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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
Arch Pediatr Adolesc Med. 2001;155:718-722.
ABSTRACT
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Background Currently available clinical tools cannot accurately identify the extent
of perinatal hypoxic injuries. During hypoxia, reactive oxygen species cause
lipid peroxidation of cell membranes, yielding oxidation products that constitute
thiobarbituric acid–reacting substances (TBARS).
Objective To see if the concentrations of TBARS excreted in urine would be elevated
during the first day of life in term and preterm infants following chronic
hypoxia or acute asphyxia.
Design Thiobarbituric acid–reacting substances levels were measured by
a spectrophotometric assay in urine samples collected from term and near-term
( 34 weeks gestation, n = 22), and preterm (<34 weeks gestation, n
= 52) infants on the first day of life.
Patients Infants were admitted to the St Peter's University Hospital (New Brunswick,
NJ) neonatal intensive care unit from July 1997 to January 1999. Acute asphyxia was defined as umbilical cord blood pH values less 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) associated with pregnancy-induced hypertension
or reversal of umbilical arterial blood flow.
Results Among term infants, urinary TBARS levels were significantly increased
following acute asphyxia (P = .02). Levels of TBARS
also tended to be elevated following chronic hypoxia. Urinary TBARS levels
in term infants tended to be increased in those requiring mechanical ventilation
(P = .05) or delivery room resuscitation (P = .15), as well as in those passing intrauterine meconium (P = .13) or having clinical evidence of hypoxic-ischemic
encephalopathy (P = .24).
Conclusions The results show a correlation between elevated urinary TBARS levels
in term and near-term infants, and perinatal hypoxia (as determined by low
Apgar scores or umbilical cord blood acidosis). We speculate that TBARS concentrations
may be useful as a biomarker for perinatal hypoxic injury in newborns. Further
studies are needed to determine whether elevations in TBARS levels are better
predictors of the extent of hypoxic injury than existing markers.
INTRODUCTION
INTRAUTERINE HYPOXIA is a major risk factor for an abnormal outcome
in the neonatal period. Approximately 700 neonatal deaths following intrauterine
hypoxia are reported in the United States each year, comprising more than
2% of total infant mortality.1 The neurodevelopmental
sequelae exhibited by survivors can be severe, constituting a large part of
pediatric health care expenditures. Despite the potential severity and prevalence
of this condition, the diagnosis is usually based on nonspecific clinical
criteria, since no reliable markers have previously been demonstrated to correlate
with the extent of intrauterine hypoxic insult. Such biomarkers, if available,
would be particularly helpful in identifying infants exposed to chronic hypoxia
in utero. Examination of these infants has not often revealed pathognomonic
findings, and the identification of such findings is necessary to provide
postnatal medical management that optimizes the medical and neurologic outcome.
In addition, several specific interventions are currently under investigation
to limit neurologic injury resulting from hypoxic-ischemic insult, including
cerebral hypothermia2 and antioxidant therapies.3 These appear to be most effective when applied early
in the course of this process. Therefore, the identification of a rapid and
early marker of the extent of perinatal asphyxia is an important step in identifying
patients eligible for prospective clinical trials and, ultimately, in devising
specific preventive interventions.
Clinical and laboratory criteria that are currently used to identify
asphyxiated or hypoxic infants, including fetal heart rate patterns, umbilical
cord blood pH values, and Apgar scores, are known to be insensitive and/or
nonspecific. Electronic fetal monitoring during labor was introduced to identify
fetuses at risk for hypoxic injury, but abnormal fetal heart rate patterns
have not been shown to predict an abnormal outcome.4
Using a large cohort of more than 50 000 high- and low-risk infants,
Shy et al5 showed that electronic fetal monitoring
had no advantage over auscultation in predicting perinatal mortality, morbidity,
Apgar scores, cord blood gases, and long-term outcome. Subsequently, in 1989,
a technical bulletin of the American College of Obstetrics and Gynecology6 stated that, within specified intervals, intermittent
auscultation is equivalent to continuous electronic fetal monitoring in detecting
fetal compromise. Thus, fetal heart rate monitoring lacks both sensitivity
and specificity in identifying birth asphyxia.
Umbilical cord blood pH might be expected to be predictive of injury
associated with perinatal hypoxic insult. When gas exchange across the placenta
is compromised, tissue hypoxia leads to anaerobic metabolism and lactic acidosis.
In addition, carbon dioxide accumulation further reduces fetal arterial pH.
Nevertheless, Winkler et al7 found no differences
between infants with cord pH values above or below 7.20 with respect to the
development of clinical signs of asphyxia (seizures, persistent hypotonia,
and renal or cardiac dysfunction). Fee et al8
noted that among 110 term infants with a cord blood pH less than 7.05 and
a base deficit greater than 10 mEq/L, 73% were admitted to regular nurseries
and were discharged as normal. Seven of 9 infants who had abnormal neurological
features at birth had no abnormal characteristics at follow-up. In another
study,9 no infant with an umbilical cord blood
pH less than 7.00, but with a 1-minute Apgar score greater than 3 had seizures
or hypotonia. Dennis et al10 found no association
between developmental outcome at 4.5 years, and acidosis at birth (defined
as an umbilical cord blood pH less than 7.10). Among a large random cohort
of normal full-term newborns, 10% were shown to have umbilical arterial base
deficits greater than 18 mEq/L.11 Only the
most severe base deficits (>20 mEq/L) in severely affected infants have been
associated with adverse neurological outcomes. These data indicate that umbilical
cord blood pH values are neither sensitive nor specific in identifying birth
asphyxia and its sequelae. Similarly, Apgar scores have been used to identify
infants with birth asphyxia. However, the 1-minute Apgar score has poor sensitivity
and positive predictive value in detecting acidosis, and 25% to 75% of infants
with acidosis are assigned normal Apgar scores.12
Therefore, current methods have been inadequate in identifying infants with
hypoxic injury. This is most likely because all of these assessments are indirect
reflections of physiologic phenomena that may follow hypoxic insult, rather
than direct measurements of the pathophysiologic progression of asphyxia at
the cellular level.
Hypoxia induces cellular damage, in part by the generation of reactive
oxygen species (ROS), including hydrogen peroxide, singlet oxygen, superoxide
anion radical, hydroxyl radical, and lipid hydroperoxides in affected tissues.
Therefore, we hypothesized that ROS that are generated in the fetus can serve
as a biomarker of intrauterine or perinatal hypoxia. In order to quantify
ROS in infants, we measured concentrations of urinary thiobarbituric acid–reacting
substances (TBARS). The most abundant of the TBARS is malondialdehyde (MDA),
an aldehydic lipid peroxidation product formed by the action of ROS on lipid
membranes. In these studies, we found that the quantity of urinary TBARS,
measured shortly after birth, correlates with the severity of intrauterine
hypoxic insult as determined by the patient history and clinical criteria.
PATIENTS AND METHODS
PATIENT CHARACTERISTICS
All inborn infants admitted to the neonatal intensive care unit at St
Peter's University Hospital (New Brunswick, NJ) during the period between
July 1997 and January 1999 were screened for eligibility, excluding those
from multiple gestations or with major congenital anomalies (1471 term and
602 preterm infants were screened). Of these, 6 term infants (34 or more weeks'
gestation) and no surviving preterm infants met the criteria for acute asphyxia.
Six term infants and 15 preterm infants met the criteria for chronic hypoxia.
All of these infants were enrolled. At the time that each eligible term infant
was enrolled, a newborn infant of comparable gestational age but with no signs
or symptoms of hypoxia, asphyxia, or growth retardation was selected as a
control. Two potential term control infants were not included because consent
could not be obtained. Therefore, 10 term infants served as controls. At the
time of enrollment of each preterm infant, 2 to 3 infants of comparable gestational
age were selected as controls. More preterm controls per subject were selected
because of anticipated difficulties in obtaining consent and urine samples
within 24 hours in this group. Therefore, 40 infants were recruited as controls.
Of these infants, consent was not obtained from 2, urine was not available
from 1, and 37 served as controls for the analysis. Study personnel obtained
demographic and medical information from maternal and infant medical records.
For analysis, infants were divided into 3 groups. Infants with acute asphyxia were defined as those with umbilical cord
blood pH values less than 7.05, or 5-minute Apgar scores of less than 5. Chronic hypoxia was defined as intrauterine growth retardation
or low birth weight (small for gestational age) associated with either pregnancy-induced
hypertension (PIH) or reversal of umbilical arterial blood flow. Control infants
were those of appropriate size for gestational age who did not exhibit any
signs of fetal or neonatal hypoxia. Preterm infants (<34 weeks gestation)
and term or near-term infants (34 weeks gestation) were analyzed separately.
DETERMINATION OF URINARY TBARS
Urine samples were obtained from study infants on the first day of life.
Informed consent was obtained from parents for the acquisition of samples,
and these studies were approved by the Committee for the Protection of Human
Subjects in Research at St Peter's University Hospital. All specimens were
collected into sterile containers and stored at -70°C for batched
analysis. Thiobarbituric acid–reacting substances levels were measured
as previously described.13, 14
Briefly, 200 µL of urine was combined with 10 µL of 5% butylated
hydroxytoluene (BHT, in glacial acetic acid) and 300 µL of a 0.5% aqueous
thiobarbituric acid (TBA) solution. The samples were then vortexed and incubated
at 100°C for 30 minutes. After cooling to room temperature, the absorbance
of samples at 532 nm was measured using a Lamba 3B spectrophotometer (Perkin
Elmer Corp, Baden Seewerk, Germany). Samples were blanked against reference
cuvettes containing reagents without urine. The concentration of TBARS was
calculated from the absorbance at 532 nm, using 156 000 as the molar
extinction coefficient. The quantity of TBARS is proportionate to the amount
of MDA, a lipid peroxidation product generated by the oxidation of membrane
lipids by ROS. Malondialdehyde reacts with TBA to form a 1:2 MDA-TBA adduct,
which absorbs at 532 nm. In the present study, MDA was confirmed to be the
predominant TBA-reacting adduct by high-performance liquid chromatography
analysis of representative samples.
To control for urine concentration, data were normalized to urine creatinine
concentrations. Urinary creatinine was measured using the revised Jaffe method.15 Briefly, alkaline picrate (formed by combining picric
acid and 10% sodium hydroxide in a ratio of 5:1) was added to serial dilutions
of urine. Samples were incubated at room temperature for 15 minutes, and the
absorbance at 500 nm was measured.
DATA ANALYSIS
Results are expressed as means ± SDs. As urinary TBARS concentrations
were not normally distributed, we transformed them to their natural logs,
which were normally distributed. Group means were compared using unpaired t tests. A P value less than .05
was considered significant.
RESULTS
Among term and near-term infants (n = 22), those with 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
(Table 1). Infants with acute
asphyxia had significantly lower 1-minute and 5-minute Apgar scores than controls.
The log concentration of urinary TBARS in term infants with acute asphyxia
(9.06 ± 0.60) was significantly greater than that of controls (7.97
± 0.88) (P = .02). Chronically hypoxic term
infants also exhibited elevated urinary TBARS concentrations (8.33 ±
1.38), but this did not differ statistically from levels in control infants
(Table 1). Using a urinary TBARS
threshold of 5500 ng/mg creatinine, the sensitivity of this measurement for
identifying acute asphyxia in term and near-term infants was 67%, and its
specificity was 90%. This resulted in a positive predictive value of 80% and
a negative predictive value of 82%. Urinary TBARS levels in term and near-term
infants tended to be increased in those requiring mechanical ventilation (P = .05) or delivery room resuscitation (P = .15), as well as those passing intrauterine meconium (P = .13) or with clinical evidence of hypoxic-ischemic encephalopathy
(P = .24) (Table
2). Urinary TBARS were not correlated with chorioamnionitis, PIH,
umbilical artery blood flow, or mode of delivery.
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Table 1. Demographic Variables and Urinary TBARS Levels in Term and
Near-Term Infants*
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Table 2. Association of Urinary TBARS Levels With Clinical Indicators
in Term and Near-Term Infants*
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Among preterm infants (<34 weeks gestation) there were 15 infants
with chronic hypoxia and 37 control infants. The mean gestational ages, birth
weights, and Apgar scores were not significantly different between these groups
(Table 3). Urinary TBARS levels
were significantly greater in control preterm infants than in control term
and near-term infants (P = .01), but were not further
increased following chronic intrauterine hypoxia. Acute asphyxia could not
be studied in preterm infants because of insufficient numbers.
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Table 3. Demographic Variables and Urinary TBARS Levels in Preterm
Infants*
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COMMENT
In this study, we have shown that urinary TBARS, measured during the
first day of life, are elevated in term and near-term infants following acute
asphyxia, as defined by low Apgar scores or acidemia. Full-term infants also
tend to exhibit higher urinary excretion of TBARS following chronic low-grade
oxygen deprivation, as indicated by intrauterine growth retardation in the
presence of PIH or reversed umbilical arterial blood flow. Urinary TBARS were
not directly correlated with PIH (defined by maternal blood pressure), suggesting
that the association of TBARS levels with chronic hypoxia occurs primarily
in infants with the most severely compromised uteroplacental blood flow (reversed
umbilical arterial blood flow or severe PIH). We also found that preterm infants
exhibit elevated urinary TBARS levels relative to term infants, possibly reflecting
stresses inherent in preterm delivery.
This study is limited by the small sample size (term subjects, n = 12),
a consequence of the low incidence of perinatal hypoxia and asphyxia during
this period at our neonatal intensive care unit. Nevertheless, the physiology
of hypoxic-ischemic injury supports the potential role of early measurement
of TBARS levels in the identification of affected infants. The generation
of free radicals during tissue hypoxia plays a major role in the pathogenesis
of tissue injury. Newborns are particularly susceptible to such injury because
they exhibit an imbalance between antioxidant and oxidant-generating systems.
Free radicals, in excess, inactivate proteins, disrupt DNA, and oxidize lipids.16, 17 The oxidation of lipids by radicals
has been long regarded as a critical event leading to cellular injury. Cell
membranes contain a high proportion of polyunsaturated lipids and are susceptible
to peroxidation, resulting in the formation of hydroperoxides, the most abundant
product being MDA.18, 19 Malondialdehyde
has a long half-life (>20 days) in neutral or acidic solutions, but is metabolised
in vivo by reaction with tissue proteins and nucleic acids.20
Although its biological half-life is likely to be variable and has not been
well described, MDA and TBARS have been used in animals and humans as reliable
indicators of the response to pro-oxidant provocations.21
Conditions initiating lipid peroxidation in the perinatal period (eg, uteroplacental
restriction) are likely to be ongoing (acute or chronic) in the presence of
unlimited substrate.
Therefore, TBARS measurements, which are proportional to MDA content,
may provide a direct assessment of the progression of hypoxic injury at the
cellular level. Consistent with our data, several previous studies have demonstrated
that serum MDA levels in healthy full-term infants are low at birth and rise
postnatally in response to the stress of abdominal delivery and/or to the
events of respiratory transition during the first days of life.22, 23
Umbilical cord blood MDA levels are increased following fetal hypoxia, suggesting
that MDA reflects the extent of lipid peroxidation in vivo.24, 25
Hasegawa et al26 observed that the content
of TBARS in the brains of newborn mice increased during reoxygenation after
20 minutes of hypoxia. Malondialdehyde levels are also elevated in preterm
infants, particularly those receiving oxygen and mechanical ventilation.27 Buonocore et al28
showed that umbilical cord blood total lipid hydroperoxides are increased
in preterm infants following fetal hypoxia. Furthermore, high umbilical cord
blood hydroperoxide levels in preterm infants have been correlated with adverse
outcomes in the newborn period that are reflective of ROS-mediated oxidative
injury (death, severe intraventricular hemorrhage, necrotizing enterocolitis,
or pulmonary hemorrhage).29
In this study, we have defined acute and chronic perinatal asphyxia
using existing standards that are available at the time of birth (ie, Apgar
scores, umbilical cord blood pH, and intrauterine growth). Although these
measures are known to be unreliable in determining the severity of hypoxic
injury, no other early parameters have been established as gold standards
for diagnosing hypoxic injury in newborns. Further studies will be required
to validate TBARS as a true biomarker for perinatal hypoxic-ischemic injury.
To be useful, such a biomarker must do the following: (1) be scientifically
sound, (2) be available rapidly during the early hours of life, (3) concur
with more easily obtained or established clinical and laboratory indicators,
and (4) provide further information or precision not provided by the other
methods of predicting outcomes of interest. Previous studies have validated
the first criterion by establishing that MDA (measured by TBARS) is produced
as a direct product of lipid peroxidative damage to cell membranes, and that
hypoxia induces free radical generation, oxidative stress, and lipid peroxidation.
In this study, we found that early TBARS measurements are correlated with
perinatal stress and conventional measures of hypoxia, suggesting that the
second and third criteria are valid. Further studies will be required to validate
the final criterion by establishing the predictive value of elevated urinary
TBARS with regard to long-term medical and neurologic prognoses. As we enter
an era during which specific therapies may become available to avert the course
of hypoxic-ischemic encephalopathy, the early identification of patients at
risk will gain urgent importance.
AUTHOR INFORMATION
Accepted for publication February 2, 2001.
From the Division of Neonatology,
Department of Pediatrics, UMDNJ–Robert Wood Johnson Medical School, St
Peter's University Hospital, New Brunswick, NJ (Drs Siciarz,
Weinberger, Hiatt, and Hegyi), and the Department of Environmental and
Community Medicine, UMDNJ–Robert Wood Johnson Medical School,
Piscataway, NJ (Dr Witz).
Corresponding author and reprints: Barry Weinberger, MD, Division
of Neonatology, St Peter's University Hospital, 254 Easton Ave, New Brunswick,
NJ 08903 (e-mail: barryw{at}pol.net).
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ABSTRACT
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