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Oxidative stress in asphyxiated term infants resuscitated with 100% oxygen
ORIGINAL ARTICLES
OXIDATIVE STRESS IN ASPHYXIATED TERM INFANTS RESUSCITATED WITH 100% OXYGEN MÁXIMO VENTO, PHD, MD, MIGUEL ASENSI, PHD, JUAN SASTRE, PHD, ANA LLORET, PHD, FERNANDO GARCÍA-SALA, MD, AND JOSÉ VIÑA, PHD, MD
Objective tive stress.
To test the hypothesis that resuscitation of asphyxiated infants with pure oxygen causes hyperoxemia and oxida-
Study design Asphyxiated term newborn infants (n = 106) were randomly resuscitated with room air (RAR = 51) or 100% oxygen (OxR = 55). The Apgar score, time of the first cry, and establishment of a sustained pattern of respiration were recorded. Assays performed included: blood gases; reduced glutathione (GSH) and oxidized glutathione (GSSG) in whole blood; glutathione-related enzyme activities; and superoxide dismutase activity (SOD) in erythrocytes. Results The RAR group needed less time of ventilation for resuscitation (5.3 ± 1.5 vs 6.8 ± 1.2 min; P < .05). Pure oxygen caused hyperoxemia (PO2, 126.3 ± 21.8 mm Hg) that did not occur with the use of room air (PO2, 72.2 ± 6.8 mm Hg). GSH was decreased and GSSG, the glutathione cycle enzymes, and SOD activities were increased in both asphyxiated groups. However, the 100% oxygen-resuscitated group showed significantly greater alterations that correlated positively with hyperoxemia. Conclusions Asphyxia causes oxidative stress in the perinatal period, and resuscitation with 100% oxygen causes hyperoxemia and increased oxidative stress. Because there are no advantages to resuscitation with 100% oxygen, room air may be preferred under certain circumstances for the resuscitation of asphyxiated neonates. (J Pediatr 2003;142:240-6)
he fetal-to-neonatal transition is characterized by physiologic and metabolic changes that are accompanied by a marked increase in the availability of oxygen to the body.1 As a consequence, the pro-oxidant status may cause oxidative stress.2 Indeed, under physiologic conditions, there is a remarkable increase in oxidized glutathione levels in rat liver and erythrocytes from human neonates.3-5 Intrapartum asphyxia is characterized by transient periods of hypoxia during the ischemic phase followed by reperfusion.6 Upon reperfusion and re-oxygenation a flood of oxygen-free radicals are generated by the xanthine oxidase system. The amount of oxygen radicals produced is directly dependent on the oxygen as well as the hypoxanthine and other purine concentrations in the tissue.7,8 Oxygen-free radical generation causes endothelial cell damage and abnormalities in n-methyl-D-aspartate receptors, synaptosome structure, and astrocyte function, thus contributing to the development of brain injury after a hypoxic-ischemic episode.9 Guidelines recommend the use of 100% oxygen for the resuscitation of the asphyctic newly born infant.10 However, the use of room air has proved to be efficient for newborn resuscitation, and no differences regarding mortality or short-term morbidity were detected with the use or room air compared with pure oxygen.11,12 In addition, the use of pure oxygen for resuscitation in animal experiments increased the arterial partial pressure of oxygen above the physiologic range.13 Hyperoxemia has been associated with numerous negative side effects, including delayed initiation of spontaneous respiration, increased oxygen consumption, and irregularities in the cerebral circulation.14
T
GPx GR GSH GSH-s-tr GSSG
Glutathione peroxidase Glutathione reductase Reduced glutathione Glutathione-s-transferase Oxidized glutathione
OxR RAR ROS SOD
100% oxygen resuscitated newly born infants Room air resuscitated newly born infants Reactive oxygen species Superoxide dismutase
See editorial, p 221.
From School of Medicine, University of Alicante, Department of Pediatrics and Neonatology and Division of Neonatology, Hospital Virgen del Consuelo, and the Department of Physiology, School of Medicine, University of Valencia, Spain. Supported in part by the Annual Research Grant (1999) of the Sociedad Española de Neonatologia (to M.V.). Submitted for publication Jan 28, 2002; revisions received July 1, 2002. and Oct 24, 2002; accepted Nov 22, 2002. Reprint requests: Máximo Vento, PhD, MD, Servicio de Pediatría, Hospital Virgen del Consuelo, Callosa de Ensarriá, 15 bajo, E-46007 Valencia, Spain. E-mail: [email protected]. Copyright © 2003, Mosby, Inc. All rights reserved. 0022-3476/2003/$30.00 + 0
10.1067/mpd.2003.91
240
Fig 1. A, Linear regression analysis correlating paO2 with GSSG in newly born asphyxiated infants independently of the gas source (room air or 100% oxygen) used for resuscitation. Values plotted represent the combined data obtained at birth, during active resuscitation and at clinical stabilization. B, Linear regression analysis correlating the time of positive pressure ventilation employed for resuscitation of the asphyxiated infants, expressed in minutes with GSSG.
Previously, we found that the use of 100% oxygen for resuscitation of asphyctic neonates caused prolonged oxidation of blood glutathione compared with room air.12 The two distinctive features between both groups were: a 5-fold greater oxygen concentration in the 100% oxygen group (OxR) (100% vs 21%) and a more prolonged time of exposure to oxygen in the 100% oxygen group, compared with the room air group (RAR) (7.5 ± 1.8 vs 4.6 ± 0.7 min).12 The aim of the current study was to test whether the greater amount of oxygen delivered to the OxR group could be the triggering factor for oxidative stress. To test this hypothesis, we have performed a randomized clinical trial of the relationship between the duration of oxygen administration and the degree of hyperoxia with markers of oxidative stress at the end of active resuscitation and at the time clinical stabilization after resuscitation.
MATERIAL AND METHODS Design This is a randomized clinical trial, blinded for the gas source during resuscitation. Randomization was performed by assigning a sealed enveloped containing a computer generated random number plus a statement indicating the corresponding group (100% oxygen or room air) to each mother’s record at admission to the obstetric ward before delivery. Eligible patients were recruited among term neonates (37-40 weeks’ gestation) born at the Hospital Virgen del Consuelo that showed evident signs of asphyxia. Thus, they were hypotonic and apneic, nonresponsive to external stimuli, pale, bradycardic (<80 bpm), and acidotic (pH ≤7.05). The neonatologist evaluated Oxidative Stress in Asphyxiated Term Infants Resuscitated With 100% Oxygen
the infants’ clinical statuses immediately after birth, and if they were asphyxiated, obtained a first blood sample from the umbilical cord. A nurse opened the sealed envelope and switched the gas source in accordance to randomization. The resuscitating team was not aware of the oxygen concentration they were using, but the gas mixture could be changed if requested. The Ethical Committee of the hospital approved the study protocol. Parents were informed of the ongoing trial in the hospital by the attending obstetrician. The parents’ written informed consent was required in all cases and obtained before delivery after admission to the obstetric ward.
Population Of 151 eligible neonates, only 106 met the requirements of the study: 51 in the RAR group and 55 in the OxR group. Controls consisted of 22 nonasphyxiated term neonates. A total of 45 neonates were excluded, because of not fulfilling the biochemical requirements (n = 10), having insufficient blood for analytical purposes (n = 14), because they were switched from room air to 100% oxygen (n = 7) or vice-versa (n = 5), or because they were not blindly resuscitated (n = 9). The decision to switch the gas source was made by the neonatologist in charge when evident clinical signs of asphyxia were present and after several minutes of active ventilation. Major confounders for both experimental groups are described in Table I and clinical variables recorded are shown in Table II.
Clinical Proceedings The resuscitation team consisted of a neonatologist, an anesthesiologist, and two nurses. Immediately after birth, as241
Table I. Clinical variables of the newly born population
Gestational age (wk) Birth weight (g) Vaginal/Cesarean delivery Epidural analgesia/General anesthesia* Fetal bradycardia (<80 beats/min) Meconium-stained amniotic fluid Intubation (for suctioning and/or ventilation)‡
Non-asphyxiated † control (n = 22)
Asphyxiated RAR (n = 51)
Asphyxiated OxR (n = 55)
39.5 ± 1.9 3310 ± 190 15/7 20/2 0 2 0
38.9 ± 1.6 3160 ± 240 16/35b 16/35b 34b 10a 5a
40.5 ± 1.1 3220 ± 168 14/41b 14/41b 32b 13a 7a
*General anesthesia included sedation, relaxation, intubation, and/or bag and mask ventilation. Pre-oxygenation was used before intubation, and supplemental oxygen was administered as required during the inhalational anesthesia, always under pulseoximetry control. †Control indicates nonasphyxiated newly born term infants. ‡The difference between the number of infants with meconium-stained amniotic fluid and intubations is because several infants were suctioned with a suction catheter and ventilated with bag and mask, and thus were not intubated. a, RAR/OxR versus control P < .05; b, RAR/OxR versus control P < .01.
Table II. Clinical information
1-min Apgar score median 5-min Apgar score median 10-min Apgar score median Onset of first cry (min)* Onset of spontaneous respiration (min)† Hemoglobin saturation >90% (reached at min)
Nonasphyxiated controls (n = 22)
Asphyxiated RAR (n = 55)
Asphyxiated OxR (n = 52)
8 (7-9) 9 (8-10) 9 (8-10) 0.5 ± 0.1 0.6 ± 0.2 0.9 ± 0.4 (n = 18)
3 (1-4)b 6 (5-8)a 8 (7-9) 1.4 ± 0.3b 5.3 ± 1.5b 2.0 ± 0.7b (n = 48)
3 (1-4)b 6 (4-8)a 8 (6-9) 1.9 ± 0.4b,c 6.8 ± 1.2b,c 1.8 ± 0.9b (n = 43)
Apgar scores are expressed as median with 5th to 95th percentiles in parentheses. Other values are expressed as mean ± SD. Control indicates normal nonasphyxiated newly born term infants. n = number of infants to whom a specific variable was recorded. Significance is expressed as follows: a, P < .05; b, P < .01 versus control group; c, P < .05 versus RAR group. *Time elapsed until the occurrence of the first audible cry. †Time elapsed until the onset of a regular, spontaneous respiration that did not need additional intervention of the resuscitation team.
phyxiated infants were resuscitated following standard procedures, which included thermal stabilization, aspiration of the upper respiratory airway, and positive-pressure ventilation with bag and mask, as recently described.10 Meconium-stained amniotic fluid was directly suctioned from the trachea. In cases of ineffective ventilation (poor color, poor chest expansion, and sustained bradycardia) the resuscitation team proceeded to endotracheal intubation. Neonatal nurses placed probes to monitor the infants’ clinical variables (temperature, heart rate, respiratory frequency, and pulseoximetry). Heart rate and respiratory frequency also were evaluated by direct auscultation of the infant. Nurses also recorded the Apgar scores at 1, 5, and 10 minutes of postnatal life, the time elapsed until the first audible cry, and the time elapsed until the onset of spontaneous respiration (Table II). This was defined, as the time needed to attain a respiratory pattern without the intervention of the resuscitation team.14,15 Two infants in the OxR group needed supplemental oxygen therapy for approximately 3 hours (maximal Fi02 of 30%). Non of the infants in the RAR group needed supplemental oxygen. No infant had hyperthermia. 242 Vento et al
The first blood sample was obtained in all infants from umbilical vessels just before detachment from the placenta. Thereafter, in asphyxiated infants, two more samples were obtained from an umbilical artery catheter or from the right radial artery. The first sample was obtained just after the onset of spontaneous respiration (Table II). The second sample was obtained 10 minutes thereafter when clinical recovery (defined as spontaneous respiration, normal heart rate, good response to stimuli, and normal oxygen saturation) was evident. A total of 6 mL of blood was required to complete all measurements. Umbilical artery canalization is not a routine procedure in our nursery and was specifically performed to analyze blood gases in the current study. In case of failure to canalize, we used the right radial artery (Table III). After clinical stabilization umbilical catheters were immediately removed. There were no complications derived from the use of the catheters. A structured neurologic evaluation, electroencephalogram, and ultrasonographic imaging were performed at the end of the first week of life and repeated 4 weeks later. The Journal of Pediatrics • March 2003
chloride. The supernatant was aspirated, and the cell pellet was hemolyzed with distilled water. Enzyme activities were assayed in the hemolysate and were expressed per gram of hemoglobin content.
Statistical Analysis
Fig 2. Multiple regression analysis representing the correlation between paO2 (mm Hg) as independent variable and glutathione redox cycle enzymes (glutathione reductase, glutathione peroxidase, glutathione—s-transferase) as dependent variables in asphyxiated newly born infants, independently from the type of gas (room air or 100% oxygen) used for resuscitation. Values data plotted represent the combined data obtained at birth, during active resuscitation, and at clinical stabilization.
Analytical Assays Blood gases were processed immediately after blood withdrawal (Radiometer, Copenhagen). GSH and GSSG were determined in whole blood as we have previously described.15,16 Glutathione peroxidase, glutathione reductase and glutathione-S-transferase, and superoxide dismutase activities were determined as described by Flohé and Güzzler,17 Akerboom and Sies,18 Habig et al,19 and Flohé and Otting,20 respectively. Blood in heparinized tubes was immediately centrifuged for 10 minutes at 500 g and 4°C. Plasma was removed, and erythrocytes were washed twice with 0.9% sodium Oxidative Stress in Asphyxiated Term Infants Resuscitated With 100% Oxygen
All calculations and graphs were made using the GBSTAT computer program (Dynamics Microsystems, Inc, Silver Spring, Md). Nonparametric statistics were used because the distribution of the variables was not normal. We used the Mann-Whitney U test for nonpaired samples and the Kruskall-Wallis test for >2 nonpaired (independent) samples. Both simple and multiple regression analyses were performed to assess the effect of variations in the arterial paO2 (independent variable) on GSH, GSSG, and the activity of glutathione redox cycle enzymes and superoxide dismutase (dependent variables). The effect of time of positive pressure ventilation (independent variable) on GSSG (dependent variable) was evaluated by simple regression analysis.
RESULTS There was an increased rate of cesarean delivery among the asphyxiated infants (72%) compared with the control group (32%) (Table I). In addition, significant differences because of the presence of fetal bradycardia, meconium-stained amniotic fluid, and the need for intubation occurred in the asphyxiated neonates compared with the control group. The median Apgar score at 1 and 5 minutes was significantly lower in the asphyxiated newly born infants than in the control nonasphyxiated group (Table II). The time elapsed until the onset of the first cry, and for the onset of a sustained respiratory pattern, was significantly shorter in the control group than in both experimental groups (P < .01). However, the RAR group needed significantly less time of ventilation to initiate the first cry (P < .05) and for the onset of a sustained respiratory pattern (P < .05) than the OxR group (RAR, 5.3 ± 1.5 minutes; OxR, 6.8 ± 1.2 minutes). 243
Table III. Blood gases in asphyxiated newly born infants resuscitated with room air or 100% oxygen Control (n = 22) Sampling (min) Umbilical artery Radial artery pH pCO2 (mm Hg) pO2 (mm Hg)
Birth 22 0 7.38 ± 0.06 51.0 ± 9.9 30.3 ± 4.5
Asphyxiated RAR (n = 51) Birth 51 0 7.05 ± 0.07† 57.3 ± 9.1* 27.8 ± 5.2
5.3 ± 1.5 38 13 7.24 ± 0.9 46.4 ± 6.8 72.2 ± 6.8
Asphyxiated OxR (n = 55) 15 41 10 7.38 ± 0.5 37.4 ± 9.2 82.7 ± 5.2
Birth 55 0 7.02 ± 0.3† 59.3 ± 6.9* 30.0 ± 6.8
6.8 ± 1.2 40 15 7.27 ± 0.5 49.5 ± 5.3 126.3 ± 21.8‡
15 43 12 7.32 ± 0.4 40.2 ± 7.6 110.5 ± 15.8‡
Control indicates nonasphyxiated newly born term infants. RAR indicates asphyxiated newly born term infants resuscitated with room-air. OxR indicates asphyxiated newly born term infants resuscitated with 100% oxygen. pH comparisons are used based on the –log [H+]. Values are expressed as mean ± SD. Significance is expressed as follows: *P < .05; †P < .01 versus control. ‡P < .01 versus RAR.
The control group needed significantly less time than the asphyxiated groups to attain a hemoglobin saturation of >90% (P < .01). The timing of the blood sampling for analytical purposes was as follows: immediately after birth (time 0), at the end of the resuscitating maneuvers coinciding with the onset of spontaneous respiration, and 10 minutes thereafter when clinical stabilization was achieved. The pH values in the RAR and OxR groups immediately after birth were significantly lower than the pH of the control group (P < .01), but there were no differences between asphyxiated infants and the control group at the end of resuscitation, and when clinical stabilization was achieved (Table III). The experimental groups had significantly increased paCO2 values at birth compared with the control group. However, at the onset of spontaneous respiration, both experimental groups had normal paCO2 values. No significant difference between paO2 in the control and asphyxiated groups were detected at birth. Positive pressure ventilation raised paO2 in both asphyxiated groups. However, paO2 was higher in the OxR group than in the RAR group at the end of resuscitation (P < .01). Moreover, when both groups reached clinical stabilization, paO2 in the OxR group was still higher than in the RAR group (P < .01). GSH concentration decreased in a similar manner in the first 15 minutes of postnatal life in both asphyxiated groups (Table IV). GSSG levels in umbilical cord increased significantlt in both experimental groups (P < .01) relative to the control group. However, there was a significantly higher GSSG concentration in OxR than in RAR group at the end of resuscitation and at 15 minutes life (P < .05). The activity of GPx, GSH-s-transferase and GR increased similarly in both asphyxiated groups at the end of the resuscitation compared with the control group (P < .01). However, at 15 minutes of life, these enzymes showed a greater activity in the OxR group than in the RAR group (P < .05). There was also a significantly higher activity of SOD in both asphyxiated groups at birth than in the control nonasphyxiated group (P < .05). However, at 15 minutes of life, the activity of SOD in the OxR group was significantly greater than in the RAR group (P < .05). 244 Vento et al
The paO2 for OxR and RAR correlated with GSSG concentration (P < .05) (Fig 1, A). There was also a significant correlation between the time of ventilation and GSSG concentration. Furthermore, the slope for the OxR group was much higher than that of the RAR group (P < .01). The activities of the glutathione redox cycle enzymes, at birth, at the onset of spontaneous respiration, and when clinical stabilization was attained, correlated significantly with paO2 (Fig 2). Follow-up studies at the end of the first week of life and 4 weeks of age did not reveal any differences between either experimental groups.
DISCUSSION Oxidative stress is a physiologic event in the fetal-toneonatal transition. In isolated hepatocytes from fetal and newborn rats, we found a dramatic decrease (15- to 20-fold) in the GSH/GSSG ratio, mainly because of a marked increase in the GSSG concentration in the fetal-to-neonatal transition.3 In addition, all the enzyme activities involved in the glutathione redox cycle increased during that transition, especially glutathione peroxidase and glutathione-s-transferase.3 Similar results were found during normal fetal-to-neonatal transition in human erythrocytes. However, by 3 days after birth, GSSG concentration and the activity of the enzymes returned to normal in healthy neonates.4 The oxygen shortage during asphyxia leads to an impairment of mitochondrial function, energy failure, accumulation of purine derivatives, and increased generation of reactive oxygen species.6 In the current study, we found significantly increased GSSG concentrations, as well as increased SOD activity in asphyxiated infants immediately at birth, compared with nonasphyxiated controls reflecting oxidative stress. Upon reperfusion, reactive species of oxygen are generated through a mechanism dependent on the concentration of oxygen and purine derivatives accumulated in the tissue.6 The damaging effects of ROS on brain cells constitute the principal hypothesis put forward to explain brain damage during the asphyxia-reperfusion process.21 In fact, in the current study, asphyxiated infants showed increased GSSG levels and activities of SOD and of the glutathione The Journal of Pediatrics • March 2003
Table IV. Reduced GSH, GSSG, and glutathione cycle enzyme activities in asphyxiated newly born infants resuscitated either with RAR or OxR Umbilical vessel
GSH (µM) GSSG (µM) GPx (U/gHb) GR (U/gHb) GSH-s-tr (U/gHb) SOD (U/gHb)
End of resuscitation
Clinical stabilization
Control (n = 22)
RAR (n = 51)
OxR (n = 55)
RAR
OxR
RAR
OxR
1025 ± 166 22.4 ± 6.7 45 ± 12.0 8.1 ± 2.6 6.6 ± 1.5 1.3 ± 0.5
955 ± 134 61.2 ± 7.5† 51 ± 7.9 8.8 ± 3.1 5.9 ± 2.0 2.2 ± 0.5*
1130 ± 140 67.4 ± 9.2† 52.5 ± 8.8 7.6 ± 2.7 7.0 ± 0.8 2.6 ± 0.7*
760 ± 110* 82.5 ± 10.1† 65.7 ± 12.0* 12.5 ± 2.9* 10.4 ± 3.1† 2.5 ± 0.3
835 ± 96* 102 ± 13.8†‡ 71.2 ± 13.4* 15.2 ± 3.9* 12.4 ± 2.1† 3.1 ± 0.7
720 ± 75* 83.1 ± 12.5† 67.8 ± 9.5* 16.2 ± 5.1† 11.3 ± 2.6† 2.8 ± 0.4
640 ± 90† 110.7 ± 21.4†‡ 79.6 ± 11.8†‡ 20.2 ± 4.3†‡ 16.5 ± 3.2†‡ 3.5 ± 0.8‡
Control indicates nonasphyxiated newly born infants. Values are expressed as mean ± SD. Significance is expressed as: *P < .05; †P < .01 versus control group; ‡P < .05 versus RAR group.
redox cycle enzymes at the end of ventilation than were present immediately after birth. Thus, during reperfusion biochemical indicators of oxidative stress increased significantly in both asphyxiated groups, independently of the use of room air and 100% oxygen. We hypothesized that there were two factors responsible for the prolonged oxidative stress in the OxR group. First, the OxR group received a significantly larger amount of oxygen than the RAR group. The amount of oxygen received by each infant during resuscitation is the result of multiplying the tidal volume respiratory rate duration of resuscitation expressed in minutes (RAR, 5.3 ± 1.5 minutes; OxR, 6.8 ± 1.2 minutes) oxygen concentration in the breathing gas (RAR, 0.21; OxR, 1.0). Thus, the OxR infants received an average of 350 mL more oxygen than infants in the RAR group. Second, the OxR group was exposed to supraphysiologic concentrations of oxygen for a more prolonged time. High paO2 is known to increase the production of ROS.25 Under these circumstances, the antioxidant defenses of the newborn infant may be incapable of coping with this pro-oxidant challenge. In our current study, the OxR group had higher paO2 than the RAR group at the end of positive pressure ventilation and thereafter. In addition, the OxR group was ventilated for a longer time than the RAR group. Hyperoxemia and the time of positive pressure ventilation significantly correlated with the GSSG levels. Hence, the GSH/GSSG ratio in the OxR group was significantly lower than in the RAR group. In the current study, GPx, GR, and GSH-s-tr, had an increased activity in erythrocytes of both asphyxiated groups during resuscitation, and 15 minutes after birth, their activity was significantly higher in the OxR group. Previous studies have reported that an increase in the activities of erythrocyte glutathione redox cycle enzyme is indicative of an adaptation to oxidative stress, as seen during fetal-toneonatal transition.26,27 Our results show that hyperoxia increases the activities of glutathione-related enzymes. The expression of these enzymes might be up-regulated by hyOxidative Stress in Asphyxiated Term Infants Resuscitated With 100% Oxygen
peroxia, thus acting as sensors of oxidative stress. Therefore, our results suggest that oxidative stress modulate the antioxidant cellular defenses as an adaptive response in newly born infants. Blood GSH/GSSG ratio and erythrocyte enzymes activities should be considered as general indicators of systemic oxidative stress.3,5 Thus, measurements of the glutathione redox ratio and other oxidative stress indicators in specific tissues, such as brain and lung, would need to be performed to demonstrate oxidative damage. Glutathione is considered to be the major thioldisulfide redox buffer of mammalian cells, and the GSH-toGSSG ratio has been used to estimate the redox environment of a cell. An increase in the GSSG concentration is associated with a pro-oxidant challenge.28 Changes in the cellular redox environment can alter signal transduction, DNA and RNA synthesis, protein synthesis, enzyme activation, and even regulation of the cell cycle.29 In addition, overexpression of antioxidant enzymes, such as the increase of SOD activity, may inhibit cell growth.30 Hence the increased activity of SOD in the OxR group may be of relevance regarding cell proliferation. The changes in the glutathione redox cycle and the antioxidant enzyme activities associated with high oxygen concentrations deserve further study. The use of pure oxygen during resuscitation needs to be reconsidered. An appropriate redox environment is a sine qua non for optimal cellular metabolism. The consequences of its alterations may not be predictable. Room air resuscitation may have advantages over pure oxygen. We thank Marilyn R. Noyes for revising the manuscript.
REFERENCES 1. Dejour P. Principles of comparative respiratory physiology. Amsterdam: Elsevier/North Holland; 1991. p. 147-71. 2. Saugstad OD. Mechanism of tissue injury by oxygen radicals: implications for neonatal disease. Acta Paediatr 1986;85:1-4.
245
3. Pallardó FV, Sastre J, Asensi M, Rodrigo F, Estrela JM, Viña J. Physiological changes in glutathione metabolism in fetal and newborn rat liver. Biochem J 1991;274:891-3. 4. Vento M, Asensi M, García-Sala F, Catalá J, Viña J. Oxidative stress caused by fetal to neonatal transition in humans [abstract]. Ped Res 1995;38:460. 5. Sastre J, Asensi M, Rodrigo F, Pallardó FV, Vento M, Viña J. Antioxidant administration to the mother prevents oxidative stress associated with birth. Life Sci 1994;54:2055-9. 6. Fellman V, Raivio K. Reperfusion injury as the mechanism of brain damage after perinatal asphxyia. Ped Res 1997;41:599-606. 7. Saugstad OD, Aasen AO. Plasma hypoxanthine levels as a prognostic aid of tissue hypoxia. Eur Surg Res 1980;12:123-9. 8. Fridovich I. Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase. J Biol Chem 1968;243:5753-60. 9. Bracci R. Red blood cell involvement in fetal/neonatal hypoxia. Biol Neonate 2001;79:210-2. 10. International Guidelines for Neonatal Resuscitation. An Excerpt from the Guidelines 2000 for Cardiopulmonary Resuscitation and Emergency. Cardiovascular care: international consensus on science. Pediatrics 2000;106:e29. 11. Saugstad OD, Rootweelt T, Aalen O, and the Resair 2 group. Resuscitation of asphyxiated newborn infants with room air or oxygen: an international controlled trial: the Resair 2 Study. Pediatrics 1998;102:1-7e. 12. Vento M, Asensi M, Sastre J, Pallardo FV, García-Sala F, Viña J. Resuscitation with room air instead of 100% oxygen prevents oxidative stress in moderately asphyxiated term neonates. Pediatrics 2001;107:642-7. 13. Poulsen JP, Øyasaeter S, Saugstad OD. Hypoxanthine, xanthine, and uric acid in newborn pigs during hypoxemia, followed by resuscitation with room air or 100% oxygen. Crit Care Med 1993;21:1058-65. 14. Saugstad OD. Resuscitation with room air or with oxygen supplementation. Clin Perinatol 1998;25:741-56. 15. Asensi M, Sastre J, Pallardó FV, Lloret A, Lehner M, Garcia-de-laAsunción J, et al. Ratio of reduced to oxidized glutathione as indicator of oxidative stress status and DNA damage. Methods Enzymol 1999;299:267-76. 16. Asensi M, Sastre J, Pallardó FV, García de la Asunción J, Estrela JM, Viña J. A high-performance liquid chromatography method for measure-
246 Vento et al
ment of oxidized glutathione in biological samples. Ann Biochem 1994;217:323-8. 17. Flohé L, Güzzler WA. Assays on glutathione peroxidase. Methods Enzymol 1984;105:114-21. 18. Akerboom TPM, Sies H. Glutathione reductase assay. Methods Enzymol 1981;77:373-82. 19. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974;249:7130-9. 20. Flohé L, Otting F. Superoxide dismutase assays. Methods Enzymol 1984;105:93-104. 21. Siesjö BK, Siesjö P. Mechanisms of secondary brain injury. Eur J Anaesthesiol 1996;13:247-68. 22. Peeters C, Van Bel F. Pharmacotherapeutical reduction of post-hypoxic-ischemic brain injury in the newborn. Biol Neonate 2001;79:274-80. 23. Kondo M, Itoh S, Isobe K, Kondo M, Kunikata T, Imai T, et al. Chemiluminescence because of the production of reactive oxygen species in the lungs of newborn piglets during resuscitation periods after asphyxiation load. Ped Res 2000;47:524-7. 24. Kutzsche S, Ilves P, Kirkeby OJ, Saugstad OD. Hydrogen peroxide production in leukocytes during cerebral hypoxia and reoxygenation with 100% or 21% oxygen in newborn piglets. Ped Res 2001;49:834-42. 25. Jamieson D, Chance B, Cadenas E, Boveris A. The relation of free radicals production to hyperoxia. Annu Rev Physiol 1986;48:703-19. 26. Neefjes VME, Evelo CTA, Baars, LGM, Blanco CE. Erythrocyte glutathione-s-transferase as a marker of oxidative stress at birth. Arch Dis Child Fetal Neonatal Ed 1999;81:F130-3. 27. Frank L, Groseclose EE. Preparation for birth into an O2-rich environment: the antioxidant enzymes in the developing rabbit lung. Ped Res 1984;18:240-4. 28. Schafer FQ, Buettner GR. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Rad Biol Med 2001;30:1191-212. 29. Suzuki YJ, Forman HJ, Sevanian A. Oxidants as stimulators of signal transduction. Free Rad Biol Med 1997;22:269-85. 30. Nakamura H, Nakamura K, Yodoi J. Redox regulation of cellular activation. Annu Rev Immunol 1997;15:351-69.
The Journal of Pediatrics • March 2003
OXIDATIVE STRESS IN ASPHYXIATED TERM INFANTS RESUSCITATED WITH 100% OXYGEN MÁXIMO VENTO, PHD, MD, MIGUEL ASENSI, PHD, JUAN SASTRE, PHD, ANA LLORET, PHD, FERNANDO GARCÍA-SALA, MD, AND JOSÉ VIÑA, PHD, MD
Objective tive stress.
To test the hypothesis that resuscitation of asphyxiated infants with pure oxygen causes hyperoxemia and oxida-
Study design Asphyxiated term newborn infants (n = 106) were randomly resuscitated with room air (RAR = 51) or 100% oxygen (OxR = 55). The Apgar score, time of the first cry, and establishment of a sustained pattern of respiration were recorded. Assays performed included: blood gases; reduced glutathione (GSH) and oxidized glutathione (GSSG) in whole blood; glutathione-related enzyme activities; and superoxide dismutase activity (SOD) in erythrocytes. Results The RAR group needed less time of ventilation for resuscitation (5.3 ± 1.5 vs 6.8 ± 1.2 min; P < .05). Pure oxygen caused hyperoxemia (PO2, 126.3 ± 21.8 mm Hg) that did not occur with the use of room air (PO2, 72.2 ± 6.8 mm Hg). GSH was decreased and GSSG, the glutathione cycle enzymes, and SOD activities were increased in both asphyxiated groups. However, the 100% oxygen-resuscitated group showed significantly greater alterations that correlated positively with hyperoxemia. Conclusions Asphyxia causes oxidative stress in the perinatal period, and resuscitation with 100% oxygen causes hyperoxemia and increased oxidative stress. Because there are no advantages to resuscitation with 100% oxygen, room air may be preferred under certain circumstances for the resuscitation of asphyxiated neonates. (J Pediatr 2003;142:240-6)
he fetal-to-neonatal transition is characterized by physiologic and metabolic changes that are accompanied by a marked increase in the availability of oxygen to the body.1 As a consequence, the pro-oxidant status may cause oxidative stress.2 Indeed, under physiologic conditions, there is a remarkable increase in oxidized glutathione levels in rat liver and erythrocytes from human neonates.3-5 Intrapartum asphyxia is characterized by transient periods of hypoxia during the ischemic phase followed by reperfusion.6 Upon reperfusion and re-oxygenation a flood of oxygen-free radicals are generated by the xanthine oxidase system. The amount of oxygen radicals produced is directly dependent on the oxygen as well as the hypoxanthine and other purine concentrations in the tissue.7,8 Oxygen-free radical generation causes endothelial cell damage and abnormalities in n-methyl-D-aspartate receptors, synaptosome structure, and astrocyte function, thus contributing to the development of brain injury after a hypoxic-ischemic episode.9 Guidelines recommend the use of 100% oxygen for the resuscitation of the asphyctic newly born infant.10 However, the use of room air has proved to be efficient for newborn resuscitation, and no differences regarding mortality or short-term morbidity were detected with the use or room air compared with pure oxygen.11,12 In addition, the use of pure oxygen for resuscitation in animal experiments increased the arterial partial pressure of oxygen above the physiologic range.13 Hyperoxemia has been associated with numerous negative side effects, including delayed initiation of spontaneous respiration, increased oxygen consumption, and irregularities in the cerebral circulation.14
T
GPx GR GSH GSH-s-tr GSSG
Glutathione peroxidase Glutathione reductase Reduced glutathione Glutathione-s-transferase Oxidized glutathione
OxR RAR ROS SOD
100% oxygen resuscitated newly born infants Room air resuscitated newly born infants Reactive oxygen species Superoxide dismutase
See editorial, p 221.
From School of Medicine, University of Alicante, Department of Pediatrics and Neonatology and Division of Neonatology, Hospital Virgen del Consuelo, and the Department of Physiology, School of Medicine, University of Valencia, Spain. Supported in part by the Annual Research Grant (1999) of the Sociedad Española de Neonatologia (to M.V.). Submitted for publication Jan 28, 2002; revisions received July 1, 2002. and Oct 24, 2002; accepted Nov 22, 2002. Reprint requests: Máximo Vento, PhD, MD, Servicio de Pediatría, Hospital Virgen del Consuelo, Callosa de Ensarriá, 15 bajo, E-46007 Valencia, Spain. E-mail: [email protected]. Copyright © 2003, Mosby, Inc. All rights reserved. 0022-3476/2003/$30.00 + 0
10.1067/mpd.2003.91
240
Fig 1. A, Linear regression analysis correlating paO2 with GSSG in newly born asphyxiated infants independently of the gas source (room air or 100% oxygen) used for resuscitation. Values plotted represent the combined data obtained at birth, during active resuscitation and at clinical stabilization. B, Linear regression analysis correlating the time of positive pressure ventilation employed for resuscitation of the asphyxiated infants, expressed in minutes with GSSG.
Previously, we found that the use of 100% oxygen for resuscitation of asphyctic neonates caused prolonged oxidation of blood glutathione compared with room air.12 The two distinctive features between both groups were: a 5-fold greater oxygen concentration in the 100% oxygen group (OxR) (100% vs 21%) and a more prolonged time of exposure to oxygen in the 100% oxygen group, compared with the room air group (RAR) (7.5 ± 1.8 vs 4.6 ± 0.7 min).12 The aim of the current study was to test whether the greater amount of oxygen delivered to the OxR group could be the triggering factor for oxidative stress. To test this hypothesis, we have performed a randomized clinical trial of the relationship between the duration of oxygen administration and the degree of hyperoxia with markers of oxidative stress at the end of active resuscitation and at the time clinical stabilization after resuscitation.
MATERIAL AND METHODS Design This is a randomized clinical trial, blinded for the gas source during resuscitation. Randomization was performed by assigning a sealed enveloped containing a computer generated random number plus a statement indicating the corresponding group (100% oxygen or room air) to each mother’s record at admission to the obstetric ward before delivery. Eligible patients were recruited among term neonates (37-40 weeks’ gestation) born at the Hospital Virgen del Consuelo that showed evident signs of asphyxia. Thus, they were hypotonic and apneic, nonresponsive to external stimuli, pale, bradycardic (<80 bpm), and acidotic (pH ≤7.05). The neonatologist evaluated Oxidative Stress in Asphyxiated Term Infants Resuscitated With 100% Oxygen
the infants’ clinical statuses immediately after birth, and if they were asphyxiated, obtained a first blood sample from the umbilical cord. A nurse opened the sealed envelope and switched the gas source in accordance to randomization. The resuscitating team was not aware of the oxygen concentration they were using, but the gas mixture could be changed if requested. The Ethical Committee of the hospital approved the study protocol. Parents were informed of the ongoing trial in the hospital by the attending obstetrician. The parents’ written informed consent was required in all cases and obtained before delivery after admission to the obstetric ward.
Population Of 151 eligible neonates, only 106 met the requirements of the study: 51 in the RAR group and 55 in the OxR group. Controls consisted of 22 nonasphyxiated term neonates. A total of 45 neonates were excluded, because of not fulfilling the biochemical requirements (n = 10), having insufficient blood for analytical purposes (n = 14), because they were switched from room air to 100% oxygen (n = 7) or vice-versa (n = 5), or because they were not blindly resuscitated (n = 9). The decision to switch the gas source was made by the neonatologist in charge when evident clinical signs of asphyxia were present and after several minutes of active ventilation. Major confounders for both experimental groups are described in Table I and clinical variables recorded are shown in Table II.
Clinical Proceedings The resuscitation team consisted of a neonatologist, an anesthesiologist, and two nurses. Immediately after birth, as241
Table I. Clinical variables of the newly born population
Gestational age (wk) Birth weight (g) Vaginal/Cesarean delivery Epidural analgesia/General anesthesia* Fetal bradycardia (<80 beats/min) Meconium-stained amniotic fluid Intubation (for suctioning and/or ventilation)‡
Non-asphyxiated † control (n = 22)
Asphyxiated RAR (n = 51)
Asphyxiated OxR (n = 55)
39.5 ± 1.9 3310 ± 190 15/7 20/2 0 2 0
38.9 ± 1.6 3160 ± 240 16/35b 16/35b 34b 10a 5a
40.5 ± 1.1 3220 ± 168 14/41b 14/41b 32b 13a 7a
*General anesthesia included sedation, relaxation, intubation, and/or bag and mask ventilation. Pre-oxygenation was used before intubation, and supplemental oxygen was administered as required during the inhalational anesthesia, always under pulseoximetry control. †Control indicates nonasphyxiated newly born term infants. ‡The difference between the number of infants with meconium-stained amniotic fluid and intubations is because several infants were suctioned with a suction catheter and ventilated with bag and mask, and thus were not intubated. a, RAR/OxR versus control P < .05; b, RAR/OxR versus control P < .01.
Table II. Clinical information
1-min Apgar score median 5-min Apgar score median 10-min Apgar score median Onset of first cry (min)* Onset of spontaneous respiration (min)† Hemoglobin saturation >90% (reached at min)
Nonasphyxiated controls (n = 22)
Asphyxiated RAR (n = 55)
Asphyxiated OxR (n = 52)
8 (7-9) 9 (8-10) 9 (8-10) 0.5 ± 0.1 0.6 ± 0.2 0.9 ± 0.4 (n = 18)
3 (1-4)b 6 (5-8)a 8 (7-9) 1.4 ± 0.3b 5.3 ± 1.5b 2.0 ± 0.7b (n = 48)
3 (1-4)b 6 (4-8)a 8 (6-9) 1.9 ± 0.4b,c 6.8 ± 1.2b,c 1.8 ± 0.9b (n = 43)
Apgar scores are expressed as median with 5th to 95th percentiles in parentheses. Other values are expressed as mean ± SD. Control indicates normal nonasphyxiated newly born term infants. n = number of infants to whom a specific variable was recorded. Significance is expressed as follows: a, P < .05; b, P < .01 versus control group; c, P < .05 versus RAR group. *Time elapsed until the occurrence of the first audible cry. †Time elapsed until the onset of a regular, spontaneous respiration that did not need additional intervention of the resuscitation team.
phyxiated infants were resuscitated following standard procedures, which included thermal stabilization, aspiration of the upper respiratory airway, and positive-pressure ventilation with bag and mask, as recently described.10 Meconium-stained amniotic fluid was directly suctioned from the trachea. In cases of ineffective ventilation (poor color, poor chest expansion, and sustained bradycardia) the resuscitation team proceeded to endotracheal intubation. Neonatal nurses placed probes to monitor the infants’ clinical variables (temperature, heart rate, respiratory frequency, and pulseoximetry). Heart rate and respiratory frequency also were evaluated by direct auscultation of the infant. Nurses also recorded the Apgar scores at 1, 5, and 10 minutes of postnatal life, the time elapsed until the first audible cry, and the time elapsed until the onset of spontaneous respiration (Table II). This was defined, as the time needed to attain a respiratory pattern without the intervention of the resuscitation team.14,15 Two infants in the OxR group needed supplemental oxygen therapy for approximately 3 hours (maximal Fi02 of 30%). Non of the infants in the RAR group needed supplemental oxygen. No infant had hyperthermia. 242 Vento et al
The first blood sample was obtained in all infants from umbilical vessels just before detachment from the placenta. Thereafter, in asphyxiated infants, two more samples were obtained from an umbilical artery catheter or from the right radial artery. The first sample was obtained just after the onset of spontaneous respiration (Table II). The second sample was obtained 10 minutes thereafter when clinical recovery (defined as spontaneous respiration, normal heart rate, good response to stimuli, and normal oxygen saturation) was evident. A total of 6 mL of blood was required to complete all measurements. Umbilical artery canalization is not a routine procedure in our nursery and was specifically performed to analyze blood gases in the current study. In case of failure to canalize, we used the right radial artery (Table III). After clinical stabilization umbilical catheters were immediately removed. There were no complications derived from the use of the catheters. A structured neurologic evaluation, electroencephalogram, and ultrasonographic imaging were performed at the end of the first week of life and repeated 4 weeks later. The Journal of Pediatrics • March 2003
chloride. The supernatant was aspirated, and the cell pellet was hemolyzed with distilled water. Enzyme activities were assayed in the hemolysate and were expressed per gram of hemoglobin content.
Statistical Analysis
Fig 2. Multiple regression analysis representing the correlation between paO2 (mm Hg) as independent variable and glutathione redox cycle enzymes (glutathione reductase, glutathione peroxidase, glutathione—s-transferase) as dependent variables in asphyxiated newly born infants, independently from the type of gas (room air or 100% oxygen) used for resuscitation. Values data plotted represent the combined data obtained at birth, during active resuscitation, and at clinical stabilization.
Analytical Assays Blood gases were processed immediately after blood withdrawal (Radiometer, Copenhagen). GSH and GSSG were determined in whole blood as we have previously described.15,16 Glutathione peroxidase, glutathione reductase and glutathione-S-transferase, and superoxide dismutase activities were determined as described by Flohé and Güzzler,17 Akerboom and Sies,18 Habig et al,19 and Flohé and Otting,20 respectively. Blood in heparinized tubes was immediately centrifuged for 10 minutes at 500 g and 4°C. Plasma was removed, and erythrocytes were washed twice with 0.9% sodium Oxidative Stress in Asphyxiated Term Infants Resuscitated With 100% Oxygen
All calculations and graphs were made using the GBSTAT computer program (Dynamics Microsystems, Inc, Silver Spring, Md). Nonparametric statistics were used because the distribution of the variables was not normal. We used the Mann-Whitney U test for nonpaired samples and the Kruskall-Wallis test for >2 nonpaired (independent) samples. Both simple and multiple regression analyses were performed to assess the effect of variations in the arterial paO2 (independent variable) on GSH, GSSG, and the activity of glutathione redox cycle enzymes and superoxide dismutase (dependent variables). The effect of time of positive pressure ventilation (independent variable) on GSSG (dependent variable) was evaluated by simple regression analysis.
RESULTS There was an increased rate of cesarean delivery among the asphyxiated infants (72%) compared with the control group (32%) (Table I). In addition, significant differences because of the presence of fetal bradycardia, meconium-stained amniotic fluid, and the need for intubation occurred in the asphyxiated neonates compared with the control group. The median Apgar score at 1 and 5 minutes was significantly lower in the asphyxiated newly born infants than in the control nonasphyxiated group (Table II). The time elapsed until the onset of the first cry, and for the onset of a sustained respiratory pattern, was significantly shorter in the control group than in both experimental groups (P < .01). However, the RAR group needed significantly less time of ventilation to initiate the first cry (P < .05) and for the onset of a sustained respiratory pattern (P < .05) than the OxR group (RAR, 5.3 ± 1.5 minutes; OxR, 6.8 ± 1.2 minutes). 243
Table III. Blood gases in asphyxiated newly born infants resuscitated with room air or 100% oxygen Control (n = 22) Sampling (min) Umbilical artery Radial artery pH pCO2 (mm Hg) pO2 (mm Hg)
Birth 22 0 7.38 ± 0.06 51.0 ± 9.9 30.3 ± 4.5
Asphyxiated RAR (n = 51) Birth 51 0 7.05 ± 0.07† 57.3 ± 9.1* 27.8 ± 5.2
5.3 ± 1.5 38 13 7.24 ± 0.9 46.4 ± 6.8 72.2 ± 6.8
Asphyxiated OxR (n = 55) 15 41 10 7.38 ± 0.5 37.4 ± 9.2 82.7 ± 5.2
Birth 55 0 7.02 ± 0.3† 59.3 ± 6.9* 30.0 ± 6.8
6.8 ± 1.2 40 15 7.27 ± 0.5 49.5 ± 5.3 126.3 ± 21.8‡
15 43 12 7.32 ± 0.4 40.2 ± 7.6 110.5 ± 15.8‡
Control indicates nonasphyxiated newly born term infants. RAR indicates asphyxiated newly born term infants resuscitated with room-air. OxR indicates asphyxiated newly born term infants resuscitated with 100% oxygen. pH comparisons are used based on the –log [H+]. Values are expressed as mean ± SD. Significance is expressed as follows: *P < .05; †P < .01 versus control. ‡P < .01 versus RAR.
The control group needed significantly less time than the asphyxiated groups to attain a hemoglobin saturation of >90% (P < .01). The timing of the blood sampling for analytical purposes was as follows: immediately after birth (time 0), at the end of the resuscitating maneuvers coinciding with the onset of spontaneous respiration, and 10 minutes thereafter when clinical stabilization was achieved. The pH values in the RAR and OxR groups immediately after birth were significantly lower than the pH of the control group (P < .01), but there were no differences between asphyxiated infants and the control group at the end of resuscitation, and when clinical stabilization was achieved (Table III). The experimental groups had significantly increased paCO2 values at birth compared with the control group. However, at the onset of spontaneous respiration, both experimental groups had normal paCO2 values. No significant difference between paO2 in the control and asphyxiated groups were detected at birth. Positive pressure ventilation raised paO2 in both asphyxiated groups. However, paO2 was higher in the OxR group than in the RAR group at the end of resuscitation (P < .01). Moreover, when both groups reached clinical stabilization, paO2 in the OxR group was still higher than in the RAR group (P < .01). GSH concentration decreased in a similar manner in the first 15 minutes of postnatal life in both asphyxiated groups (Table IV). GSSG levels in umbilical cord increased significantlt in both experimental groups (P < .01) relative to the control group. However, there was a significantly higher GSSG concentration in OxR than in RAR group at the end of resuscitation and at 15 minutes life (P < .05). The activity of GPx, GSH-s-transferase and GR increased similarly in both asphyxiated groups at the end of the resuscitation compared with the control group (P < .01). However, at 15 minutes of life, these enzymes showed a greater activity in the OxR group than in the RAR group (P < .05). There was also a significantly higher activity of SOD in both asphyxiated groups at birth than in the control nonasphyxiated group (P < .05). However, at 15 minutes of life, the activity of SOD in the OxR group was significantly greater than in the RAR group (P < .05). 244 Vento et al
The paO2 for OxR and RAR correlated with GSSG concentration (P < .05) (Fig 1, A). There was also a significant correlation between the time of ventilation and GSSG concentration. Furthermore, the slope for the OxR group was much higher than that of the RAR group (P < .01). The activities of the glutathione redox cycle enzymes, at birth, at the onset of spontaneous respiration, and when clinical stabilization was attained, correlated significantly with paO2 (Fig 2). Follow-up studies at the end of the first week of life and 4 weeks of age did not reveal any differences between either experimental groups.
DISCUSSION Oxidative stress is a physiologic event in the fetal-toneonatal transition. In isolated hepatocytes from fetal and newborn rats, we found a dramatic decrease (15- to 20-fold) in the GSH/GSSG ratio, mainly because of a marked increase in the GSSG concentration in the fetal-to-neonatal transition.3 In addition, all the enzyme activities involved in the glutathione redox cycle increased during that transition, especially glutathione peroxidase and glutathione-s-transferase.3 Similar results were found during normal fetal-to-neonatal transition in human erythrocytes. However, by 3 days after birth, GSSG concentration and the activity of the enzymes returned to normal in healthy neonates.4 The oxygen shortage during asphyxia leads to an impairment of mitochondrial function, energy failure, accumulation of purine derivatives, and increased generation of reactive oxygen species.6 In the current study, we found significantly increased GSSG concentrations, as well as increased SOD activity in asphyxiated infants immediately at birth, compared with nonasphyxiated controls reflecting oxidative stress. Upon reperfusion, reactive species of oxygen are generated through a mechanism dependent on the concentration of oxygen and purine derivatives accumulated in the tissue.6 The damaging effects of ROS on brain cells constitute the principal hypothesis put forward to explain brain damage during the asphyxia-reperfusion process.21 In fact, in the current study, asphyxiated infants showed increased GSSG levels and activities of SOD and of the glutathione The Journal of Pediatrics • March 2003
Table IV. Reduced GSH, GSSG, and glutathione cycle enzyme activities in asphyxiated newly born infants resuscitated either with RAR or OxR Umbilical vessel
GSH (µM) GSSG (µM) GPx (U/gHb) GR (U/gHb) GSH-s-tr (U/gHb) SOD (U/gHb)
End of resuscitation
Clinical stabilization
Control (n = 22)
RAR (n = 51)
OxR (n = 55)
RAR
OxR
RAR
OxR
1025 ± 166 22.4 ± 6.7 45 ± 12.0 8.1 ± 2.6 6.6 ± 1.5 1.3 ± 0.5
955 ± 134 61.2 ± 7.5† 51 ± 7.9 8.8 ± 3.1 5.9 ± 2.0 2.2 ± 0.5*
1130 ± 140 67.4 ± 9.2† 52.5 ± 8.8 7.6 ± 2.7 7.0 ± 0.8 2.6 ± 0.7*
760 ± 110* 82.5 ± 10.1† 65.7 ± 12.0* 12.5 ± 2.9* 10.4 ± 3.1† 2.5 ± 0.3
835 ± 96* 102 ± 13.8†‡ 71.2 ± 13.4* 15.2 ± 3.9* 12.4 ± 2.1† 3.1 ± 0.7
720 ± 75* 83.1 ± 12.5† 67.8 ± 9.5* 16.2 ± 5.1† 11.3 ± 2.6† 2.8 ± 0.4
640 ± 90† 110.7 ± 21.4†‡ 79.6 ± 11.8†‡ 20.2 ± 4.3†‡ 16.5 ± 3.2†‡ 3.5 ± 0.8‡
Control indicates nonasphyxiated newly born infants. Values are expressed as mean ± SD. Significance is expressed as: *P < .05; †P < .01 versus control group; ‡P < .05 versus RAR group.
redox cycle enzymes at the end of ventilation than were present immediately after birth. Thus, during reperfusion biochemical indicators of oxidative stress increased significantly in both asphyxiated groups, independently of the use of room air and 100% oxygen. We hypothesized that there were two factors responsible for the prolonged oxidative stress in the OxR group. First, the OxR group received a significantly larger amount of oxygen than the RAR group. The amount of oxygen received by each infant during resuscitation is the result of multiplying the tidal volume respiratory rate duration of resuscitation expressed in minutes (RAR, 5.3 ± 1.5 minutes; OxR, 6.8 ± 1.2 minutes) oxygen concentration in the breathing gas (RAR, 0.21; OxR, 1.0). Thus, the OxR infants received an average of 350 mL more oxygen than infants in the RAR group. Second, the OxR group was exposed to supraphysiologic concentrations of oxygen for a more prolonged time. High paO2 is known to increase the production of ROS.25 Under these circumstances, the antioxidant defenses of the newborn infant may be incapable of coping with this pro-oxidant challenge. In our current study, the OxR group had higher paO2 than the RAR group at the end of positive pressure ventilation and thereafter. In addition, the OxR group was ventilated for a longer time than the RAR group. Hyperoxemia and the time of positive pressure ventilation significantly correlated with the GSSG levels. Hence, the GSH/GSSG ratio in the OxR group was significantly lower than in the RAR group. In the current study, GPx, GR, and GSH-s-tr, had an increased activity in erythrocytes of both asphyxiated groups during resuscitation, and 15 minutes after birth, their activity was significantly higher in the OxR group. Previous studies have reported that an increase in the activities of erythrocyte glutathione redox cycle enzyme is indicative of an adaptation to oxidative stress, as seen during fetal-toneonatal transition.26,27 Our results show that hyperoxia increases the activities of glutathione-related enzymes. The expression of these enzymes might be up-regulated by hyOxidative Stress in Asphyxiated Term Infants Resuscitated With 100% Oxygen
peroxia, thus acting as sensors of oxidative stress. Therefore, our results suggest that oxidative stress modulate the antioxidant cellular defenses as an adaptive response in newly born infants. Blood GSH/GSSG ratio and erythrocyte enzymes activities should be considered as general indicators of systemic oxidative stress.3,5 Thus, measurements of the glutathione redox ratio and other oxidative stress indicators in specific tissues, such as brain and lung, would need to be performed to demonstrate oxidative damage. Glutathione is considered to be the major thioldisulfide redox buffer of mammalian cells, and the GSH-toGSSG ratio has been used to estimate the redox environment of a cell. An increase in the GSSG concentration is associated with a pro-oxidant challenge.28 Changes in the cellular redox environment can alter signal transduction, DNA and RNA synthesis, protein synthesis, enzyme activation, and even regulation of the cell cycle.29 In addition, overexpression of antioxidant enzymes, such as the increase of SOD activity, may inhibit cell growth.30 Hence the increased activity of SOD in the OxR group may be of relevance regarding cell proliferation. The changes in the glutathione redox cycle and the antioxidant enzyme activities associated with high oxygen concentrations deserve further study. The use of pure oxygen during resuscitation needs to be reconsidered. An appropriate redox environment is a sine qua non for optimal cellular metabolism. The consequences of its alterations may not be predictable. Room air resuscitation may have advantages over pure oxygen. We thank Marilyn R. Noyes for revising the manuscript.
REFERENCES 1. Dejour P. Principles of comparative respiratory physiology. Amsterdam: Elsevier/North Holland; 1991. p. 147-71. 2. Saugstad OD. Mechanism of tissue injury by oxygen radicals: implications for neonatal disease. Acta Paediatr 1986;85:1-4.
245
3. Pallardó FV, Sastre J, Asensi M, Rodrigo F, Estrela JM, Viña J. Physiological changes in glutathione metabolism in fetal and newborn rat liver. Biochem J 1991;274:891-3. 4. Vento M, Asensi M, García-Sala F, Catalá J, Viña J. Oxidative stress caused by fetal to neonatal transition in humans [abstract]. Ped Res 1995;38:460. 5. Sastre J, Asensi M, Rodrigo F, Pallardó FV, Vento M, Viña J. Antioxidant administration to the mother prevents oxidative stress associated with birth. Life Sci 1994;54:2055-9. 6. Fellman V, Raivio K. Reperfusion injury as the mechanism of brain damage after perinatal asphxyia. Ped Res 1997;41:599-606. 7. Saugstad OD, Aasen AO. Plasma hypoxanthine levels as a prognostic aid of tissue hypoxia. Eur Surg Res 1980;12:123-9. 8. Fridovich I. Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase. J Biol Chem 1968;243:5753-60. 9. Bracci R. Red blood cell involvement in fetal/neonatal hypoxia. Biol Neonate 2001;79:210-2. 10. International Guidelines for Neonatal Resuscitation. An Excerpt from the Guidelines 2000 for Cardiopulmonary Resuscitation and Emergency. Cardiovascular care: international consensus on science. Pediatrics 2000;106:e29. 11. Saugstad OD, Rootweelt T, Aalen O, and the Resair 2 group. Resuscitation of asphyxiated newborn infants with room air or oxygen: an international controlled trial: the Resair 2 Study. Pediatrics 1998;102:1-7e. 12. Vento M, Asensi M, Sastre J, Pallardo FV, García-Sala F, Viña J. Resuscitation with room air instead of 100% oxygen prevents oxidative stress in moderately asphyxiated term neonates. Pediatrics 2001;107:642-7. 13. Poulsen JP, Øyasaeter S, Saugstad OD. Hypoxanthine, xanthine, and uric acid in newborn pigs during hypoxemia, followed by resuscitation with room air or 100% oxygen. Crit Care Med 1993;21:1058-65. 14. Saugstad OD. Resuscitation with room air or with oxygen supplementation. Clin Perinatol 1998;25:741-56. 15. Asensi M, Sastre J, Pallardó FV, Lloret A, Lehner M, Garcia-de-laAsunción J, et al. Ratio of reduced to oxidized glutathione as indicator of oxidative stress status and DNA damage. Methods Enzymol 1999;299:267-76. 16. Asensi M, Sastre J, Pallardó FV, García de la Asunción J, Estrela JM, Viña J. A high-performance liquid chromatography method for measure-
246 Vento et al
ment of oxidized glutathione in biological samples. Ann Biochem 1994;217:323-8. 17. Flohé L, Güzzler WA. Assays on glutathione peroxidase. Methods Enzymol 1984;105:114-21. 18. Akerboom TPM, Sies H. Glutathione reductase assay. Methods Enzymol 1981;77:373-82. 19. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974;249:7130-9. 20. Flohé L, Otting F. Superoxide dismutase assays. Methods Enzymol 1984;105:93-104. 21. Siesjö BK, Siesjö P. Mechanisms of secondary brain injury. Eur J Anaesthesiol 1996;13:247-68. 22. Peeters C, Van Bel F. Pharmacotherapeutical reduction of post-hypoxic-ischemic brain injury in the newborn. Biol Neonate 2001;79:274-80. 23. Kondo M, Itoh S, Isobe K, Kondo M, Kunikata T, Imai T, et al. Chemiluminescence because of the production of reactive oxygen species in the lungs of newborn piglets during resuscitation periods after asphyxiation load. Ped Res 2000;47:524-7. 24. Kutzsche S, Ilves P, Kirkeby OJ, Saugstad OD. Hydrogen peroxide production in leukocytes during cerebral hypoxia and reoxygenation with 100% or 21% oxygen in newborn piglets. Ped Res 2001;49:834-42. 25. Jamieson D, Chance B, Cadenas E, Boveris A. The relation of free radicals production to hyperoxia. Annu Rev Physiol 1986;48:703-19. 26. Neefjes VME, Evelo CTA, Baars, LGM, Blanco CE. Erythrocyte glutathione-s-transferase as a marker of oxidative stress at birth. Arch Dis Child Fetal Neonatal Ed 1999;81:F130-3. 27. Frank L, Groseclose EE. Preparation for birth into an O2-rich environment: the antioxidant enzymes in the developing rabbit lung. Ped Res 1984;18:240-4. 28. Schafer FQ, Buettner GR. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Rad Biol Med 2001;30:1191-212. 29. Suzuki YJ, Forman HJ, Sevanian A. Oxidants as stimulators of signal transduction. Free Rad Biol Med 1997;22:269-85. 30. Nakamura H, Nakamura K, Yodoi J. Redox regulation of cellular activation. Annu Rev Immunol 1997;15:351-69.
The Journal of Pediatrics • March 2003