving iNO for greater than 24 hours compared to all other treated with NO for 24 hours or greater but the oxygen-exposed groups, but further exposure to iNO did not further increase LPO. infants also showed an increased protein nitrosylation. The This would suggest that iNO has acute pro-oxidative effects, but decreasing trend in the protein carbonyls and nitrotyrosine residues that these are short lived. with longer iNO exposure suggests that the oxidative stress

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resulting from iNO exposure is perhaps short-lived and overcome 4. Roberts JD, Fineman JR, Morin FC, et al. Inhaled nitric oxide and persistent by endogenous mechanisms not identified here. pulmonary hypertension of the newborn. N Engl J Med 1997;336:605–610. Elevated nitration products correlates with the development of 5. The Neonatal Inhaled Nitric Oxide Study Group. Inhaled nitric oxide in CLD in ventilated preterm newborns not exposed to iNO.29 In this full-term and nearly full-term infants with hypoxic respiratory failure. N study, we did observe an increased incidence of CLD in the iNO- Engl J Med 1997;336:597–604. treated infants as compared to infants treated with oxygen only. 6. Clark RH, Kueser TJ, Walker MW, et al. Low-dose nitric oxide therapy for However, there was no difference in the duration of oxygen persistent pulmonary hypertension of the newborn. Clinical Inhaled Nitric Oxide Research Group. N Engl J Med 2000;342:469–74. exposure between the two groups and there was no correlation 7. Austin AT. The chemistry of the higher oxides of nitrogen as related to the between cumulative iNO exposure and CLD. This suggests that the manufacture, storage and administration of nitrous oxide. Br J Anaesth severity of illness rather than the exposure to oxygen or iNO 1967;39:345–50. correlates with the development of CLD. In addition, there was no 8. Foubert L, Fleming B, Latimer R, et al. Safety guidelines for use of nitric correlation of LPO, GSH or TAS seen in individual patients and the oxide [letter] [see comments]. Lancet 1992;339:1615–6. development of CLD. 9. Robbins CG, Horowitz S, Merritt TA, et al. Recombinant human superoxide Others have implicated iNO exposure and subsequent increased dismutase reduces lung injury caused by inhaled nitric oxide and nitrosylation of serum proteins in the onset of CLD in a small hyperoxia. Am J Physiol 1997;272:L903–7. group of premature infants.12 The present study involved term 10. Gutierrez HH, Nieves B, Chumley P, Rivera A, Freeman BA. Nitric oxide infants who have a considerably lower incidence of CLD than regulation of superoxide-dependent lung injury: oxidant-protective actions preterms. Demonstrating an effect of iNO on CLD in term infants of endogenously produced and exogenously administered nitric oxide. Free would require a large patient population beyond the scope of this Radic Biol Med 1996;21:43–52. study. It is likely that we did not have sufficient power to detect a 11. Storme L, Zerimech F, Riou Y, et al. Inhaled nitric oxide neither alters oxidative stress parameters nor induces lung inflammation in premature difference in CLD rates between the iNO and oxygen groups given lambs with moderate hyaline membrane disease. Biol. Neonate the small size of the study. To this effect, three large randomized 1998;73:172–81. trials of iNO therapy in term and near-term infants have not 12. Lorch SA, Banks BA, Christie J, et al. Plasma 3-nitrotyrosine and outcome in demonstrated an increased incidence of CLD in the iNO-treated neonates with severe bronchopulmonary dysplasia after inhaled nitric oxide. 4–6 groups. Furthermore, a single large randomized trial Free Radic Biol Med 2003;34:1146–52. demonstrated a lower incidence of CLD in the iNO-treated group 13. Haddad IY, Pataki G, Hu P, et al. Quantitation of nitrotyrosine levels in corroborating the absence of significant NO-mediated oxidative lung sections of patients and animals with acute lung injury. J Clin Invest stress.6 1994;94:2407–13. In conclusion, in term infants, we observed increased markers 14. Ohishi N, Ohkawa H, Miike A, Tatana T, Yagi K. A new assay method for of oxidative stress with iNO exposure suggesting that iNO results in lipid peroxides using a methylene blue derivative. Biochem Int increased oxidative injury. The lack of a sustained increase in these 1985;10:205–11. markers except LPO suggests that these effects may be transient 15. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent and not likely to result in chronic oxidative injury. hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 1990;87:1620–4. 16. Halliwell B. Oxidants and human disease: some new concepts. FASEB J Acknowledgements 1987;1:358–64. The authors thank Patricia Hartsell, RN and Dorothy Inguilo, RN for their 17. Kinsella JP, Parker TA, Galan H, Sheridan BC, Halbower AC, Abman SH. invaluable assistance in collecting samples. This work was funded in part by the Effects of inhaled nitric oxide on pulmonary edema and lung neutrophil Child Health Research Fund of Lucile Packard Children’s Hospital at Stanford accumulation in severe experimental hyaline membrane disease. Pediatr University, and in part by Grant 5 M01-RR00070 from the National Center for Res 1997;41:457–63. Research Resources, National Institutes of Health. 18. Ben Baouali A, Aube H, Maupoil V, Blettery B, Rochette L. Plasma lipid peroxidation in critically ill patients: importance of mechanical ventilation. Free Radic Biol Med 1994;16:223–7. References 19. Kokk T, Talvik R, Zilmer M, Kutt E, Talvik T. Markers of oxidative stress 1. Davis JM, Spitzer AR, Cox C, Fox WW. Predicting survival in infants with before and after exchange transfusion for neonatal sepsis. Acta Paediatr persistent pulmonary hypertension of the newborn. Pediatr Pulmonol 1996;85:1244–6. 1988;5:6–9. 20. Dominguez C, Ruiz E, Gussinye M, Carrascosa, A. Oxidative stress at onset 2. Hageman JR, Dusik J, Keuler H, Bregman J, Gardner TH. Outcome of and in early stages of type 1 diabetes in children and adolescents. Diabetes persistent pulmonary hypertension in relation to severity of presentation. Care 1998;21:1736–42. Am J Dis Child 1988;142:293–6. 21. Inouye M, Hashimoto H, Mio T, Sumino K. Levels of lipid peroxidation 3. Gersony WM. Neonatal pulmonary hypertension: pathophysiology, product and glycated hemoglobin A1c in the erythrocytes of diabetic classification, and etiology. Clin Perinatol 1984;11:517–24. patients. Clin Chim Acta 1998;276:163–72.

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22. Jornot L, Junod AF. Variable glutathione levels and expression of 26. Gladstone Jr IM, Levine RL. Oxidation of proteins in neonatal lungs. antioxidant enzymes in human endothelial cells. Am J Physiol 1993; Pediatrics 1994;93:764–8. 264:L482–9. 27. Varsila E, Pitkanen O, Hallman M, Andersson S. Immaturity-dependent free 23. Kennedy KA, Lane NL. Effect of in vivo hyperoxia on the radical activity in premature infants. Pediatr Res 1994;36:55–9. glutathione system in neonatal rat lung. Exp Lung Res 1994;20: 28. Winterbourn CC, Chan T, Buss IH, Inder TE, Mogridge N, Darlow BA. 73–83. Protein carbonyls and lipid peroxidation products as oxidation markers in 24. Ho YS, Dey MS, Crapo JD. Antioxidant enzyme expression in rat lungs preterm infant plasma: associations with chronic lung disease and retino- during hyperoxia. Am J Physiol 1996;270:L810–8. pathy and effects of selenium supplementation. Pediatr Res 2000;48:84–90. 25. Silvers KM, Gibson AT, Russell JM, Powers HJ. Antioxidant activity, packed 29. Banks BA, Ischiropoulos H, McClelland M, Ballard PL, Ballard RA. Plasma cell transfusions, and outcome in premature infants. Arch Dis Child Fetal 3-nitrotyrosine is elevated in premature infants who develop bronchopul- Neonatal Ed 1998;78:F214–9. monary dysplasia. Pediatrics 1998;101:870–4.

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