Predictive Role of F2-Isoprostanes as Biomarkers for Brain Damage after Neonatal Surgery.

OBJECTIVE: Neonates have a high risk of oxidative stress during anesthetic procedures. The predictive role of oxidative stress biomarkers on the occurrence of brain injury in the perioperative period has not been reported before. METHODS: A prospective cohort study of patients requiring major surgery in the neonatal period was conducted. Biomarker levels of nonprotein-bound iron (NPBI) in plasma and F2-isoprostane in plasma and urine before and after surgical intervention were determined. Brain injury was assessed using postoperative MRI. RESULTS: In total, 61 neonates were included, median gestational age at 39 weeks (range 31-42) and weight at 3000 grams (1400-4400). Mild to moderate brain lesions were found in 66%. Logistic regression analysis showed a significant difference between plasma NPBI in patients with nonparenchymal injury versus no brain injury: 1.34 umol/L was identified as correlation threshold for nonparenchymal injury (sensitivity 67%, specificity 91%). In the multivariable analysis, correcting for GA, no other significant relation was found with the oxidative stress biomarkers and risk factors. CONCLUSION: Oxidative stress seems to occur during anaesthesia in this cohort of neonates. Plasma nonprotein-bound iron showed to be associated with nonparenchymal injury after surgery, with values of 1.34 umol/L or higher. Risk factors should be elucidated in a more homogeneous patient group.

Maternal obesity and perinatal oxidative stress: the strength of the association.

Maternal obesity is a chronic inflammatory state, which has been shown to induce increased levels of free fatty acids, reactive oxygen species and inflammatory cells. Recent evidence reveals increased levels of lipid peroxidation products in the plasma of obese women during pregnancy. The aim of this study was to test the hypothesis that maternal overweight or obesity is associated with increased oxidative stress (OS) in offspring. Two hundred and forty-five pregnant women and their newborns were prospectively enrolled. Mothers were divided in two groups: lean control - LC (n=175, Group I); overweight or obese (n=70, Group II) according to BMI ≥ 25 before pregnancy. Cord blood F2-isoprostanes (F2-IsoPs), as reliable markers of OS, were measured in all newborns. Lower 1 minute APGAR score and higher weight at discharge were found in Group II neonates, compared to those of Group I (p less than 0.05). Small for gestational age (SGA) newborns of both groups showed increased levels of F2-IsoPs than appropriate (AGA) or large (LGA) for gestational age (GA) (p less than 0.01). SGA newborns of Group II had higher F2-IsoPs levels compared to SGA of Group I (p less than 0.01), which were significantly correlated to maternal BMI at the end of pregnancy (r=0.451, p less than 0.01). Multivariate regression analysis corrected for confounding factors, showed that maternal overweight or obesity was significantly associated with high F2-IsoPs levels in SGA offspring (p less than 0.01). Maternal overweight or obesity is associated with increased OS in their SGA newborns. Data suggest the need of antioxidant protection for both mothers during pregnancy and infants soon after birth.

Oxidative Stress as a Physiological Pain Response in Full-Term Newborns.

This research paper aims to investigate if oxidative stress biomarkers increase after a painful procedure in term newborns and if nonpharmacological approaches, or sex, influence pain degree, and the subsequent OS. 83 healthy term newborns were enrolled to receive 10% oral glucose or sensorial saturation (SS) for analgesia during heel prick (HP). The ABC scale was used to score the pain. Advanced oxidation protein products (AOPP) and total hydroperoxides (TH) as biomarkers of OS were measured at the beginning (early-sample) and at the end (late-sample) of HP. The early-sample/late-sample ratio for AOPP and TH was used to evaluate the increase in OS biomarkers after HP. Higher levels of both AOPP and TH ratio were observed in high degree pain (4-6) compared with low degree pain score (0-3) (AOPP: p = 0.049; TH: p = 0.001). Newborns receiving SS showed a significantly lower pain score (p = 0.000) and AOPP ratio levels (p = 0.021) than those without. Males showed higher TH levels at the end of HP (p = 0.005) compared to females. The current study demonstrates that a relationship between pain degree and OS exists in healthy full-term newborns. The amount of OS is gender related, being higher in males. SS reduces pain score together with pain-related OS in the newborns.

Neonatal oxidative stress depends on oxygen blood pressure in umbilical artery.

With advancing gestation, partial pressure of oxygen (pO2) and pH fall significantly. Hypoxia is a main factor inducing free radical generation and thereby oxidative stress (OS). Placental and fetal tissue response when oxygen becomes restricted is complex and partially known. We tested the hypothesis that changes in umbilical artery and vein blood gas concentrations modulate OS occurrence in the newborn. Seventy umbilical artery and vein plasma samples were collected from healthy term newborns immediately after delivery. F2 Isoprostanes (F2-Isop) were measured in all samples as reliable markers of lipid peroxidation. Significantly lower pCO2 and higher pO2 and pH were found in umbilical vein than in artery, as expected. A positive correlation was detected between pH and pO2 only in umbilical artery (p=0.019). F2-Isop levels were no different between artery and vein in cord blood. Significant correlations were found between F2-Isop and pCO2 (p=0.025) as well as between F2-Isop and pH in umbilical vein (p=0.027). F2-Isop correlated with pCO2 (p=0.007) as well as with pO2 values (p=0.005) in umbilical artery blood. Oxidative stress (OS) in newborns depends on oxygen concentrations in umbilical artery. OS biomarkers significantly correlate with pO2 and in umbilical artery but not in umbilical vein. In normoxic conditions fetal-maternal gas exchanges occurring in placenta re-establish normal higher oxygen levels in umbilical vein than artery, with a normal production of free radicals without any deleterious effects.

A comparative study on oxidative stress role in nasal breathing impairment and obstructive sleep apnoea syndrome.

Obstructive sleep apnoea syndrome (OSAS) is a sleep disorder that leads to metabolic abnormalities and increased cardiovascular risk. This study aimed to define the expression and clinical significance of biomarkers involved in oxidative stress in patients with OSAS. A prospective study was designed to compare outcomes of oxidative stress laboratory tests in three groups of subjects. The study involved the recruitment of three groups of subjects, 10 patients with obstructive sleep apnoea syndrome with AHI > 30; 10 patients suffering from snoring at night with AHI < 15; 10 patients with nasal respiratory impairment with AHI < 5. Patients were subjected to skin prick tests for common aero-allergens, nasal endoscopy, active anterior rhinomanometry, fibrolaryngoscopy and polysomnography; and extra-routine diagnostic tests and procedures; analysis of oxidative and antioxidant (plasma thiol groups) biomarkers in blood and urine samples. No statistical differences in age, sex distribution or body mass index were present between the three groups (p > 0.05). There were significant differences in AHI among the three groups of patients (p < 0.05). No statistical significance was found in the Analysis of Variance (ANOVA) test (p > 0.05) between the levels of biomarkers of oxidative stress in the three populations studied. The results of our study show that the nose can play a role in the pathogenesis of OSAS through the production of biomarkers of oxidative stress.

Resuscitating preterm infants with 100% oxygen is associated with higher oxidative stress than room air.

AIM: The starting fraction of inspired oxygen for preterm resuscitation is a matter of debate, and the use of room air in full-term asphyxiated infants reduces oxidative stress. This study compared oxidative stress in preterm infants randomised for resuscitation with either 100% oxygen or room air titrated to internationally recommended levels of preductal oxygen saturations. METHODS: Blood was collected at birth, two and 12 hours of age from 119 infants <32 weeks of gestation randomised to resuscitation with either 100% oxygen (n = 60) or room air (n = 59). Oxidative stress markers, including advanced oxidative protein products (AOPP) and isoprostanes (IsoP), were measured with high-performance liquid chromatography and mass spectrometry. RESULTS: Significantly higher levels of AOPP were found at 12 hours in the 100% oxygen group (p < 0.05). Increases between two- and 12-hour AOPP (p = 0.004) and IsoP (p = 0.032) concentrations were significantly higher in the 100% oxygen group. CONCLUSION: Initial resuscitation with room air versus 100% oxygen was associated with lower protein oxidation at 12 hour and a lower magnitude of increase in AOPP and IsoP levels between two and 12 hours of life. Correlations with clinical outcomes will be vital to optimise the use of oxygen in preterm resuscitation.

Proton nuclear magnetic resonance spectroscopy of urine samples in preterm asphyctic newborn: a metabolomic approach.

In order to highlight differences in the metabolic profile of healthy (control) compared with asphyxiated newborns, by using untargeted metabolomic approach coupled with (1)H NMR spectroscopy, we evaluated the effects of asphyxia on newborn urine metabolites. Our results showed that lactate, glucose and TMAO, together with threonine plus 3-hydroxyisovalerate are the metabolites more characterizing the asphyxiated group; lower contribute to discrimination is related to other metabolites such as dimethylglycine, dimethylamine, creatine, succinate, formate, urea and aconitate. After 24-48h from resuscitation preterm asphyctic neonates showed their recovery pattern that still can be differentiated by the controls.

Oxidative stress in patients with obstructive sleep apnoea syndrome.

Obstructive sleep apnoea syndrome (OSAS) is a disorder that leads to metabolic abnormalities and increased cardiovascular risk. The aim of this study was to identify early laboratory markers of cardiovascular disease through analysis of oxidative stress in normal subjects and patients with OSAS. A prospective study was designed to compare outcomes of oxidative stress laboratory tests in 20 adult patients with OSAS and a control group of 20 normal subjects. Laboratory techniques for detecting and quantifying free radical damage must be targeted to assess the pro-oxidant component and the antioxidant in order to obtain an overall picture of oxidative balance. No statistical differences in age, sex distribution, or BMI were found between the two groups (p>0.05). There were significant differences in the apnoea/hypopnoea index (AHI) between OSAS patients and the control group (p<0.05). Statistically significant differences in isoprostane, advanced oxidation protein products (AOPP) and non-protein bound iron (NPBI) levels were found between the study and control groups. No significant difference in the levels of thiol biomarkers was found between the two groups. The main finding of the present study was increased production of oxidative stress biomarkers in OSAS patients. The major difference between thiols and other oxidative stress biomarkers is that thiols are antioxidants, while the others are expressions of oxidative damage. The findings of the present study indicate that biomarkers of oxidative stress in OSAS may be used as a marker of upper airway obstructive episodes due to mechanical trauma, as well as a marker of hypoxaemia causing local oropharyngeal inflammation.

Lipid and protein oxidation in newborn infants after lutein administration.

OBJECTIVES: To test the hypothesis that neonatal supplementation with lutein in the first hours of life reduces neonatal oxidative stress (OS) in the immediate postpartum period. METHODS: A randomized controlled, double-blinded clinical trial was conducted among 150 newborns divided into control group, not supplemented (n = 47), and test group, supplemented with lutein on the first day postpartum (n = 103). Blood Samples were collected at birth from cord and at 48 hrs postpartum while routine neonatal metabolic screenings were taking place. Total hydroperoxide (TH), advanced oxidation protein products (AOPP), and biological antioxidant potential (BAP) were measured by spectrophotometry and data were analyzed by Wilcoxon rank sum test and by multivariate logistic regression analysis. RESULTS: Before lutein supplementation, the mean blood concentrations of AOPP, TH, and BAP were 36.10 umol/L, 156.75 mmol/H2O2, and 2361.04 umol/L in the test group. After lutein supplementation, significantly higher BAP increment (0.17 ± 0.22 versus 0.06 versus ± 0.46) and lower TH increment (0.46 ± 0.54 versus 0.34 ± 0.52) were observed in the test group compared to controls. CONCLUSION: Neonatal supplementation with lutein in the first hours of life increases BAP and reduces TH in supplemented babies compared to those untreated. The generation of free radical-induced damage at birth is reduced by lutein. This trial is registered with ClinicalTrials.gov NCT02068807.

Antioxidant effects of potassium ascorbate with ribose therapy in a case with Prader Willi Syndrome.

Oxidative stress (OS) is involved in several human diseases, including obesity, diabetes, atherosclerosis, carcinogenesis, as well as genetic diseases. We previously found that OS occurs in Down Syndrome as well as in Beckwith-Wiedemann Syndrome (BWS). Here we describe the clinical case of a female patient with Prader Willi Syndrome (PWS), a genomic imprinting disorder, characterized by obesity, atherosclerosis and diabetes mellitus type 2, pathologies in which a continuous and important production of free radicals takes place. We verified the presence of OS by measuring a redox biomarkers profile including total hydroperoxides (TH), non protein-bound iron (NPBI), thiols (SH), advanced oxidation protein products (AOPP) and isoprostanes (IPs). Thus we introduced in therapy an antioxidant agent, namely potassium ascorbate with ribose (PAR), in addition to GH therapy and we monitored the redox biomarkers profile for four years. A progressive decrease in OS biomarkers occurred until their normalization. In the meantime a weight loss was observed together with a steady growth in standards for age and sex.

The use of melatonin in hypoxic-ischemic brain damage: an experimental study.

OBJECTIVE: Oxidative stress (OS) plays a key role in perinatal brain damage. The aim of this study is to evaluate the effectiveness of melatonin as a neuroprotective drug by investigating the influence of melatonin on OS and inflammation biomarkers in an animal model of cerebral hypoxia-ischemia. METHODS: Five minutes after hypoxic-ischemic (HI) injury melatonin was administered to 28 rats (HI-Mel group). At the same time, 28 hypoxic-ischemic rats were vehicle-treated (V-HI group). Five rats were used as sham operated controls (CTL). OS biomarkers: isoprostanes (IsoPs), neuroprostanes (NPs) and neurofurans (NFs), and microglial activation markers (glial fibrillary acidic protein [GFAP] and monoclonal antirat CD68 [ED1]) were measured in the cerebral cortex of the two lobes. RESULTS: A significant increase of IsoPs on the left lobe was observed in V-HI after 1 hour (h) from HI injury (p < 0.001); a significant increase of NPs on both side (p < 0.05) and a significant increase of NFs on the left (p < 0.05) were also observed in V-HI after 24 h. A significant increase of IsoPs on the left (p < 0.05) and of NPs on both lobes (p < 0.05) were observed in HI-Mel after 48 h. The ED1 and GFAP expression was lower in the HI-Mel brain tissue. CONCLUSIONS: Melatonin reduces OS and inflammatory cells recruitment and glial cells activation in cerebral cortex after neonatal HI damage. These results lay the groundwork for future clinical studies in infants.

Inhibition of rapamycin-induced autophagy causes necrotic cell death associated with Bax/Bad mitochondrial translocation.

Rapamycin, a lipophilic macrolide antibiotic, has been found to reduce injury in different models of neurodegenerative disorders. We have previously shown that in neonatal rats subjected to hypoxia-ischemia (HI) the neuroprotective effect of rapamycin was associated with increased autophagy and decreased caspase-3 activation. We show here that the strong reduction of caspase-3 activation after rapamycin was due, at least in part, to its effect on the intrinsic apoptotic mitochondrial pathway because after rapamycin treatment there was a marked reduction of Bax and Bad translocation to mitochondria, cytochrome c release, and caspase-3 activation. Poly (ADP-ribose) polymerase 1 (PARP-1) cleavage and the number of terminal dUDP nick-end labeling (TUNEL)-positive cells were also reduced. To assess how the antiapoptotic effect of rapamycin was linked to the strong autophagy signal induced by the drug, we blocked the formation of autophagosomes with 3-methyladenine (3MA). 3MA administered 10 min after rapamycin, elicited again Bax and Bad translocation to the mitochondria but did not cause cytochrome c release and caspase-3 activation. After 3MA treatment, cells underwent necrotic cell death. These data indicate that rapamycin administered before HI prevents the apoptotic signaling taking place through the mitochondrial pathway. We hypothesize that rapamycin confers a preconditioning-like protection and suggest that caution is necessary before using pharmacological agents targeting autophagy in neuroprotection because they could interfere with endogenous protective mechanisms.

Markers di stress ossidativo nei liquidi biologici. (Markers of oxidative stress in biological fluids).

The damaging effects of free radicals (FR) in the perinatal period may be detected by testing specific markers of oxidative stress (OS) in newborn's biological fluids. Currently, more than 70 different markers are used to assess both the damage caused by excessive production of FR and body's antioxidant response. Our goal is to identify reliable markers for the detection of OS in physiology and pathology. These biomarkers can be useful for several clinical purpose such as to monitor the response to any antioxidant intervention and then develop preventive strategies. The standardization and a large clinical use of these biomarkers should contribute to the daily neonatologists work in the early detection, management and therapy of the several neonatal FR diseases.

Even routine painful procedures can be harmful for the newborn.

Stressful events can damage neonatal brain through a complexity of events including free radical (FR) generation. We examined whether pain provoked by a routine heel prick can generate an increase in potentially harmful FR in neonatal blood. To this aim, advanced oxidation protein products (AOPP) and total hydroperoxide (TH) concentrations were measured at the beginning (sample A) and at the end (sample B) of each sampling in 64 babies (corrected age: 37.2+/-2.7 weeks) who underwent heel prick for routine blood tests. We scored pain of every procedure in all newborns. No differences were detected between AOPP and TH blood concentrations at the beginning and at the end of heel prick sampling, considering the whole cohort of babies. Conversely, a significant increase was observed between AOPP and TH blood concentrations considering only those babies who showed the highest pain intensity. When babies' pain was high (ABC score >or=4), mean AOPP and TH blood levels increased significantly; in this case, mean AOPP values increased from 53.5microm/l (SD=41.6) to 63.2microm/l (SD=44.3) and TH values from 218.3UCarr (SD=89.2) to 228.7UCarr (SD=93.3), with a significant p value of 0.02 and 0.036, respectively. A significant correlation was also found between AOPP blood levels ratio (sample B/sample A) in each baby, and the correspondent level of pain. These data show that even common routine procedures can be potentially harmful for the newborn if they provoke a high level of pain.

Early oxidative stress in amniotic fluid of pregnancies with Down syndrome.

OBJECTIVE: Some evidence suggests that oxidative stress, due to an imbalance between oxidants and antioxidants, occurs in babies with Down syndrome (DS). This study tests the hypothesis that oxidative stress occurs early in DS pregnancies. DESIGN AND METHODS: Isoprostanes (IPs), a new marker of free radical-catalyzed lipid peroxidation, were measured in amniotic fluid from pregnancies with normal, growth restricted and DS fetuses, diagnosed by karyotype analysis of amniotic cells cultured. RESULTS: A nine-fold increase in IP concentrations was found in amniotic fluid of pregnancies with DS fetuses. This increase (595.15; 542.96-631.64 pg/ml, median; 95% CI), was greater than in pregnancies with fetal growth-restricted fetuses (155; 130.57-172.23 pg/ml, median; 95% CI) and normal fetuses (67; 49.82-98.38 pg/ml, median; 95% CI; p<0.0001). CONCLUSIONS: The study reveals that oxidative stress occurs early in pregnancy and supports the idea of testing whether prenatal antioxidant therapy may prevent or delay the onset of oxidative stress diseases in the DS population.

Non protein bound iron concentrations in amniotic fluid.

OBJECTIVES: To investigate whether amniotic fluid concentrations of non protein bound iron (NPBI) vary with growth in healthy fetuses and also offer a reference curve in the second trimester of pregnancy. DESIGN AND METHODS: Amniotic fluid concentrations of NPBI were measured by HPLC in 118 women with physiological singleton pregnancies, who underwent amniocentesis for fetal karyotype between weeks 15 and 18 of gestation. RESULTS: NPBI increased progressively from weeks 14--15 to weeks 15--16, peaking at 17--18 weeks of gestation. NPBI values regressed positively with gestational age (GA). Multiple linear regression analysis between NPBI, as dependent variable, and various fetal parameters, as independent variables, showed a statistically significant regression coefficient with GA, bi-parietal diameter and transverse cerebellar diameter. CONCLUSIONS: The present data constitutes the first quantification of NPBI concentrations in amniotic fluid under physiological conditions. Correlations with GA and ultrasound fetal biometry suggest that NPBI may play a role in fetal growth.

Nucleated red blood cell count in term and preterm newborns: reference values at birth.

The prognostic value of nucleated red blood cell count at birth in relation to neonatal outcome has been established. However, reference values were needed to usefully interpret this variable. The normal range of reference values for absolute nucleated red blood cell count in 695 preterm and term newborns is reported.

Blood pressure in the small-for-gestational age newborn.

AIM: The aim of the paper is to verify the existence of an inverse correlation between birth weight and blood pressure (BP) in neonates, infants and adolescents. METHODS: BP was measured at 7 days, 3, 6, 9, 12 months and 7-18 years in 432 subjects born at term at the Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena; 228 of these subjects were small for gestational age (SGA) and 204 appropriate for gestational age (AGA). For small babies, BP was measured with a DYNAMAP oscillometer which provides digital visualisation of systolic, diastolic and mean arterial pressure and heart rate. In older children, a mercury sphygmomanometer was used. Statistical analysis was carried out with SPSS 8.01 software using the Kolmogorov-Smirnov test for normality of populations. RESULTS: Statistical analysis did not reveal any significant differences between SGA and AGA subjects in the various age classes of the first 12 months of life. Significant correlation was found between 7 and 18 years with differences in the various age classes for systolic pressure. Subjects with normal birthweight had lower systolic and diastolic BP. SGA males had higher risk of high systolic and diastolic pressure, whereas SGA females were only at higher risk for elevated diastolic pressure. CONCLUSIONS: SGA subjects should be monitored for BP and life-style between 7 and 18 years to risk of cardiovascular disease.

Perfluorocarbons attenuate oxidative lung damage.

The aim of this study was to investigate the effect of tidal liquid ventilation (TLV) compared to conventional mechanical ventilation (CMV) on oxidative lung damage in the setting of acute respiratory distress syndrome (ARDS). After repeated lung lavages, 10 minipigs were treated with CMV or TLV for 4 hr before the animals were sacrificed. Samples for blood gas analysis and bronchial aspirate samples were withdrawn before the induction of lung injury, and at 10 min, 2 hr, and 4 hr after the beginning of ventilatory support. To assess lung oxidative damage, total hydroperoxide (TH) and advanced oxidation protein product (AOPP) concentrations were measured in bronchial aspirate samples. After 2 and 4 hr of ventilatory support, partial oxygen tension (PaO(2)) and base excess (BE) were significantly higher in the TLV group than in the CMV group, while PaCO(2) was slightly higher, but with no statistical significance. In the CMV group, the AOPP level was significantly higher at 4 hr than at baseline. TH and AOPP bronchial aspirate concentrations were higher in the CMV group than in the TLV group at 2 and 4 hr of ventilation. We conclude that animals treated with TLV showed lower oxidative lung damage compared to animals treated with CMV.

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.