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Servicio de Neonatología, Hospital Universitario Materno-Infantil La Fe
Departamento de Fisiología, Faculty of Medicine, University of Valencia, Valencia, Spain
ABSTRACT
Rationale: Pure oxygen causes more oxidative stress than room air in resuscitation of asphyctic neonates, and consequently could be associated with increased tissue damage.
Objectives: To compare damage caused to heart and kidneys on reoxygenation in severely asphyctic term neonates resuscitated with room air (RAR) or 100% oxygen (OxR). Nonasphyctic term newborn infants served as a control group.
Methods and Measurements: This is a prospective randomized clinical trial masked for the gas mixture. Reduced glutathione (GSH), oxidized glutathione (GSSG), and superoxide dismutase (SOD) activity were measured to assess oxidative stress. Plasma cardiac troponin T (cTnT) and urinary N-acetyl-glucosaminidase (NAG) assessed cardiac and renal damage, respectively. Daily determinations of NAG for a 2-wk period were performed to monitor postasphyctic renal damage.
Main Results: Both asphyctic groups showed oxidative stress when compared with the control group as evidenced by diminished GSH/GSSG ratios, adaptive increases in SOD activity, and higher values of NAG and cTnT (markers of tissue damage). However, the OxR group showed significantly higher values of NAG and cTnT, lower GSH/GSSG ratios, and higher SOD activity than the RAR group. Moreover, NAG values persisted in being higher than normal in the OxR group for 2 wk after birth, whereas NAG in the RAR group dropped to normal within the first week. A linear correlation between cTnT or NAG and GSSG was found.
Conclusions: The use of room air on resuscitation causes less oxidative stress and damage to heart and kidney than pure oxygen.
Key Words: birth asphyxia glutathione oxidative stress room-air resuscitation
Perinatal asphyxia is one of the leading causes of morbidity and mortality in the neonatal period. In fact, approximately 4 to 7 million newborn infants suffer every year from birth asphyxia, and despite recommended resuscitation maneuvers, 1 million of them die (1). Among the survivors, clinical manifestations of organ damage, other than the central nervous system, associated with hypoxic-ischemic insults have been described (2). For this purpose, tissue-specific markers have been used. Thus, cardiac troponin T (cTnT), a contractile protein located in the actin filament, has been used as a reliable index of myocardial damage (3). Plasma cTnT levels are not influenced by gestational age, type of delivery, or sex, rendering this marker especially suitable for the perinatal period (4, 5). Ischemic renal damage has been assessed by the urinary concentration of N-acetyl-glucosaminidase (NAG), an enzyme present in the lysosomes of the proximal tubular cells, not filtered through the glomerulus but rapidly cleared from the circulation by the liver (6) and especially reliable for detecting tubular damage after neonatal anoxia (7, 8).
Asphyxia, which is characterized by prolonged periods of ischemia and hypoxia, may lead to exhaustion of intracellular reserves of ATP and accumulation of purine derivatives (9). During the postasphyctic reperfusion phase, xanthine oxidase uses oxygen as substrate to generate a burst of oxygen free radicals. These reactive oxygen species are capable of damaging nearby cellular components, such as nucleic acids, membranes, and structural proteins or enzymes (10).
In previous studies, room-air resuscitation has proved to be as efficient as 100% oxygen in asphyxiated newly born infants (11–15), and has caused less oxidative stress as measured by the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in whole blood (13). Indeed, a significant correlation has been found between blood GSSG concentrations and PaO2, leading to the highest GSSG levels when hyperoxia (PaO2 > 100 mm Hg) was present in infants resuscitated with 100% oxygen (14). The follow-up study of these infants at 2 yr of life did not show neurodevelopmental or physical differences between room-air– and pure oxygen–resuscitated infants (16). More important are the findings of recent meta-analyses, which show less mortality in resuscitation with room air compared with pure oxygen (17, 18).
We hypothesized that, if oxidative stress was attenuated in asphyxiated neonates with the use of room air instead of 100% oxygen for resuscitation, use of room air should similarly diminish damage to tissues. To test this hypothesis, we launched a prospective, randomized clinical trial with a masked gas source and compared serial oxidative stress (GSH, GSSG, and superoxide dismutase activity) and biochemical markers of tissue injury (plasma cTnT and urinary NAG), determined at birth and thereafter in asphyxiated newborn infants resuscitated with either room air or pure oxygen. Some of the results of this study have been previously reported as an abstract (19).
METHODS
Design
This is a prospective randomized clinical trial, blinded for the gas mixture subjects received during resuscitation. A random number was assigned to each record, which stated whether room air or 100% oxygen was used.
Eligible infants were recruited from among severely asphyxiated neonates born in our hospital during a 4-yr period (1999–2002). Severe asphyxia was defined as pale color, presence of bradycardia (< 80 beats/min), nonresponsiveness to stimuli, a cord pH of 7.0 or less at birth, and an Apgar score of 5 or less for more than 5 min (20).
The attending team evaluated the neonate immediately after birth, and if it was asphyxiated, a first blood sample was obtained from the umbilical cord and resuscitation was initiated according to randomization, which was blinded for the gas mixture. However, the gas composition could be changed at request if ventilation proved unsuccessful. Informed parental consent was obtained on admission before the delivery. The ethics committee of the Hospital Virgen del Consuelo approved the study protocol.
Population
Seventeen of the 56 eligible newly born infants did not qualify (see Table 1), leaving 39 to be studied. Of these, 17 were assigned to the room-air group (RAR) and 22 to the 100% oxygen group (OxR). Infants for the control group were selected from among nonasphyxiated term neonates who were born on the same day as asphyxiated infants included in the trial.
Clinical Proceedings
Asphyxiated neonates were resuscitated according to established procedures (21). Infants were monitored for heart and respiratory rate, skin temperature, and oxygen saturation by pulse oximetry. Apgar score at 1, 5, and 10 min; time to first audible cry; and the duration of the resuscitation procedure were recorded.
The first blood sample was obtained from the umbilical cord and two further samples were obtained 24 and 48 h thereafter.
An ECG of all asphyxiated infants was performed within the first 48 h after birth. An echocardiogram was only taken of infants with ECG or clinical signs of myocardial damage (22).
Analytic Assays
GSH was determined by spectrophometry (23) and GSSG by high- performance liquid chromatography of the dinitrobenzene derivatives. To avoid autooxidation of GSH, the thiol group was blocked with N-ethylmaleimide (24, 25). SOD activity was determined by the method of Flohe and Otting, and expressed per gram of hemoglobin content (26).
cTnT was determined by enzyme immunoassay with a detection limit of 0.010 ng/ml (4, 5).
Single-spot urine samples were collected every 24 h for 2 wk, and frozen at –80°C until processed. NAG was determined by colorimetry measuring the release of 3-cresol purple from the substrate 3-cresolsulphonphthaleinyl-N-acetyl-B-D-glucosamine (7). Results have been expressed as IU/mmol creatinine. Creatinine was measured in urine and plasma using the universally accepted Jaffe method.
Statistical Analysis
The statistical analysis has been performed using nonparametric statistics. Thus, we used Mann-Whitney's test for the comparison of nonpaired samples. However, the Kruskal-Wallis test was used for paired comparisons of the evolution of daily urinary NAG excretion in the first 2 wk of life. Simple regression was used to correlate GSSG as an independent variable with cTnT and NAG concentrations as dependent variables (27).
RESULTS
The main clinical variables at birth characterizing both RAR and OxR infants did not show any significant differences (see Tables 2 and 3). However, neonates resuscitated with room air cried earlier and needed significantly less time of resuscitation. Moreover, room-air–resuscitated infants needed significantly less time of resuscitation maneuvers to achieve clinical stabilization than the infants resuscitated with 100% oxygen (see Table 3).
Table 4 and Figure 1 show the biochemical parameters related to oxidative stress analyzed at birth (umbilical vessel), at 24 and 48 h in nonasphyxiated control infants and in both asphyxiated groups. GSSG values were significantly higher in both asphyxiated groups than in the control group at birth and at 24 and 48 h after birth, indicating that asphyxia and reperfusion caused GSH oxidation in these groups of infants, independently of the type of resuscitation. However, at 48 h of life, the OxR group showed significantly higher GSSG values than the RAR group. Oxidative stress was evidenced by a lower GSH/GSSG ratio at birth and at 24 and 48 h in both asphyxiated groups compared with the nonasphyxiated control group (Figure 1A). However, Figure 1A shows that, at 48 h, the OxR group had a significantly lower GSH/GSSG ratio (p < 0.05) than the RAR group, indicating a more prolonged oxidative stress in this group. Concomitantly, Figure 1B shows that the OxR group exhibited significantly higher SOD activity at 48 h of life than the RAR group (OxR vs. RAR: 3.4 ± 1.0 vs. 1.9 ± 0.5 IU/g hemoglobin; p < 0.05).
Table 4 also shows the biochemical parameters associated with tissue damage, specifically cTnT and NAG. The OxR group exhibited significantly higher plasmatic levels of cTnT at 24 and 48 h than the RAR group. Similar results were obtained for NAG urinary levels. Thus, both experimental groups showed significantly higher NAG excretion levels at 24 and 48 h of life than the control group. However, the OxR group excreted significantly higher amounts of NAG at 48 h of life than the RAR group. Significant linear correlations between blood GSSG and plasma cTnT levels (Figure 2A) and urinary NAG (Figure 2B) were found.
Figure 3 shows that the OxR group eliminated significantly higher quantities of urinary NAG than the RAR group during the first week of life. Moreover, although NAG elimination in the RAR group approached normal values at the end of the first week of life, it was not until the second week that NAG elimination in the OxR group merged with that of the control values.
ECG was abnormal in 87% of the infants in the RAR group and in 82% in the OxR group at 24 to 48 h after birth, including the infants who died in the first 4 wk of life (two in the RAR group and four in the OxR group). The final results were not influenced by the exclusion of those subjects who died during the study.
DISCUSSION
Birth asphyxia is one of the main causes of mortality and morbidity in the perinatal period (1). Organs such as brain, heart, liver, and kidney may suffer from structural or functional alterations, leading to acute or long-term dysfunction (2). Hypoxia and subsequent reoxygenation are the cornerstones of the pathophysiology of perinatal asphyctic organ damage. General evidence for involvement of reactive oxygen species in ischemia-reoxygenation injury includes detection of lipid peroxidation and protein nitration products in reperfused tissues, protection of reperfused organs by antioxidant enzymes including SOD, and inhibition of postischemic injury by allopurinol, an inhibitor of xanthine oxidase (28). Moreover, there is a significant in vivo correlation between oxygen partial pressure in tissues such as lungs and the amount of anion superoxide and hydrogen peroxide produced (29).
Numerous experiments in different animal models of asphyxia have shown an increased oxidative stress on resuscitation with pure oxygen as compared with room air, and have compared different therapeutic approaches with a varying degree of success (30). However, so far, clinical experience in the prevention of asphyctic damage in the human newborn is still very limited. Notwithstanding, studies performed on asphyxiated neonates have shown that the use of room air instead of pure oxygen has favored a prompter clinical recovery (11–15). In addition, room-air–resuscitated infants have lower oxidative stress in the immediate postnatal period and even several weeks thereafter as compared with infants resuscitated with 100% oxygen (12).
We report here that asphyxiated newborns showed evident signs of oxidative stress at birth and after initial resuscitation. Thus, they showed greater activity of SOD and higher concentration of whole blood GSSG compared with nonasphyxiated control infants. Overexpression of intracellular SOD on reoxygenation has been considered an adaptive mechanism to protect heart and lungs from reperfusion injury from an excess of free radicals (31). Furthermore, higher GSSG concentration and a lower GSH/GSSG ratio reflect the presence of oxidative stress associated with birth asphyxia (13, 14).
In addition, infants resuscitated with pure oxygen exhibited higher plasma cTnT concentration at 24 and 48 h after birth than did infants resuscitated with room air. cTnT is considered a reliable marker for assessing myocardial damage due to ischemic episodes in the perinatal period (3–5). Our findings are in accordance with recent experiments performed in a piglet model of neonatal asphyxia, in which myocardial damage on resuscitation with room air or pure oxygen was assessed measuring tissue concentration and activity of matrix metalloproteinases (32, 33). Piglets resuscitated with 100% oxygen showed significantly greater concentration and activity of myocardial matrix metalloproteinases than room-air–resuscitated piglets. There was also greater depletion in the total antioxidant capacity in piglets resuscitated with pure oxygen than in those resuscitated with room air (32, 33). Both of these findings coincide with our own results for cTnT and GSH/GSSG ratio. Reperfusion injury to the myocardium has been mainly attributed to the action of free radicals whose generation is closely related with high PaO2 on reoxygenation (34). Thus, it is conceivable that limiting oxygen availability could reduce the production of oxygen-derived free radicals in the myocardium. In our study, GSSG concentration in room-air–resuscitated infants was always lower than in the group resuscitated with 100% oxygen, indicating less oxidative stress in the former group of infants. These findings are coincident with lower cTnT plasmatic values in the room-air–resuscitated infants, reflecting a lesser degree of myocardial damage in this group. Accordingly, a significant correlation was found between plasmatic cTnT concentration and blood GSSG.
In acute ischemic renal failure, loss of renal blood supply results in tissue hypoxia, leading to necrosis of renal tubular cells mediated by free radicals (35, 36). Thus, urine samples collected during the first 48 h after birth revealed elevated concentrations of NAG in asphyxiated newborns. NAG is a specific enzyme present in the proximal tubular cells of the kidney, and its appearance in urine is a highly sensitive marker of tubular cell necrosis (6–8). In previous studies, newborn infants presented elevated urinary concentrations of NAG after birth anoxia (8). Moreover, the intensity of the asphyctic insult correlated with the urinary excretion of NAG (7, 8). Although it has been postulated that immature renal cells are more resistant to hypoxia than mature cells and recover more rapidly after reoxygenation (36), our asphyctic subjects showed significantly higher levels of NAG in urine in the first 2 d as compared with nonasphyxiated infants in the control group. In addition, we found significantly higher values of NAG in neonates resuscitated with 100% oxygen as compared with those resuscitated with room air, especially at 48 h of life. Infants with higher urinary NAG excretion showed higher blood GSSG concentrations and higher erythrocyte SOD activity. These findings reveal increased and more prolonged damage to the proximal tubular cells in infants who received higher oxygen concentration on reoxygenation in the delivery room and suffered from a more intense oxidative stress.
We conclude that the use of room air is as effective as 100% oxygen in severely asphyctic newborn infants. In addition, room-air resuscitation diminishes ischemia-reperfusion–derived acute damage to myocardium and kidney as compared with pure oxygen. Consequently, resuscitation with pure oxygen should be reevaluated (37).
FOOTNOTES
Supported in part by the Annual Research Award for Outstanding Research 2003–2004 of the Asociacion Espaola de Pediatría to M.V.
Originally Published in Press as DOI: 10.1164/rccm.200412-1740OC on September 1, 2005
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.
REFERENCES
World Health Organization. Child health and development: health of the newborn. Geneva, Switzerland: World Health Organization; 1991.
Shah P, Riphagen S, Beyene J, Perlman M. Multiorgan dysfunction in infants with post-asphyxial hypoxic-ischemic encephalopathy. Arch Dis Child Fetal Neonatal Ed 2004;89:F152–F155.
Sato Y, Kita T, Takatsu Y, Kimura T. Biochemical markers of myocite injury in heart failure. Heart 2004;90:1110–1113.
Clark SJ, Newland P, Yoxall CW, Subhedar NV. Cardiac troponin T in cord blood. Arch Dis Child Fetal Neonatal Ed 2001;84:F34–F37.
Trevisanauto D, Lachin M, Zaninotto M, Pellegrino PA, Plebani M, Cantarutti F, Zanardo V. Cardiac Troponin T in newborn infants with transient myocardial ischemia. Biol Neonate 1998;73:161–165.
Westhuyzen J, Endre ZH, Reece G, Reith DM, Saltissi D, Morgan TJ. Measurement of tubular enzymuria facilitates early detection of acute renal impairment in the intensive care unit. Nephrol Dial Transplant 2003;18:543–551.
Willis F, Summers J, Minutillo C, Hewitt I. Indices of renal tubular function in perinatal asphyxia. Arch Dis Child 1997;77:F57–F60.
Cataldi L, Mussap M, Verlato G, Plebani M, Fanos V; Neonatal Nephrology Study Group of the Italian Society of Neonatology. Netilmicin effect on urinary retinol binding protein (RBP) and N-acetyl-beta- D-glucosaminidase (NAG) in preterm newborn with and without anoxia. J Chemother 2002;14:76–83.
Saugstad OD. Role of xanthine oxidase and its inhibitor in hypoxia: reoxygenation injury. Pediatrics 1996;98:103–107.
Bowler RP, Crapo JD. Oxidative stress in airways: is there a role for extracellular superoxide dismutase. Am J Respir Crit Care Med 2002;166:S38–S43.
Saugstad OD, Rootwelt T, Aalen O. Resuscitation of asphyxiated newborn infants with room air or oxygen: an international controlled trial: the Resair 2 study. Pediatrics 1998;102(1). Available from: http://www.pediatrics.org/cgi/content/full/102/1/e1 [Last accessed October 14, 2005].
Vento M, Asensi M, Sastre J, García-Sala F, Via J. Six years' experience in the use of room air for the resuscitation of asphyxiated newly born term infants. Biol Neonat 2001;79:261–267.
Vento M, Asensi M, Sastre J, García-Sala F, Pallardo FV, Via J. Resuscitation with room air instead of 100% oxygen prevents oxidative stress in moderately asphyxiated term infants. Pediatrics 2001;107:642–647.
Vento M, Sastre J, Asensi M, Lloret A, García-Sala F, Via J. Oxidative stress in asphyxiated term infants resuscitated with 100% oxygen. J Pediatr 2003;142:242–248.
Ramji S, Rasaily R, Mishra P, Narang A, Jayam S, Kapoor AN, Kamba I, Mathur A, Saxena BN. Resuscitation of asphyxiated newborns with room air or 100% oxygen at birth: a multicentric clinical trial. Indian Pediatr 2003;40:510–517.
Saugstad O, Ramji S, Irani S, El-Meneza S, Hernandez E, Vento M, Talvik T, Solberg R, Rootwelt T, Aalen OO. Resuscitation of newborn infants with 21% or 100% oxygen: follow-up at 18 to 24 months. Pediatrics 2003;112:296–300.
Saugstad O, Ramji S, Vento M. Resuscitation of depressed newborn infants with ambient air or pure oxygen: a meta-analysis. Biol Neonate 2005;746:1–8.
Davis PG, Tan A, O'Donnell CPF, Schulze A. Resuscitation of newborn infants with 100% oxygen or air: a systematic review and meta-analysis. Lancet 2004;364:1329–1333.
Vento M, Sastre J, Asensi M, Gomez C, Lloret A, Via J, Borras C. Asphyctic renal damage is increased by the use of pure oxygen upon resuscitation. Pediatr Res 2004;56:508A.
Leuthner SR, Das UG. Low Apgar scores and the definition of birth asphyxia. Pediatr Clin N Am 2004;51:737–746.
Kattwinkel J, Niermeyer S, Nadkarni V, Tibballs J, Phillips B, Zideman D, Van Reempts P, Osmond M. An advisory statement from the Pediatric Working Group of the International Liaison Committee on Resuscitation: resuscitation of the newly born infant. Pediatrics 1999;103:e56.
Sahn DJ, DeMaria A, Kisslo J, Weyman A. The committee of M-mode standardization by the American Society of Echocardiography: results of a survey of echocardiographic measurements. Circulation 1978;58:1072–1083.
Via J, Sastre J, Asensi M, Packer L. Assay of blood glutathione oxidation during physical exercise. Methods Enzymol 1995;251:237–243.
Asensi M, Sastre J, Pallardo FV, García de la Asuncion J, Estrela JM, Via J. A high-performance liquid chromatography method for measurement of oxidized glutathione in biological samples. Anal Biochem 1994;217:323–328.
Asensi M, Sastre J, Pallardo FV, Lloret A, Lehner M, García de la Asuncion J, Via J. Ratio of reduced to oxidized glutathione as indicator of oxidative stress status and DNA damage. Methods Enzymol 1999;299:267–276.
Flohe L, Otting F. Superoxide dismutase assays. Methods Enzymol 1984;105:93–104.
Conover WJ. Practical non-parametric statistics. New York: Wiley & Sons; 2001.
Warner DS, Sheng H, Batinie-Haberle I. Oxidants, antioxidants and the ischemic brain. J Exp Biol 2004;207:3221–3231.
Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol 2003;552:335–344.
Grow J, Barks JDE. Pathogenesis of hypoxic-ischemic cerebral injury in the term infant: current concepts. Clin Perinatol 2002;29:585–602.
Li C, Jackson RM. Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol 2002;282:C227–C241.
Munkeby BH, Borke WB, Bjornland K, Sikkeland LI, Borge GI, Halvorsen B, Saugstad OD. Resuscitation with 100% oxygen increases cerebral injury in hypoxemic piglets. Pediatr Res 2004;56:783–790.
Borke WB, Munkeby BH, Halvorsen B, Bjornland K, Tunheim SH, Borge I, Thaulow E, Saugstad OD. Increased myocardial matrix metalloproteinases in hypoxic newborn pigs during resuscitation: effects of oxygen and carbon dioxide. Eur J Clin Invest 2004;34:459–466.
Kaneda T, Ku K, Inoue T, Onoe M, Oku H. Postischemic reperfusion injury can be attenuated by oxygen tension control. Jpn Circ J 2001;65:213–218.
Deng J, Hu X, Yuen PST, Star RA. -Melanocyte-stimulating hormone inhibits lung injury after renal ischemia/reperfusion. Am J Respir Crit Care Med 2004;169:749–756.
Adachi S, Zelenin S, Matsuo Y, Holtback U. Cellular response to renal hypoxia is different in adolescent and infant rats. Pediatr Res 2004;55:485–491.
Hansmann G. Neonatal resuscitation on air: it is time to turn down the oxygen tanks Lancet 2004;364:1293–1294