Urinary Thiobarbituric Acid Reacting Substances As Potential Biomarkers of Intrauterine Hypoxia

ARTICLE Urinary Thiobarbituric Acid–Reacting Substances as Potential Biomarkers of Intrauterine Hypoxia

Ann Siciarz, MD; Barry Weinberger, MD; Gisela Witz, PhD; Mark Hiatt, MD; Thomas Hegyi, MD

Background: Currently available clinical tools can- associated with pregnancy-induced hypertension or re- not accurately identify the extent of perinatal hypoxic versal of umbilical arterial blood flow. injuries. During hypoxia, reactive oxygen species cause lipid peroxidation of cell membranes, yielding Results: Among term infants, urinary TBARS levels were oxidation products that constitute thiobarbituric acid– significantly increased following acute asphyxia (P=.02). reacting substances (TBARS). Levels of TBARS also tended to be elevated following chronic hypoxia. Urinary TBARS levels in term infants Objective: To see if the concentrations of TBARS ex- tended to be increased in those requiring mechanical ven- creted in urine would be elevated during the first day of tilation (P=.05) or delivery room resuscitation (P=.15), life in term and preterm infants following chronic hy- as well as in those passing intrauterine meconium (P=.13) poxia or acute asphyxia. or having clinical evidence of hypoxic-ischemic encephalopathy (P=.24). Design: Thiobarbituric acid–reacting substances levels were measured by a spectrophotometric assay in urine Conclusions: The results show a correlation between samples collected from term and near-term (Ն 34 weeks elevated urinary TBARS levels in term and near-term in- gestation, n=22), and preterm (Ͻ34 weeks gestation, fants, and perinatal hypoxia (as determined by low Ap- n=52) infants on the first day of life. gar scores or umbilical cord blood acidosis). We specu- late that TBARS concentrations may be useful as a Patients: Infants were admitted to the St Peter’s Uni- biomarker for perinatal hypoxic injury in newborns. Fur- versity Hospital (New Brunswick, NJ) neonatal inten- ther studies are needed to determine whether elevations sive care unit from July 1997 to January 1999. Acute as- in TBARS levels are better predictors of the extent of hy- phyxia was defined as umbilical cord blood pH values less poxic injury than existing markers. than 7.05, or Apgar scores of less than 5 at 5 minutes. Chronic hypoxia was defined as intrauterine growth retardation or low birth weight (small for gestational age) Arch Pediatr Adolesc Med. 2001;155:718-722

NTRAUTERINE HYPOXIA is a major fants has not often revealed pathogno- risk factor for an abnormal out- monic findings, and the identification of come in the neonatal period. Ap- such findings is necessary to provide post- proximately 700 neonatal deaths natal medical management that optimizes following intrauterine hypoxia are the medical and neurologic outcome. In ad- Ireported in the United States each year, dition, several specific interventions are cur- comprising more than 2% of total infant rently under investigation to limit neuro- mortality.1 The neurodevelopmental se- logic injury resulting from hypoxic- From the Division of quelae exhibited by survivors can be se- ischemic insult, including cerebral Neonatology, Department of vere, constituting a large part of pediatric hypothermia2 and antioxidant therapies.3 Pediatrics, UMDNJ–Robert health care expenditures. Despite the po- These appear to be most effective when ap- Wood Johnson Medical School, tential severity and prevalence of this con- plied early in the course of this process. St Peter’s University Hospital, dition, the diagnosis is usually based on Therefore, the identification of a rapid and New Brunswick, NJ nonspecific clinical criteria, since no reli- early marker of the extent of perinatal as- (Drs Siciarz, Weinberger, able markers have previously been dem- phyxia is an important step in identifying Hiatt, and Hegyi), and the Department of Environmental onstrated to correlate with the extent of in- patients eligible for prospective clinical tri- and Community Medicine, trauterine hypoxic insult. Such biomarkers, als and, ultimately, in devising specific pre- UMDNJ–Robert Wood Johnson if available, would be particularly helpful ventive interventions. Medical School, Piscataway, NJ in identifying infants exposed to chronic hy- Clinical and laboratory criteria that (Dr Witz). poxia in utero. Examination of these in- are currently used to identify asphyxi-

(REPRINTED) ARCH PEDIATR ADOLESC MED/ VOL 155, JUNE 2001 WWW.ARCHPEDIATRICS.COM 718

©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 PATIENTS AND METHODS DETERMINATION OF URINARY TBARS Urine samples were obtained from study infants on the first PATIENT CHARACTERISTICS day of life. Informed consent was obtained from parents for the acquisition of samples, and these studies were ap- All inborn infants admitted to the neonatal intensive care proved by the Committee for the Protection of Human Sub- unit at St Peter’s University Hospital (New Brunswick, NJ) jects in Research at St Peter’s University Hospital. All speci- during the period between July 1997 and January 1999 mens were collected into sterile containers and stored at were screened for eligibility, excluding those from mul- −70°C for batched analysis. Thiobarbituric acid–reacting sub- tiple gestations or with major congenital anomalies (1471 stances levels were measured as previously described.13,14 term and 602 preterm infants were screened). Of these, 6 Briefly, 200 µL of urine was combined with 10 µL of 5% bu- term infants (34 or more weeks’ gestation) and no surviv- tylated hydroxytoluene (BHT, in glacial acetic acid) and 300 ing preterm infants met the criteria for acute asphyxia. Six µL of a 0.5% aqueous thiobarbituric acid (TBA) solution. The term infants and 15 preterm infants met the criteria for samples were then vortexed and incubated at 100°C for 30 chronic hypoxia. All of these infants were enrolled. At the minutes. After cooling to room temperature, the absor- time that each eligible term infant was enrolled, a newborn bance of samples at 532 nm was measured using a Lamba infant of comparable gestational age but with no signs or 3B spectrophotometer (Perkin Elmer Corp, Baden See- symptoms of hypoxia, asphyxia, or growth retardation was werk, Germany). Samples were blanked against reference cu- selected as a control. Two potential term control infants vettes containing reagents without urine. The concentra- were not included because consent could not be obtained. tion of TBARS was calculated from the absorbance at 532 Therefore, 10 term infants served as controls. At the time nm, using 156000 as the molar extinction coefficient. The of enrollment of each preterm infant, 2 to 3 infants of quantity of TBARS is proportionate to the amount of MDA, comparable gestational age were selected as controls. More a lipid peroxidation product generated by the oxidation of preterm controls per subject were selected because of membrane lipids by ROS. Malondialdehyde reacts with TBA anticipated difficulties in obtaining consent and urine to form a 1:2 MDA-TBA adduct, which absorbs at 532 nm. samples within 24 hours in this group. Therefore, 40 In the present study, MDA was confirmed to be the pre- infants were recruited as controls. Of these infants, con- dominant TBA-reacting adduct by high-performance liquid sent was not obtained from 2, urine was not available from chromatography analysis of representative samples. 1, and 37 served as controls for the analysis. Study person- To control for urine concentration, data were normal- nel obtained demographic and medical information from ized to urine creatinine concentrations. Urinary creatinine was maternal and infant medical records. measured using the revised Jaffe method.15 Briefly, alkaline For analysis, infants were divided into 3 groups. In- picrate (formed by combining picric acid and 10% sodium fants with acute asphyxia were defined as those with um- hydroxide in a ratio of 5:1) was added to serial dilutions of bilical cord blood pH values less than 7.05, or 5-minute urine. Samples were incubated at room temperature for 15 Apgar scores of less than 5. Chronic hypoxia was defined minutes, and the absorbance at 500 nm was measured. as intrauterine growth retardation or low birth weight (small for gestational age) associated with either pregnancy- DATA ANALYSIS induced hypertension (PIH) or reversal of umbilical arterial blood flow. Control infants were those of appropriate Results are expressed as means ± SDs. As urinary TBARS size for gestational age who did not exhibit any signs of fe- concentrations were not normally distributed, we trans- tal or neonatal hypoxia. Preterm infants (Ͻ34 weeks ges- formed them to their natural logs, which were normally dis- tation) and term or near-term infants (Ն34 weeks gesta- tributed. Group means were compared using unpaired t tests. tion) were analyzed separately. A P value less than .05 was considered significant.

ated or hypoxic infants, including fetal heart rate pat- Umbilical cord blood pH might be expected to be terns, umbilical cord blood pH values, and Apgar scores, predictive of injury associated with perinatal hypoxic in- are known to be insensitive and/or nonspecific. Elec- sult. When gas exchange across the placenta is compro- tronic fetal monitoring during labor was introduced to mised, tissue hypoxia leads to anaerobic metabolism and identify fetuses at risk for hypoxic injury, but abnormal lactic acidosis. In addition, carbon dioxide accumula- fetal heart rate patterns have not been shown to predict tion further reduces fetal arterial pH. Nevertheless, Win- an abnormal outcome.4 Using a large cohort of more than kler et al7 found no differences between infants with cord 50000 high- and low-risk infants, Shy et al5 showed that pH values above or below 7.20 with respect to the de- electronic fetal monitoring had no advantage over aus- velopment of clinical signs of asphyxia (seizures, persis- cultation in predicting perinatal mortality, morbidity, Ap- tent hypotonia, and renal or cardiac dysfunction). Fee gar scores, cord blood gases, and long-term outcome. Sub- et al8 noted that among 110 term infants with a cord blood sequently, in 1989, a technical bulletin of the American pH less than 7.05 and a base deficit greater than 10 mEq/L, College of Obstetrics and Gynecology6 stated that, within 73% were admitted to regular nurseries and were dis- specified intervals, intermittent auscultation is equiva- charged as normal. Seven of 9 infants who had abnor- lent to continuous electronic fetal monitoring in detect- mal neurological features at birth had no abnormal char- ing fetal compromise. Thus, fetal heart rate monitoring acteristics at follow-up. In another study,9 no infant with lacks both sensitivity and specificity in identifying birth an umbilical cord blood pH less than 7.00, but with a asphyxia. 1-minute Apgar score greater than 3 had seizures or hy-

(REPRINTED) ARCH PEDIATR ADOLESC MED/ VOL 155, JUNE 2001 WWW.ARCHPEDIATRICS.COM 719

©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 Table 1. Demographic Variables and Urinary TBARS Levels Table 3. Demographic Variables and Urinary TBARS Levels in Term and Near-Term Infants* in Preterm Infants*

Chronic Acute Control Chronic Hypoxia Control Hypoxia Asphyxia Variables (n = 37) (n = 15) Variables (n = 10) (n=6) (n=6) Birthweight, g 1794 ± 366 1691 ± 305 Birthweight, g 2786 ± 926 1987 ± 569 3418 ± 661 Gestational age, wk 31.5 ± 1.3 32.1 ± 1.4 Gestational age, wk 37.4 ± 1.6 35.2 ± 1.0† 38.3 ± 2.7 Apgar scores Apgar scores 1 min 8.1 ± 1.1 8.1 ± 0.8 1 min 7.7 ± 2.4 8.8 ± 0.4 1.0 ± 0.6† 5 min 8.9 ± 0.4 8.9 ± 0.4 5 min 8.6 ± 1.3 9.2 ± 0.4 3.3 ± 2.0† TBARS, ng/mg of creatinine 8.93 ± 0.92† 8.26 ± 1.3 TBARS, ng/mg of 7.97 ± 0.87 8.33 ± 1.38 9.06 ± 0.60† creatinine *Values are expressed as means ± SDs. TBARS indicates thiobarbituric acid–reacting substances. *Values are expressed as means ± SDs. TBARS indicates thiobarbituric †Values are significantly different from term/near-term control values; acid–reacting substances. PϽ.05. †Values significantly differ from control values; PϽ.05.

infants, we measured concentrations of urinary thiobar- Table 2. Association of Urinary TBARS Levels bituric acid–reacting substances (TBARS). The most abun- With Clinical Indicators in Term and Near-Term Infants* dant of the TBARS is malondialdehyde (MDA), an aldehydic lipid peroxidation product formed by the action TBARS (ng/mg Creatinine) of ROS on lipid membranes. In these studies, we found Indicators Yes No P that the quantity of urinary TBARS, measured shortly af- Delivery room 8.75 ± 0.81 (8) 8.11 ± 1.05 (14) .15 ter birth, correlates with the severity of intrauterine hy- resuscitation poxic insult as determined by the patient history and clini- Meconium passage 9.09 ± 0.81 (4) 8.91 ± 1.03 (18) .13 cal criteria. Hypoxic-ischemic 9.21 ± 0.91 (2) 8.29 ± 1.03 (20) .24 encephalopathy Mechanical ventilation 9.07 ± 0.61 (6) 8.11 ± 1.06 (16) .05 RESULTS

*Values are expressed as means ± SDs (number of patients requiring Among term and near-term infants (n=22), those with treatment). TBARS indicates thiobarbituric acid–reacting substances. chronic hypoxia (n=6) had a lower mean gestational age than those with acute asphyxia (n=6) or control infants (n=12), and they tended to have lower birth weights potonia. Dennis et al10 found no association between de- (Table 1). Infants with acute asphyxia had signifi- velopmental outcome at 4.5 years, and acidosis at birth cantly lower 1-minute and 5-minute Apgar scores than (defined as an umbilical cord blood pH less than 7.10). controls. The log concentration of urinary TBARS in term Among a large random cohort of normal full-term new- infants with acute asphyxia (9.06±0.60) was signifi- borns, 10% were shown to have umbilical arterial base cantly greater than that of controls (7.97±0.88) (P=.02). deficits greater than 18 mEq/L.11 Only the most severe Chronically hypoxic term infants also exhibited el- base deficits (Ͼ20 mEq/L) in severely affected infants have evated urinary TBARS concentrations (8.33±1.38), but been associated with adverse neurological outcomes. this did not differ statistically from levels in control in- These data indicate that umbilical cord blood pH values fants (Table 1). Using a urinary TBARS threshold of 5500 are neither sensitive nor specific in identifying birth as- ng/mg creatinine, the sensitivity of this measurement for phyxia and its sequelae. Similarly, Apgar scores have been identifying acute asphyxia in term and near-term in- used to identify infants with birth asphyxia. However, fants was 67%, and its specificity was 90%. This re- the 1-minute Apgar score has poor sensitivity and posi- sulted in a positive predictive value of 80% and a nega- tive predictive value in detecting acidosis, and 25% to tive predictive value of 82%. Urinary TBARS levels in term 75% of infants with acidosis are assigned normal Apgar and near-term infants tended to be increased in those re- scores.12 Therefore, current methods have been inad- quiring mechanical ventilation (P=.05) or delivery room equate in identifying infants with hypoxic injury. This resuscitation (P=.15), as well as those passing intrauter- is most likely because all of these assessments are indi- ine meconium (P=.13) or with clinical evidence of hy- rect reflections of physiologic phenomena that may fol- poxic-ischemic encephalopathy (P=.24) (Table 2). Uri- low hypoxic insult, rather than direct measurements of nary TBARS were not correlated with chorioamnionitis, the pathophysiologic progression of asphyxia at the cel- PIH, umbilical artery blood flow, or mode of delivery. lular level. Among preterm infants (Ͻ34 weeks gestation) there Hypoxia induces cellular damage, in part by the gen- were 15 infants with chronic hypoxia and 37 control in- eration of reactive oxygen species (ROS), including hy- fants. The mean gestational ages, birth weights, and Ap- drogen peroxide, singlet oxygen, superoxide anion radi- gar scores were not significantly different between these cal, hydroxyl radical, and lipid hydroperoxides in affected groups (Table 3). Urinary TBARS levels were signifi- tissues. Therefore, we hypothesized that ROS that are cantly greater in control preterm infants than in control generated in the fetus can serve as a biomarker of intra- term and near-term infants (P=.01), but were not fur- uterine or perinatal hypoxia. In order to quantify ROS in ther increased following chronic intrauterine hypoxia.

(REPRINTED) ARCH PEDIATR ADOLESC MED/ VOL 155, JUNE 2001 WWW.ARCHPEDIATRICS.COM 720

©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 Acute asphyxia could not be studied in preterm infants born mice increased during reoxygenation after 20 min- because of insufficient numbers. utes of hypoxia. Malondialdehyde levels are also elevated in preterm infants, particularly those receiving 27 28 COMMENT oxygen and mechanical ventilation. Buonocore et al showed that umbilical cord blood total lipid hydroper- In this study, we have shown that urinary TBARS, mea- oxides are increased in preterm infants following fetal hy- sured during the first day of life, are elevated in term and poxia. Furthermore, high umbilical cord blood hydroper- near-term infants following acute asphyxia, as defined oxide levels in preterm infants have been correlated with by low Apgar scores or acidemia. Full-term infants also adverse outcomes in the newborn period that are reflec- tend to exhibit higher urinary excretion of TBARS fol- tive of ROS-mediated oxidative injury (death, severe in- lowing chronic low-grade oxygen deprivation, as indi- traventricular hemorrhage, necrotizing enterocolitis, or cated by intrauterine growth retardation in the presence pulmonary hemorrhage).29 of PIH or reversed umbilical arterial blood flow. Uri- In this study, we have defined acute and chronic peri- nary TBARS were not directly correlated with PIH (de- natal asphyxia using existing standards that are available fined by maternal blood pressure), suggesting that the at the time of birth (ie, Apgar scores, umbilical cord blood association of TBARS levels with chronic hypoxia oc- pH, and intrauterine growth). Although these measures curs primarily in infants with the most severely compro- are known to be unreliable in determining the severity of mised uteroplacental blood flow (reversed umbilical ar- hypoxic injury, no other early parameters have been es- terial blood flow or severe PIH). We also found that tablished as gold standards for diagnosing hypoxic injury preterm infants exhibit elevated urinary TBARS levels rela- in newborns. Further studies will be required to validate tive to term infants, possibly reflecting stresses inherent TBARS as a true biomarker for perinatal hypoxic- in preterm delivery. ischemic injury. To be useful, such a biomarker must do This study is limited by the small sample size (term the following: (1) be scientifically sound, (2) be available subjects, n=12), a consequence of the low incidence of rapidly during the early hours of life, (3) concur with more perinatal hypoxia and asphyxia during this period at our easily obtained or established clinical and laboratory in- neonatal intensive care unit. Nevertheless, the physiol- dicators, and (4) provide further information or preci- ogy of hypoxic-ischemic injury supports the potential role sion not provided by the other methods of predicting out- of early measurement of TBARS levels in the identifica- comes of interest. Previous studies have validated the first tion of affected infants. The generation of free radicals criterion by establishing that MDA (measured by TBARS) during tissue hypoxia plays a major role in the patho- is produced as a direct product of lipid peroxidative dam- genesis of tissue injury. Newborns are particularly sus- age to cell membranes, and that hypoxia induces free radi- ceptible to such injury because they exhibit an imbal- cal generation, oxidative stress, and lipid peroxidation. In ance between antioxidant and oxidant-generating systems. this study, we found that early TBARS measurements are Free radicals, in excess, inactivate proteins, disrupt DNA, correlated with perinatal stress and conventional mea- and oxidize lipids.16,17 The oxidation of lipids by radi- sures of hypoxia, suggesting that the second and third cri- cals has been long regarded as a critical event leading to teria are valid. Further studies will be required to validate cellular injury. Cell membranes contain a high propor- the final criterion by establishing the predictive value of tion of polyunsaturated lipids and are susceptible to per- elevated urinary TBARS with regard to long-term medi- oxidation, resulting in the formation of hydroperox- cal and neurologic prognoses. As we enter an era during ides, the most abundant product being MDA.18,19 which specific therapies may become available to avert the Malondialdehyde has a long half-life (Ͼ20 days) in neu- course of hypoxic-ischemic encephalopathy, the early iden- tral or acidic solutions, but is metabolised in vivo by re- tification of patients at risk will gain urgent importance. action with tissue proteins and nucleic acids.20 Al- though its biological half-life is likely to be variable and Accepted for publication February 2, 2001. has not been well described, MDA and TBARS have been Corresponding author and reprints: Barry Wein- used in animals and humans as reliable indicators of the berger, MD, Division of Neonatology, St Peter’s University response to pro-oxidant provocations.21 Conditions ini- Hospital, 254 Easton Ave, New Brunswick, NJ 08903 tiating lipid peroxidation in the perinatal period (eg, utero- (e-mail: [email protected]). placental restriction) are likely to be ongoing (acute or REFERENCES chronic) in the presence of unlimited substrate. Therefore, TBARS measurements, which are pro- 1. Wegman ME. Annual summary of vital statistics, 1993. Pediatrics. 1994;94:792- portional to MDA content, may provide a direct assess- 803. ment of the progression of hypoxic injury at the cellular 2. Gunn AJ. Cerebral hypothermia for prevention of brain injury following perinatal level. Consistent with our data, several previous studies asphyxia. Curr Opin Pediatr. 2000;12:111-115. 3. Daneyemez M, Kurt E, Cosar A, Yuce E, Ide T. Methylprednisolone and vitamin E have demonstrated that serum MDA levels in healthy full- therapy in perinatal hypoxic-ischemic brain damage in rats. Neuroscience. 1999; term infants are low at birth and rise postnatally in re- 92:693-697. sponse to the stress of abdominal delivery and/or to the 4. National Institutes of Health. Antenatal Diagnosis: Report of a Consensus Con- events of respiratory transition during the first days of ference. Bethesda, Md: National Institutes of Health; 1979. NIH publication 79- life.22,23 Umbilical cord blood MDA levels are increased 1973. 5. Shy KK, Larson EB, Luthy DA. Evaluating a new technology: the effectiveness of following fetal hypoxia, suggesting that MDA reflects the electronic fetal heart rate monitoring. Annu Rev Public Health. 1987;8:165-190. 24,25 26 extent of lipid peroxidation in vivo. Hasegawa et al 6. American College Obstetrics of Gynecology. Intrapartum and Fetal Heart Rate observed that the content of TBARS in the brains of new- Monitoring. Technical Bulletin No 132. September 1989.

(REPRINTED) ARCH PEDIATR ADOLESC MED/ VOL 155, JUNE 2001 WWW.ARCHPEDIATRICS.COM 721

©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 7. Winkler CL, Hauth JC, Tucker M, Owen J, Tucker JM. Neonatal complications at 20. Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynon- term as related to the degree of umbilical arterial acidemia. Am J Obstet Gyne- enal, malondialdehyde and related aldehydes. Free Radic Biol Med. 1991;11:81- col. 1991;164:637-641. 128. 8. Fee SC, Malee K, Deddish R, Minogue JP, Socol ML. Severe acidosis and sub- 21. Dhanakoti SN, Draper HH. Response of urinary malondialdehyde to factors that sequent neurological status. Am J Obstet Gynecol. 1990;162:802-806. stimulate lipid peroxidation in vivo. Lipids. 1987;22:643-646. 9. Gilstrap LC III, Leveno KL, Burris J, Williams ML, Little BB. Diagnosis of birth 22. Buonocore G, Zani S, Sargentini I, Gioia D, Bracci R. Malondialdehyde as a di- asphyxia on the basis of fetal pH, Apgar score, and newborn cerebral dysfunc- agnostic index of oxidative stress in the newborn [abstract]. Pediatr Res. 1995; tion. Am J Obstet Gynecol. 1989;161:825-830. 37(4 pt 2):280. 10. Dennis J, Johnson A, Mutch L, Yudkin P, Johnson P. Acid base status at birth 23. Malamitsi-Puchner A, Economou E, Efstathopoulos T, Sevastiandou S, Nicolo- and neurodevelopment at four and one half years. Am J Obstet Gynecol. 1989; poulos D. Postnatal changes of lipid peroxide plasma levels: possible indirect 161:213-220. index of gradually increasing oxygenation. In: Abstracts of the 4th European Work- 11. Silverman F, Suidan J, Wasserman J, Antoine C, Young BK. The Apgar score: is shop on Neonatology, Rome, Italy, Sept 22, 1993:182. it enough? Obstet Gynecol. 1985;66:331-336. 24. Buonocore G, Zani S, Sargentini I, Gioia D, Signorini C, Bracci R. Intraerythro- 12. Marrin M, Paes BA. Birth asphyxia: does the Apgar score have diagnostic value? cyte non-protein-bound iron and plasma malondialdehyde in the hypoxic new- Obstet Gynecol. 1988;72:120-123. born. Free Radic Biol Med. 1998;25:766-770. 13. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;52: 25. Schmidt H, Grune T, Muller R, Siems WG, Wauer RR. Increased levels of lipid 302-310. peroxidation products malondialdehyde and 4-hydroxynonenal after perinatal hy- 14. Valenzuela A. The biological significance of malondialdehyde determination in poxia. Pediatr Res. 1996;40:15-20. the assessment of tissue oxidative stress. Life Sci. 1991;48:301-309. 26. Hasegawa K, Yoshioka H, Sawada T, Nishikawa H. Lipid peroxidation in neona- 15. Coulthard MG, Hey EN, Ruddock V. Creatinine and urea clearances compared to tal mouse brain subjected to two different types of hypoxia. Brain Dev. 1991;13: inulin clearance in preterm and mature babies. Early Hum Dev. 1985;11:11-19. 101-103. 16. Sies H, ed. Oxidative Stress. New York, NY: Academic Press; 1985 27. Schlenzig J-S, Bervoets K, von Loenwenich V, Bohles H. Urinary malondialde- 17. Davis KJA. Oxidative damage and repair: introduction and overview. In: Davis hyde concentrations in preterm neonates: is there a relationship to disease en- KJA, ed. Oxidative Damage and Repair: Chemical, Biological, and Medical As- tities in neonatal intensive care? Acta Paediatr. 1993;82:202-205. pects. Elmsford, NY: Pergamon Press; 1991:xvii-xxvii. 28. Buonocore G, Perrone S, Longini M, Terzuoli L, Bracci R. Total hydroperoxide 18. Tien M, Aust SD. Comparative aspects of several model lipid peroxidation sys- and advanced oxidation protein products in preterm hypoxic babies. Pediatr Res. tems. In: Yagi K, ed. Lipid Peroxides in Biology and Medicine. Orlando, Fla: Aca- 2000;47:221-224. demic Press; 1982:23-39. 29. Supnet MC, David-Cu R, Walther FJ. Plasma xanthine oxidase activity 19. Esterbauer H, Zollner H. Methods for determination of aldehydic lipid peroxida- and lipid hydroperoxide levels in preterm infants. Pediatr Res. 1994;36:283- tion products. Free Radic Biol Med. 1989;7:197-203. 287.

(REPRINTED) ARCH PEDIATR ADOLESC MED/ VOL 155, JUNE 2001 WWW.ARCHPEDIATRICS.COM 722