J. Escobar

Preterm Resuscitation With Low Oxygen Causes Less Oxidative Stress, Inflammation, and Chronic Lung Disease

OBJECTIVE: The goal was to reduce adverse pulmonary adverse outcomes, oxidative stress, and inflammation in neonates of 24 to 28 weeks of gestation initially resuscitated with fractions of inspired oxygen of 30% or 90%. METHODS: Randomized assignment to receive 30% (N = 37) or 90% (N = 41) oxygen was performed. Targeted oxygen saturation values were 75% at 5 minutes and 85% at 10 minutes. Blood oxidized glutathione (GSSG)/reduced glutathione ratio and urinary o-tyrosine, 8-oxo-dihydroxyguanosine, and isoprostane levels, isofuran elimination, and plasma interleukin 8 and tumor necrosis factor α levels were determined. RESULTS: The low-oxygen group needed fewer days of oxygen supplementation (6 vs 22 days; P < .01) and fewer days of mechanical ventilation (13 vs 27 days; P < .01) and had a lower incidence of bronchopulmonary dysplasia at discharge (15.4% vs 31.7%; P < .05). GSSG/reduced glutathione × 100 ratios at day 1 and 3 were significantly higher in the high-oxygen group (day 1: high-oxygen group: 13.36 ± 5.25; low-oxygen group: 8.46 ± 3.87; P < .01; day 3: high-oxygen group: 8.87 ± 4.40; low-oxygen group: 6.97 ± 3.11; P < .05). Urinary markers of oxidative stress were increased significantly in the high-oxygen group, compared with the low-oxygen group, in the first week after birth. GSSG levels on day 3 and urinary isofuran, o-tyrosine, and 8-hydroxy-2′-deoxyguanosine levels on day 7 were correlated significantly with development of chronic lung disease. CONCLUSIONS: Resuscitation of preterm neonates with 30% oxygen causes less oxidative stress, inflammation, need for oxygen, and risk of bronchopulmonary dysplasia.

Oxygen and oxidative stress in the perinatal period

Fetal life evolves in a hypoxic environment. Changes in the oxygen content in utero caused by conditions such as pre-eclampsia or type I diabetes or by oxygen supplementation to the mother lead to increased free radical production and correlate with perinatal outcomes. In the fetal-to-neonatal transition asphyxia is characterized by intermittent periods of hypoxia ischemia that may evolve to hypoxic ischemic encephalopathy associated with neurocognitive, motor, and neurosensorial impairment. Free radicals generated upon reoxygenation may notably increase brain damage. Hence, clinical trials have shown that the use of 100% oxygen given with positive pressure in the airways of the newborn infant during resuscitation causes more oxidative stress than using air, and increases mortality. Preterm infants are endowed with an immature lung and antioxidant system. Clinical stabilization of preterm infants after birth frequently requires positive pressure ventilation with a gas admixture that contains oxygen to achieve a normal heart rate and arterial oxygen saturation. In randomized controlled trials the use high oxygen concentrations (90% to 100%) has caused more oxidative stress and clinical complications that the use of lower oxygen concentrations (30–60%). A correlation between the amount of oxygen received during resuscitation and the level of biomarkers of oxidative stress and clinical outcomes was established. Thus, based on clinical outcomes and analytical results of oxidative stress biomarkers relevant changes were introduced in the resuscitation policies. However, it should be underscored that analysis of oxidative stress biomarkers in biofluids has only been used in experimental and clinical research but not in clinical routine. The complexity of the technical procedures, lack of automation, and cost of these determinations have hindered the routine use of biomarkers in the clinical setting. Overcoming these technical and economical difficulties constitutes a challenge for the immediate future since accurate evaluation of oxidative stress would contribute to improve the quality of care of our neonatal patients.

Ultra high performance liquid chromatography coupled to tandem mass spectrometry determination of lipid peroxidation biomarkers in newborn serum samples

Byproducts of arachidonic (AA) and docosahexaenoic acid (DHA) oxidation are highly relevant for the study of free radical associated conditions in the perinatal period. Plasma metabolites can provide the clinician with a snapshot of the oxidant status of patients before and after specific clinical interventions (e.g.: supplementation with oxygen). We describe a new andreliable ultra-performance liquid mass spectrometry method to determine F2-isoprostanes and other byproducts (isoprostanes, isofurans, neuroprostanes, neurofurans) in newborn serum samples. Cord blood samples were obtained from severely depressed newborn infants (Apgar score 1 min < 3; arterial cord pH < 7.00), and aliquoted for serum determination and stored at -80 °C. A UHPLC-MS/MS method was employed. It has a series of technical advantages: simple sample treatment; reduced sample volume (100 μL) which is essential for preterm neonates with low circulating blood volume, high throughput of sample analysis (96 samples in less than 24 h) and high selectivity for different isoprostanes isomers. Excellent sensitivity was achieved within limits of detection between 0.06 and 4.2 nmol L(-1), which renders this method suitable to monitoranalyte concentration in newborn samples. The methods precision was satisfactory; with coefficients of variation around 5-12% (intra-day) and 7-17% (inter-day). The reliability of the described method was assessed by analysis of spiked serum samples obtaining recoveries between 70% and 120%. The proposed method has rendered suitable for serum determination for newborn babies at risk of oxygen free radical associated conditions.

Development of a reliable analytical method to determine lipid peroxidation biomarkers in newborn plasma samples

This paper describes a reliable analytical method based on ultra-performance liquid chromatography coupled to tandem mass spectrometry to determine F2-isoprostanes and other total byproducts (isoprostanes, isofurans, neuroprostanes and neurofurans) as lipid peroxidation biomarkers in newborn plasma samples. The proposed procedure is characterized by a simple sample treatment employing a reduced sample volume (100µL). Also, it shows a high throughput and high selectivity to determine simultaneously different isoprostane isomers in a large number of samples. The reliability of the described method was demonstrated by analysis of spiked plasma samples, obtaining recoveries between 70% and 130% for most of the analytes. Taking into account the implementation of further clinical studies, it was demonstrated the proper sensitivity of the method by means of the analysis of few human newborn plasma samples. In addition to this, newborn piglet plasma samples (n=80) were analyzed observing that the developed method was suitable to determine the analyte levels present in this kind of samples. Therefore, this analytical method could be applied in further clinical research about establishment of reliable lipid peroxidation biomarkers employing this experimental model.

Detection of batch effects in liquid chromatography-mass spectrometry metabolomic data using guided principal component analysis

Metabolomics based on liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for studying dynamic responses of biological systems to different physiological or pathological conditions. Differences in the instrumental response within and between batches introduce unwanted and uncontrolled data variation that should be removed to extract useful information. This work exploits a recently developed method for the identification of batch effects in high throughput genomic data based on the calculation of a δ statistic through principal component analysis (PCA) and guided PCA. Its applicability to LC-MS metabolomic data was tested on two real examples. The first example involved the repeated analysis of 42 plasma samples and 6 blanks in three independent batches, and the second data set involved the analysis of 101 plasma and 18 blank samples in a single batch with a total runtime of 50h. The first and second data set were used to evaluate between and within-batch effects using the δ statistic, respectively. Results obtained showed the usefulness of using the δ statistic together with other approaches such as summary statistics of peak intensity distributions, PCA scores plots or the monitoring of IS peak intensities, to detect and identify instrumental instabilities in LC-MS.

PO-0414 Plasma Metabolome In A Newborn Piglet Model For Asphyxia And Resuscitation

Background and aims Post-asphyxia resuscitation with air improves survival. We aimed to find reliable biomarkers of brain injury secondary to hypoxia/ischemia in plasma in a newborn piglet model for asphyxia. Methods Hypoxia was introduced to newborn piglets (standardised model). Plasma metabolomic profiles reflecting the effects of asphyxia and resuscitation were studied, and changes in target metabolites of the Kennedy pathway were analysed by LC-MS. Results A set of metabolites reflecting metabolic changes after asphyxia and resuscitation was identified. Increased levels of choline, cytidine and uridine (Kennedy pathway) during hypoxia were observed (see Figure 1). No differences were found between resuscitation using air and air+2.1% H2. Conclusions Untargeted metabolomics enabled the monitorization of changes occurring during asphyxia and resuscitation on a molecular level. A set of candidate biomarkers was identified. In accordance to previous results, alterations in the Kennedy pathway are reported. The performance of candidate biomarkers for clinical grading will be evaluated in further studies. Acknowledgments JK and JE acknowledge Sara Borrell grants CD11/00154 and CD12/00667. MV acknowledges the FISPI11/0313 and EC11–246 grant. The Laerdal Foundation (Norway) supported this study. Abstract PO-0414 Figure 1 Choline, cytidine and uridine levels before and after hypoxia as well as after resuscitation

PS-042a Docosahexaenoic Acid (dha) Is Neuroprotective After Newborn Asphyxia Proton-magnetic-resonance-spectroscopy (h±-mrs) On Hypoxic Brain Tissue In Piglets

Background Hypothermia is an established treatment for perinatal asphyxia. Post treatment hypothermia MRI supplemented with H±-MRS is used in clinics as the best predictor for neurodevelopmental outcome. DHA is an omega-3 fatty acid thought to modify apoptosis, inflammation and reduce lipid peroxidation in face of hypoxia. We have previously shown neuroprotective effects of DHA. The current study combines DHA and hypothermia. Methods 54 newborn pigs (age 12–36 h) were randomised to undergo hypoxia (N=48) or not (Control, N=6). Hypoxia was achieved on fully anaesthetised, intubated piglets through FiO2 0.08 until bloodgases reached Base Excess -20 mmol/L or middle arterial blood pressure below 20 mmHg. Piglets were then block randomised to one of four groups: (1) Hypoxia, (2) Hypoxia + Hypothermia, (3) Hypoxia + DHA or (4) Hypoxia +DHA +Hypothermia. Piglets were mechanically ventilated 9,5 h post end hypoxia and then euthanized. Hippocampal brain tissue was immediately snap frozen in liquid nitrogen. H±-MRS measuring lactate (Lac) and glutamate (Glu) in relation to n-acetylaspartate (NAA) was conducted on frozen tissue. Piglets with autolysis of the brain and outliers over 2 standard deviations were removed from the analysis. Results Abstract PS-042a Figure 1 The only Lac/NAA ratio significantly different than control, is the hypoxia group (p = 0.016). Intervention groups show no significant changes vs controls. Group 1 vs group 3 shows a borderline significance (p = 0.073). Conclusion Hypoxia significantly increases the Lac/NAA biomarker and intervention groups are at a pre-hypoxic control level. The pattern consists through the Glutamate group. DHA may be beneficial in neuroprotection after asphyxia.

236 Intermittent Hypoxia: Effects on Brain Stem of Oxidative Stress and NRF2 Transcription Factor Activation in A Rat Pup Model

Background Apnea of prematurity which is a common condition in the neonatal period caused by immature brainstem respiratory neural output may result in intermittent hypoxia and cause of oxidative stress during this vulnerable developmental period. Objective To test if chronic intermittent hypoxia (CIH) alters oxidative metabolism and resultant redox status in the medulla of rat pups. Methods Litters of 10 rat pups and their dams were assigned to: normoxia (controls) and intermittent hypoxia (Hx). Exposure occurred from P1-P7. CIH consisted of exposing rat pups to alternating cycles of N2 and air: 45 seconds of hypoxia (nadir of 5% O2) was administered every 5 minutes for 8 hours/day. For controls, animals were kept at air. On the eighth day, brainstems were harvested, snap-frozen in liquid nitrogen. Reduced (GSH) and oxidized (GSSG) glutathione, and precursors -glutamyl-cysteine (-G-cysteine) and L-cysteine in medulla were determined by UPLC-MS/MS and MDA in medulla was determined by HPLC. Results GSH was significantly reduced in medulla of rat pups submitted to chronic intermittent hypoxic (CIH) episodes associated with reduction in GSH/GSSG ratio. GSH precursors were also significantly lower in the brainstem of the Hx group. Conclusions Intermittent hypoxic episodes in rat pups cause a significant reduction in GSH and its precursors in the developing brainstem. GSH and precursors are major determinants of redox status. These alterations may activate transcription factors relevant to the expression of antioxidant enzymes and inflammation. We speculate that oxidant stress may impair central respiratory control and contribute to further enhance recurrent apnea/impaired oxygenation.