Rodolfo Bracci

Oxidative stress in preterm neonates at birth and on the seventh day of life.

Previous studies have demonstrated increased oxidative damage to proteins and increased lipid peroxidation products in the plasma of hypoxic newborns at birth. We tested the hypothesis that hypoxic preterm newborns are at increased risk for oxidative stress in the first week of life. Heparinized blood samples of 34 hypoxic and 15 control preterm newborns were obtained at birth from the umbilical vein immediately after delivery and from a peripheral vein on postnatal d 7. Plasma levels of hypoxanthine, total hydroperoxide (TH), and advanced oxidation protein products (AOPP) were measured in cord blood and blood drawn on d 7. Hypoxanthine, TH, and AOPP levels were significantly higher in cord and d 7 blood samples of hypoxic newborn than control infants. Statistically significant correlations were observed between AOPP and hypoxanthine and between AOPP and TH plasma levels on d 7. AOPP and TH plasma levels significantly increased from cord to d 7 blood in neonates without hypoxia. These findings show that the oxidative stress observed in cord blood of hypoxic preterm newborns is still higher than control infants on d 7. The significant increase in TH and AOPP levels in nonhypoxic preterm newborns at the end of the first postnatal week indicates that damage caused by free radicals also occurs in nonhypoxic babies with normal clinical course. In summary, TH and AOPP production is prolonged for several days after birth in hypoxic preterm babies. The risk of free radical damage is lower but still exists in preterm neonates with normal clinical course.

Total hydroperoxide and advanced oxidation protein products in preterm hypoxic babies.

Previous studies have shown that plasma lipoproteins are a common target of free radical-induced oxidative stress in hypoxic newborn infants. In contrast to lipids, the reaction of proteins with various oxidants during hypoxia has not been extensively studied. We tested the hypothesis that tissue hypoxia results in increased production of protein oxidation in cord blood of preterm newborns. Heparinized blood samples of 39 hypoxic and 16 control preterm newborns were obtained from the umbilical vein, after cord clamping immediately after delivery. Plasma levels of total hydroperoxide (TH), advanced oxidation protein products (AOPP), hypoxanthine (Hx), xanthine (Xa), and uric acid (UA) were measured. Higher Hx, Xa, UA, TH, and AOPP levels were found in hypoxic newborn infants than in controls. Statistically significant correlations were observed between: TH and Hx (r = 0.54, p = 0.003, n = 28), AOPP and Hx (r = 0.64, p = 0.0001, n = 27), and TH and AOPP plasma levels (r = 0.50, p = 0.02, n = 21). In summary, TH, AOPP, Hx, Xa, and UA production is increased in fetal blood during hypoxia. The more severe the hypoxia, the higher the lipid and protein damage by free radicals.

Chorioamnionitis: A Risk Factor for Fetal and Neonatal Morbidity

Despite widespread use of drugs to arrest preterm labor, there has been no decrease in the numbers of low-birth-weight or preterm infants in the last 20 years. Evidence from many sources links preterm birth to symptomatic and subclinical infections. Recently, an increasing body of evidence has suggested that not only is subclinical infection responsible for preterm birth but also for many serious neonatal sequelae, including periventricular leukomalacia, cerebral palsy, respiratory distress and even bronchopulmonary dysplasia and necrotizing enterocolitis. Proxies of intrauterine infection include clinical chorioamnionitis, histological chorioamnionitis and intraamniotic increase in cytokines, which have been found to be associated with acute neonatal morbidity and mortality and, at least to some degree, with neurological impairment, chronic lung disease and thymus involution in the preterm infant. The infectious/inflammatory mechanisms involved are not fully understood, and the types of microbes and genetic features of host adaptive and innate immune responses need to be better characterized.

Nucleated red blood cell count at birth as an index of perinatal brain damage

OBJECTIVE The prognostic value of the nucleated red blood cell count at birth with respect to perinatal brain damage and neonatal outcome was assessed in infants at high risk of having neurologic damage. STUDY DESIGN The nucleated red blood cell count at birth, pulsed Doppler ultrasonography in the cerebral arteries, cranial fontanelle sonograms, and neurodevelopmental status were evaluated in 337 newborn infants. RESULTS The nucleated red blood cell count at birth was significantly higher (1) in neonates with abnormal Doppler ultrasonographic parameters for the cerebral arteries at 48 to 72 hours after birth than in healthy neonates, (2) in 6-month-old infants with sequelae of hypoxic-ischemic encephalopathy than in healthy infants, and (3) in 3-year-old children with abnormal developmental status than in those with no abnormalities at follow-up. Significant correlations were observed between the nucleated red blood cell count and gestational age, Apgar score at 1 and 5 minutes, pH, base deficit, fraction of inspired oxygen, blood oxygen content, and birth weight. CONCLUSIONS The nucleated red blood cell count at birth not only reflects a response of the infant to perinatal hypoxia but is also a reliable index of perinatal brain damage.

Intraerythrocyte nonprotein-bound iron and plasma malondialdehyde in the hypoxic newborn

Intraerythrocyte nonprotein-bound iron (INPBI), malondialdehyde (MDA), and hypoxanthine plasma levels (HxPL), were determined by high-pressure liquid chromatography in 138 randomly selected newborn infants with gestational ages ranging from 23 to 42 weeks at birth and on fourth day of life. MDA plasma levels were significantly higher in cord and fourth-day blood samples of preterm babies than term infants as well as babies born by emergency Caesarean section than babies born by vaginal delivery and in intubated than in nonintubated newborns. Highly significant correlations both in cord blood and fourth-day blood samples were observed between MDA plasma levels and gestational age, birth weight, Apgar score at 1 min and 5 min, HxPL, pH, base deficit, and INPBI content. Multiple regression analysis identified HxPL as the best single predictor of MDA plasma levels in cord blood, and INPBI content in fourth-day blood as the best single predictor of MDA plasma levels in fourth-day blood. The results indicate that red cells and plasma lipoproteins are a common target of free radical-induced oxidative stress during hypoxia.

Maternal and Neonatal Plasma Cytokine Levels in Relation to Mode of Delivery

After birth, host defences must be recruited to manage the transition from an almost sterile to a normal environment. The present study was undertaken to evaluate the relationship between cytokine plasma levels and phagocyte burst in mothers and neonates during the peripartal period. Plasma levels of interleukin (IL)-1, IL-6, tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF) and whole blood superoxide anion (.O2-) generation were evaluated in 27 healthy mothers, 16 undergoing vaginal delivery (VD) and 11 elective caesarean section (ECS) and in their babies. Blood specimens were taken from the mothers at the beginning of labour, during labour, immediately after delivery and 4 days later in the VD group, and before anaesthesia, immediately after delivery and 4 days later in the ECS group; neonatal samples were taken at birth (cord blood) and 4 days later. After delivery by VD, these mothers had higher plasma levels of IL-1 beta, IL-6, IFN-gamma and higher .O2- generation than those delivered by ECS. IL-6 plasma levels and .O2- generation were higher in babies born by VD than in those born by ECS. A statistically significant correlation between IL-6 plasma levels and .O2- release was observed in cord blood of babies born by VD (r = 0.69; p < 0.006). The study demonstrates that labour plays an important role in modulating host defences in the newborn.

Plasma opioids in the first hours of life.

Summary: High concentrations of ACTH are present in the neonatal circulation in response to birth stress. Since ACTH and β-lipotropin (βLPH) have a common precursor, and ACTH and β-endorphin (βEP) are released in parallel in stressful situations, we measured plasma levels of βLPH and βEP in the first 24 hours of life.Blood samples were taken from the umbilical cord at birth in 27 neonates, subdivided into four groups.A further blood sample was obtained from the jugular vein after 30 min in nine cases (group A), after 6 hours in six (group B), and after 12 hours in six (group C), and after 24 hours in six (group D).βLPH and βEP were measured by specific radioimmunoassays after silicic acid extraction of the plasma and Sephadex G-75 column chromatography.In cord plasma, the mean (±S.E.) βLPH concentrations (pg/ml) varied between 219.5 ± 84.5 (group D) and 241 ± 43.3 (group B), while those of βEP ranged between 70.2 ± 8.2 (group C) and 54.6 ± 14.4 (group D). The βLPH/βEP ratio on a molar basis ranged from 1.12 ± 0.73 (group A) to 1.37 ± 0.60 (group C).Plasma βLPH concentrations determined at 30 min were 199.1 ± 50.4, and these declined slightly in group C (after 12 hours: 168.2 ± 30.2), but only showed a significant fall after 24 hours (88.5 ± 27.0; P < 0.01 in comparison with the previous groups), reaching normal adult levels (71.7 ± 25.1).The pattern of βEP plasma levels (pg/ml) was very similar to that reported for βLPH. βEP plasma levels only showed a significant decrease after 24 hours (22.7 ± 8.9; P < 0.01) when compared to the 12-hour age group C. βEP concentrations at 30 min (76.1 ± 47.7), 6 hours (68.6 ± 29.3), and 12 hours (51.8 ± 18.1) of life did not differ significantly from those found in mixed cord blood and were constantly higher than normal adult levels [24.5 ± 12.3].Since the half-life of βLPH and βEP is respectively 45 and 37 min, the present data demonstrate that neonates are able to release βLPH and βEP during the first hours of life. Both these molecules may contribute to help the newborn in the transition from a close dependence on the mother, to an autonomous existence.Speculation: Although the physiologic role of circulating PLPH and PEP has not been fully clarified, the presence of these peptides in neonatal plasma could represent an important analgesic factor in overcoming birth stress. Thus, an abnormal presence of these substances could be related to pathological situations. However, the source, the regulatory mechanisms and the biologic effects of circulating opioids in neonatal life are still unclear and require further investigation.

Hypoxia-induced free iron release in the red cells of newborn infants*

Heparinized blood samples were obtained at birth from 164 newborn infants (101 full term; 63 preterm). Intra‐erythrocyte free iron concentration and hypoxanthine plasma levels were determined by high‐pressure liquid chromatography. Intra‐erythrocyte free iron concentration was higher in preterm than in full term babies (p < 0:0001) and adults (p < 0:0001). Statistically significant correlations were observed between intra‐erythrocyte free iron concentration and hypoxanthine levels (r = 0:66; p= 0:0001), pH (r = ‐ 0:76; p = 0:0001), base excess (r = ‐ 0:79; p= 0:0001), and gestational age (r = ‐ 0:44; p= 0:0001) in both infant populations. Multiple regression analysis between intra‐erythrocyte free iron concentration in cord blood, as an independent variable, and Apgar score at 1 min, pH, base excess, hypoxanthine values, FiO2 needed for resuscitation immediately after delivery, and gestational age, as dependent variables, identified hypoxanthine levels (p= 0:0003; partial F‐test = 15.4) as the best single predictor of intra‐erythrocyte free iron concentration. In conclusion, hypoxia induces intra‐erythrocyte free iron release, and therefore enhances the risk of oxidative injury due to hydroxyl radical generation.

The Timing of Neonatal Brain Damage

Although neonatal morbidity and mortality are less than in the past, the risk of pre-natal and neonatal brain damage has not been eliminated. In order to optimize pre-natal, perinatal and neonatal care, it is necessary to detect factors responsible for brain damage and obtain information about their timing. Knowledge of the timing of asphyxia, infections and circulatory abnormalities would enable obstetricians and neonatologists to improve prevention in pre-term and full-term neonates. Cardiotocography has been criticized as being too indirect a sign of fetal condition and as having various technical pitfalls, though its reliability seems to be improved by association with pulse oximetry, fetal blood pH and electrocardiography. Neuroimaging is particularly useful to determine the timing of hypoxic-ischemic brain damage. Cranial ultrasound has been used to determine the type and evolution of brain damage. Magnetic resonance has also been used to detect antenatal, perinatal and neonatal abnormalities and timing on the basis of standardized assessment of brain maturation. Advances in the interpretation of neonatal electroencephalograms have also made this technique useful for determining the timing of brain lesions. Nucleated red blood cell count in cord blood has been recognized as an important indication of the timing of pre-natal hypoxia, and even abnormal lymphocyte and thrombocyte counts may be used to establish pre-natal asphyxia. Cord blood pH and base excess are well-known markers of fetal hypoxia, but are best combined with heart rate and blood pressure. Other markers of fetal and neonatal hypoxia useful for determining the timing of brain damage are assays of lactate and markers of oxidative stress in cord blood and neonatal blood. Cytokines in blood and amniotic fluid may indicate chorioamnionitis or post-natal infections. The determination of activin and protein S100 has also been proposed. Obstetricians and neonatologists can therefore now rely on various methods for monitoring the risk of brain damage in the antenatal and post-natal periods.

Iron release in erythrocytes and plasma non protein-bound iron in hypoxic and non hypoxic newborns

Iron is released in a desferrioxamine (DFO)-chelatable form (DCI) when erythrocytes are challenged by an oxidative stress. In g -thalassemic erythrocytes, both DCI content and release (after aerobic incubation for 24 h) are increased and correlated with the fetal hemoglobin (HbF) levels. Since erythrocytes from newborns have an extremely high content of HbF and are exposed to conditions of oxidative stress, the release of iron in these erythrocytes was investigated. The erythrocyte DCI content was increased in preterm but not in term newborns as compared to adults, while the release was increased in both preterm and term erythrocytes. The level of plasma non protein-bound iron (NPBI), which was not detectable in adults, was much higher in preterm than in term newborns. When term plus preterm newborns were divided in two groups, normoxic and hypoxic, according to cord blood pH, it was found that both iron release and NBPI were markedly higher in the hypoxic newborns compared to normoxic ones. Similar results were also obtained when the preterm and term infants were considered separately on the basis of cord blood pH. Therefore, iron release and NPBI are higher when conditions of hypoxia occur. In fact, when the values for iron release and NPBI were separately plotted against cord blood pH values, significant negative correlations were seen in both cases. NPBI was correlated with iron release seen in all the newborns and a significant part of the released iron could be recovered into the incubation medium at the end of the incubation.