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Inhaled nitric oxide (iNO) is a selective pulmonary vasodilator that is used as a treatment for persistent pulmonary hypertension in neonates (PPHN) with hypoxic respiratory failure. The generation of reactive oxygen and nitrogen species might induce oxidative/nitrosative damage to multiple organs. There is an increasing scientific and clinical interest in the determination of specific biomarkers to measure the degree of oxidative/nitrosative stress in non-invasively collected biofluids. A method for the simultaneous detection of a panel of oxidative and nitrosative stress-related biomarkers for quantifying damage to proteins and DNA/RNA in 20 μL of infant urine samples based on reversed-phase ultra-performance liquid chromatography coupled to tandem mass spectrometry operating in positive electrospray ionization mode (ESI + ) was optimized and validated. Infant urine samples from two different studies were analyzed: (i) term and preterm infants from a nutrition study (Nutrishield, N = 50) and (ii) infants with respiratory insufficiency, including infants with PPHN (N = 16) that required iNO treatment and a control group without treatment (N = 14). Eleven of 14 metabolites were detected in >50 % of infant urine samples, with ranges between 0.008 and 1400 μmol/g creatinine. When comparing across groups, differences in samples collected after iNO treatment in comparison to the rest of the groups were found for m-tyrosine (m-Tyr and m-Tyr/Phe) and ortho-tyrosine (o-Tyr and o-Tyr/Phe) (p-values <0.001, Wilcoxon rank-sum test). Positive linear relationships were found with NO exposure corrected by infant weight for m-Tyr, m-Tyr/Phe, o-Tyr, o-Tyr/Phe and 3-nitrotyrosine. Future studies will focus on the evaluation of the impact of iNO treatment on health and oxidative/nitrosative stress-related morbidities associated with prematurity., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Julia Kuligowski reports financial support was provided by Carlos III Health Institute. Abel Albiach-Delgado reports financial support was provided by Carlos III Health Institute. Aljandro Pinilla-González reports financial support was provided by Carlos III Health Institute. Inmaculada Lara Cantón reports financial support was provided by Carlos III Health Institute. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Glutathione (GSH) is a non-protein tripeptide thiol that plays a prominent role in oxidative stress defense. GSH concentration is particularly critical in the neonatal period, especially for premature newborns that face increased susceptibility to oxidative stress. Monitoring GSH levels provides valuable insights into newborn health, helping to tailor care to their specific needs. The aim of this study was the development of a sensor specifically targeted for its use in neonatology, enabling GSH determination in only 2 μL of whole blood. The newly developed sensing system simplifies sample processing, addressing a critical need in clinical applications. Unlike current methods that demand fast pre-processing of relatively large sample volumes, expensive equipment, and skilled personnel, the developed approach streamlines the analytical process. By using 2 μL of whole blood, a single syringe filter for sample treatment, a deuterated internal standard (IS) for signal normalization, and Surface Enhanced Raman Scattering (SERS) spectroscopy with a silver colloid substrate for GSH detection, the set-up's characteristics are compatible with point-of-care applications. The analytical procedure was validated and applied to diverse populations including healthy adults (N = 63) and newborns (N = 35), yielding GSH concentration values ranging from 0.6 to 1.8 and 0.8-2.1 mM, respectively. This new optical sensor offers a quick and cost-effective solution to support the assessment of GSH levels in newborns that can greatly benefit not only neonatal care, but also the study of adult populations for health monitoring., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Julia Kuligowski reports financial support was provided by Carlos III Health Institute. Abel Albiach-Delgado reports financial support was provided by Carlos III Health Institute. Jose Luis Moreno-Casillas reports financial support was provided by Spain Ministry of Science and Innovation. Julia Kuligowski reports financial support was provided by Government of Valencia. Guillermo Quintas reports financial support was provided by Spain Ministry of Science and Innovation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

An early prediction of outcomes of neonatal hypoxic-ischemic encephalopathy (NE) is of key importance in reducing neonatal mortality and morbidity. The objectives were (i) to analyze the characteristics of miRNA expression and metabolic patterns of neonates with NE and (ii) to assess their predictive performance for neurodevelopmental outcomes. Plasma samples from moderate/severe NE patients (N = 92) of the HYPOTOP study were collected before, during, and after therapeutic hypothermia (TH) and compared to a control group (healthy term infants). The expression of miRNAs and concentrations of metabolites (hypoxia-related and energy, steroid, and tryptophan metabolisms) were analyzed. Neurodevelopmental outcomes were evaluated at 24 months postnatal age using Bayley Scales of Infant Development, ed. III, BSID-III. Differences in miRNA and metabolic profiles were found between NE vs. control infants, abnormal (i.e., mildly and moderately abnormal and severe) vs. normal, and severe vs. non-severe (i.e., normal and mildly and moderately abnormal) BSID-III. 4-Androstene-3,17-dione, testosterone, betaine, xanthine, and lactate were suitable for BSID-III outcome prediction (receiver operating characteristic areas under the curve (AUCs) ≥ 0.6), as well as 68 miRNAs (AUCs of 0.5-0.9). Significant partial correlations of xanthine and betaine levels and the expression of several miRNAs with BSID-III sub-scales were found. Conclusion: We have identified metabolites/miRNAs that might be useful to support the prediction of middle-term neurodevelopmental outcomes of NE. What is known and what is new: • The early prediction of outcomes of neonatal hypoxic-ischemic encephalopathy (NE) is of key importance in reducing neonatal mortality and morbidity. • Alterations of the metabolome and miRNAs had been observed in NE. • We performed miRNA sequencing and quantified selected metabolites (i.e., lactate, pyruvate, ketone bodies, Krebs cycle intermediates, tryptophan pathway, hypoxia-related metabolites, and steroids) by GC- and LC-MS. • Specific miRNAs and metabolites that allow prediction of middle-term neurodevelopmental outcomes of newborns with NE undergoing hypothermia treatment were identified., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Small Extracellular Vesicles (sEVs) are nano-sized vesicles that are present in all biofluids including human milk (HM) playing a crucial role in cell-to-cell communication and the stimulation of the neonatal immune system. Oxylipins, which are bioactive lipids formed from polyunsaturated fatty acids, have gained considerable attention due to their potential role in mitigating disease progression and modulating the inflammatory status of breastfed infants. This study aims at an in-depth characterization of the oxylipin profiles of HM and, for the first time, of HM-derived sEVs (HMEVs) employing an ad-hoc developed and validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method., Results: The UPLC-MS/MS method covered a panel of 13 oxylipins for quantitation and 93 oxylipins for semi-quantitation. In 200 μL of HM and HMEV isolates of 15 individuals, 42 out of 106 oxylipins were detected in either HM or HMEVs, with 38 oxylipins being detected in both matrices. Oxylipins presented distinct profiles in HM and HMEVs, suggesting specific mechanisms responsible for the encapsulation of target molecules in HMEVs. Ten and eight oxylipins were quantified with ranges between 0.03 - 73 nM and 0.30 pM-0.07 nM in HM and HMEVs, respectively. The most abundant oxylipins found in HMEVs were docosahexaenoic acid derivatives (17-HDHA and 14-HDHA) with known anti-inflammatory properties, and linoleic acid derivatives (9-10-DiHOME and 12,13-DiHOME) in HM samples., Significance and Novelty: This is the first time a selective, relative enrichment of anti-inflammatory oxylipins in HMEVs has been described. Future studies will focus on the anti-inflammatory and pro-healing capacity of oxylipins encapsulated in HMEVs, with potential clinical applications in the field of preterm infant care, specifically the prevention of severe intestinal complications including necrotizing enterocolitis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Julia Kuligowski reports financial support was provided by Carlos III Health Institute. Abel Albiach-Delgado reports financial support was provided by Carlos III Health Institute. Pilar Sepulveda reports financial support was provided by Carlos III Health Institute. Jose L Moreno-Casillas reports financial support was provided by Government of Valencia. Julia Kuligowski reports financial support was provided by Government of Valencia. Julia Kuligowski reports financial support was provided by Valencia Innovation Agency. Pilar Sepulveda reports financial support was provided by Valencia Innovation Agency. Julia Kuligowski reports financial support was provided by Spain Ministry of Science and Innovation. Isabel Ten-Doménech reports financial support was provided by Valencia Innovation Agency. Guillermo Quintas reports financial report was provided by Spain Ministry of Science and Innovation. Abel Albiach-Delgado, Isabel Ten-Domenech, Pilar Sepulveda, and Marta Gomez-Ferrer have patent Composition comprising oxylipins present in human milk derived small extracellular vesicles and its use in the prevention and treatment of intestinal diseases (EP23382313) (pending). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Currently, the treatment of anemia in preterm infants is based on packed red blood cell (RBC) transfusions from adult donors. Oxygen (O2) is mainly transported to the tissues bound to hemoglobin (Hb). In extremely low gestational age neonates (ELGANs), fetal hemoglobin (HbF), which has a higher affinity for O2, represents up to 95% of circulating hemoglobin. During the first month of life, the majority of ELGANs will require an adult-donor RBC transfusion causing HbF levels to rapidly drop. HbA releases 50% more oxygen in peripheral tissues than HbF. Increased release of O2 in the retina is one of the main factors related to the development of retinopathy of prematurity (ROP). Collecting umbilical cord blood and using autologous umbilical cord whole blood (UCB) transfusions would contribute to maintaining physiological HbF concentrations in newborns and avoid oxygen-in-excess derived damage., Methods: This is a randomized, double-blinded, multicenter clinical trial. ELGANs ≤28 weeks of gestational age will be randomized 1:1 to receive an autologous umbilical cord blood transfusion (intervention arm) or standard transfusion of packed RBC from an adult donor (control arm) to assess ROP development. Assuming a 50% reduction in ROP incidence, 134 patients (67 per group) will be recruited. When blood transfusion is indicated, the Blook Bank will supply UCB or RCB according to the patient's group. The primary endpoint is the incidence of any ROP. Secondary endpoints are assessessment of treatment safety, results of biomarkers related to ROP and its chronology, and urine oxidative stress markers. In addition, the cellular composition of umbilical cord blood and its relationship with prematurity-related pathologies will be analyzed. All patients will be followed-up to 24 months of corrected age to evaluate their neurodevelopment., Discussion: ROP is a major cause of irreversible blindness in preterm newborns. Transfusions with adult donor blood can lead to complications, including ROP. UCB transfusions offer advantages by maintaining physiological HbF levels and potentially optimizing postnatal development. Moreover, autologous UCB transfusion could reduce risks associated with heterologous blood products, although volume collection remains challenging. UCB contains growth factors and progenitor cells that may impact ROP., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2023 Torrejon-Rodriguez, Pinilla-Gonzalez, Lara Cantón, Albiach-Delgado, Cascant-Vilaplana, Cernada, Kuligowski, Solves Alcaina, Gómez, Vento and Aguar Carrascosa.)

Background: Neonatal encephalopathy (NE) is a major cause of mortality and severe neurological disability in the neonatal period and beyond. We hypothesized that the degree of brain injury is reflected in the molecular composition of peripheral blood samples., Methods: A sub-cohort of 28 newborns included in the HYPOTOP trial was studied. Brain injury was assessed by magnetic resonance imaging (MRI) once per patient and neurodevelopment at 24 months of age was evaluated using the Bayley III Scales of Infant and Toddler Development. The nuclear magnetic resonance (NMR) profile of 60 plasma samples collected before, during, and after cooling was recorded., Results: In total, 249 molecular features were quantitated in plasma samples from newborns and postnatal age showed to affect detected NMR profiles. Lactate, beta-hydroxybutyrate, pyruvate, and three triglyceride biomarkers showed the ability to discern between different degrees of brain injury according to MRI scores. The prediction performance of lactate was superior as compared to other clinical and biochemical parameters., Conclusions: This is the first longitudinal study of an ample compound panel recorded by NMR spectroscopy in plasma from NE infants. The serial determination of lactate confirms its solid position as reliable candidate biomarker for predicting the severity of brain injury., Impact: The use of nuclear magnetic resonance (NMR) spectroscopy enables the simultaneous quantitation of 249 compounds in a small volume (i.e., 100 μL) of plasma. Longitudinal perturbations of plasma NMR profiles were linked to magnetic resonance imaging (MRI) outcomes of infants with neonatal encephalopathy (NE). Lactate, beta-hydroxybutyrate, pyruvate, and three triglyceride biomarkers showed the ability to discern between different degrees of brain injury according to MRI scores. Lactate is a minimally invasive candidate biomarker for early staging of MRI brain injury in NE infants that might be readily implemented in clinical guidelines for NE outcome prediction., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Wheat is critical for food security, and is challenged by biotic stresses, chiefly aphids and the viruses they transmit. The objective of this study was to determine whether aphids feeding on wheat could trigger a defensive plant reaction to oxidative stress that involved plant oxylipins. Plants were grown in chambers with a factorial combination of two nitrogen rates (100% N vs. 20% N in Hoagland solution), and two concentrations of CO 2 (400 vs. 700 ppm). The seedlings were challenged with Rhopalosiphum padi or Sitobion avenae for 8 h. Wheat leaves produced phytoprostanes (PhytoPs) of the F 1 series, and three types of phytofurans (PhytoFs): ent -16( RS )-13- epi -ST-Δ 14 -9-PhytoF, ent -16( RS )-9- epi -ST-Δ 14 -10-PhytoF and ent -9( RS )-12- epi -ST-Δ 10 -13-PhytoF. The oxylipin levels varied with aphids, but not with other experimental sources of variation. Both Rhopalosiphum padi and Sitobion avenae reduced the concentrations of ent -16( RS )-13- epi -ST-Δ 14 -9-PhytoF and ent -16( RS )-9- epi -ST-Δ 14 -10-PhytoF in relation to controls, but had little or no effect on PhytoPs. Our results are consistent with aphids affecting the levels of PUFAs (oxylipin precursors), which decreased the levels of PhytoFs in wheat leaves. Therefore, PhytoFs could be postulated as an early indicator of aphid hosting for this plant species. This is the first report on the quantification of non-enzymatic PhytoFs and PhytoPs in wheat leaves in response to aphids.

Human milk (HM) is a complex biofluid containing a wide cell variety including epithelial cells and leukocytes. However, the cellular compositions and their phenotypic properties over the course of lactation are poorly understood. The aim of this preliminary study was to characterize the cellular metabolome of HM over the course of lactation. Cells were isolated via centrifugation and the cellular fraction was characterized via cytomorphology and immunocytochemical staining. Cell metabolites were extracted and analyzed using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QqTOF-MS) in the positive and negative electrospray ionization modes. Immunocytochemical analysis revealed a high variability of the number of detected cells with relative median abundances of 98% of glandular epithelial cells, 1% of leukocytes, and 1% of keratinocytes. Significant correlations between the milk postnatal age with percentage of epithelial cells and leukocytes, and with total cell count were observed. Results from the Hierarchical Cluster Analysis of immunocytochemical profiles were very similar to those observed in the analysis of the metabolomic profiles. In addition, metabolic pathway analysis showed alterations in seven metabolic pathways correlating with postnatal age. This work paves the way for future investigations on changes in the metabolomic fraction of the cellular compartment of HM.

Fetuses with congenital heart disease (CHD) have circulatory changes that may lead to predictable blood flow disturbances that may affect normal brain development. Hypoxemia and hypoperfusion may alter the redox balance leading to oxidative stress (OS), that can be assessed measuring stable end-products. OS biomarkers (OSB) were measured in amniotic fluid in fetuses with ( n = 41) and without CHD ( n = 44) and analyzed according to aortic flow, expected cyanosis after birth, and a CHD classification derived from this. Birth head circumference (HC) was used as a neurodevelopment biomarker. CHD fetuses had higher levels of ortho-Tyrosine (o-Tyr) than controls ( p = 0.0003). There were no differences in o-Tyr levels considering aortic flow obstruction ( p = 0.617). Fetuses with expected extreme cyanosis presented the highest levels of o-Tyr ( p = 0.003). Among groups of CHD, fetuses without aortic obstruction and extreme cyanosis had the highest levels of o-Tyr ( p = 0.005). CHD patients had lower HC than controls ( p = 0.023), without correlation with OSB. Patients with HC < 10th percentile, presented high levels of o-Tyr ( p = 0.024). Fetuses with CHD showed increased OSB and lower HC when compared to controls, especially those with expected extreme cyanosis. Our results suggest that increased levels of OSB are more influenced by the effect of low oxygenation than by aortic flow obstruction. Future studies with larger sample size are needed to further investigate the role of OSB as an early predictor of neurodevelopmental problems in CHD survivors.