Ángel Sánchez-Illana

Urinary Lipid Peroxidation Byproducts: Are They Relevant for Predicting Neonatal Morbidity in Preterm Infants?

Preterm infants have an immature antioxidant system; however, they frequently require supplemental oxygen. Oxygen-free radicals cause both pulmonary and systemic inflammation, and they are associated with increased morbidity and mortality. Consequently, screening of metabolite profiles representing the amount of lipid peroxidation is considered of great relevance for the evaluation of in vivo oxidative stress and derived inflammation and damage. Ranges for total relative contents of isoprostanes (IsoPs), isofurans (IsoFs), neuroprostanes (NeuroPs), and neurofurans (NeuroFs) within targeted SpO2 ranges were determined in urine samples of 254 preterm infants<32 weeks of gestation within the frame of two randomized, controlled, and blinded clinical trials employing ultra-performance liquid chromatography-tandem mass spectrometry. A total of 536 serial urine samples collected during the first 4 weeks after birth in recruited infants who did not develop free radical associated conditions were analyzed. A reference range for lipid peroxidation byproducts, including isoprostanes, isofurans, neuroprostanes, and neurofurans, was calculated and possible correlations with neonatal conditions were investigated. Urinary elimination of isofurans in the first 4 days after birth correlated with later development of bronchopulmonary dysplasia. Our observations lead to the hypothesis that early urinary determination of lipid peroxidation byproducts, especially isofurans, is relevant to predict development of chronic lung conditions.

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.

Development of a reliable method based on ultra-performance liquid chromatography coupled to tandem mass spectrometry to measure thiol-associated oxidative stress in whole blood samples

The aminothiols are biological compounds with numerous vital functions. One of the most relevant is their role as antioxidant systems. The reduced to oxidized ratios are extremely useful indicators of oxidative stress and cellular redox status. We have validated an ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method to determine the levels of glutathione, cysteine, homocysteine, and their respective oxidized compounds in whole blood samples. Results showed excellent linearity for all the analytes with correlation coefficients between 0.990 and 0.997, suitable precision with intra-day coefficient of variation ≤20%, and satisfactory accuracy with recoveries between 75 and 130%. The limits of detection in whole blood samples were 1.16 nmol L(-1) for glutathione, 115.8 nmol L(-1) for oxidized glutathione, 9.3 nmol L(-1) for homocystine, 92.6 nmol L(-1) for homocysteine, 347 nmol L(-1) for cystine and 0.23 nmol L(-1) for cysteine. The suitability of the method was ascertained in whole blood samples (n=80) from a consolidated experimental model of hypoxia-reoxygenation in newborn piglets.

Changes of the plasma metabolome of newly born piglets subjected to postnatal hypoxia and resuscitation with air

Background:Perinatal hypoxic–ischemic brain damage is a major cause of mortality and morbidity in the neonatal period. Currently, limited ranges of biochemical tests assessing the intensity and duration of hypoxia are ready for clinical use. However, the need to initiate hypothermia therapy early after the clinical suspicion of hypoxic–ischemic encephalopathy requires the availability of early and reliable hypoxia markers. We have sought these biomarkers in an experimental model of hypoxia reoxygenation.Methods:Hypoxia and hypotension were induced in newborn piglets following a standardized model and reoxygenation was carried out using room air (RA). An untargeted liquid chromatography—time of flight mass spectrometry (LC-TOFMS) approach was used to assess changes in the metabolomic profile of plasma samples after intense hypoxia and upon reoxygenation.Results:At the end of hypoxia, the plasma metabolome showed an increased plasma concentration of analytes reflecting a metabolic adaptation to prolonged anaerobiosis. However, after resuscitation, metabolite levels returned to the starting values.Conclusion:Severe hypoxia induces early, significant, and transient changes of specific metabolites in the plasma metabolome, which represent a snapshot of the biochemical adaptation of mammals to intense hypoxia. These metabolites could have applicability in predicting the severity of hypoxia in the clinical setting.

Evolution of Energy Related Metabolites in Plasma from Newborns with Hypoxic-Ischemic Encephalopathy during Hypothermia Treatment

Therapeutic hypothermia (TH) initiated within 6 h from birth is the most effective therapeutic approach for moderate to severe hypoxic-ischemic encephalopathy (HIE). However, underlying mechanisms and effects on the human metabolism are not yet fully understood. This work aims at studying the evolution of several energy related key metabolites in newborns with HIE undergoing TH employing gas chromatography – mass spectrometry. The method was validated following stringent FDA requirements and applied to 194 samples from a subgroup of newborns with HIE (N = 61) enrolled in a multicenter clinical trial (HYPOTOP) for the determination of lactate, pyruvate, ketone bodies and several Krebs cycle metabolites at different sampling time points. The analysis of plasma samples from newborns with HIE revealed a decrease of lactate, pyruvate and β-hydroxybutyrate concentrations, whereas rising malate concentrations were observed. In healthy control newborns (N = 19) significantly lower levels of pyruvate and lactate were found in comparison to age-matched newborns with HIE undergoing TH, whereas acetoacetate and β-hydroxybutyrate levels were clearly increased. Access to a validated analytical method and a controlled cohort of newborns with HIE undergoing hypothermia treatment for the first time allowed the in-depth study of the evolution of key metabolites of metabolic junctions in this special population.

Plasma metabolite score correlates with Hypoxia time in a newly born piglet model for asphyxia

Hypoxic-ischemic encephalopathy (HIE) secondary to perinatal asphyxia is a leading cause of mortality and acquired long-term neurologic co-morbidities in the neonate. The most successful intervention for the treatment of moderate to severe HIE is moderate whole body hypothermia initiated within 6 h from birth. The objective and prompt identification of infants who are at risk of developing moderate to severe HIE in the critical first hours still remains a challenge. This work proposes a metabolite score calculated based on the relative intensities of three metabolites (choline, 6,8-dihydroxypurine and hypoxanthine) that showed maximum correlation with hypoxia time in a consolidated piglet model for neonatal hypoxia-ischemia. The metabolite scores performance as a biomarker for perinatal hypoxia and its usefulness for clinical grading and decision making have been assessed and compared to the performance of lactate which is currently considered the gold standard. For plasma samples withdrawn before and directly after a hypoxic insult, the metabolite score performed similar to lactate. However, it provided an enhanced predictive capacity at 2 h after resuscitation. The present study evidences the usefulness of the metabolite score for improving the early assessment of the severity of the hypoxic insult based on serial determinations in a minimally invasive biofluid. The applicability of the metabolite score for clinical diagnosis and patient stratification for hypothermia treatment has to be confirmed in multicenter trials involving newborns suffering from HIE.

Mass spectrometric detection of biomarkers for early assessment of intraamniotic fluid infection

This data article contains information on glutathione sulfonamide (GSA) structural confirmation and purity after synthesis, as well as mass spectrometry acquisition parameters for the determination of GSA and other biomarkers for the early assessment of intraamniotic fluid infection in amniotic fluid samples (Cháfer-Pericás et al., 2015) [1]. GSA standards were synthesized and structural confirmation was carried out employing time-of-flight mass spectrometry (TOF-MS); purity was assessed by high performance liquid chromatography (HPLC) with UV detection. For optimization of the acquisition parameters of GSA and other biomarkers, individual analytical standard solution at a concentration of 1 µmol L−1 was injected into an Acquity – Xevo TQ liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS) system from Waters (Milford, MA, USA) operating in the positive electrospray (ESI+) mode. Mass spectrometric detection of 3-nitro-tyrosine (3NO2-Tyr), 3-chloro-tyrosine (3Cl-Tyr), 8-hydroxy-2′-deoxyguanosine (8OHdG), GSA and oxidized glutathione (GSSG) was carried out by multiple reaction monitoring (MRM). Linear response curves were calculated for each analyte normalizing the signal with peak areas of internal standards.

Assessment of phospholipid synthesis related biomarkers for perinatal asphyxia: a piglet study

The prompt and reliable identification of infants at risk of hypoxic-ischemic encephalopathy secondary to perinatal asphyxia in the first critical hours is important for clinical decision-making and yet still remains a challenge. This work strives for the evaluation of a panel of metabolic biomarkers that have been associated with the hypoxic-ischemic insult in the perinatal period. Plasma and urine samples from a consolidated newborn piglet model of hypoxia and withdrawn before and at different time points after a hypoxic insult were analyzed and compared to a control group. Time-dependent metabolic biomarker profiles were studied and observed patterns were similar to those of lactate levels, which are currently considered the gold standard for assessing hypoxia. Class prediction performance could be improved by the use of a combination of the whole panel of determined metabolites in plasma as compared to lactate values. Using a multivariate model including lactate together with the studied metabolic biomarkers allowed to improve the prediction performance of duration of hypoxia time, which correlates with the degree of brain damage. The present study evidences the usefulness of choline and related metabolites for improving the early assessment of the severity of the hypoxic insult.

On-Capillary Surface-Enhanced Raman Spectroscopy: Determination of Glutathione in Whole Blood Microsamples

Oxidative stress monitoring in the neonatal period supports early outcome prediction and treatment. Glutathione (GSH) is the most abundant antioxidant in most cells and tissues, including whole blood, and its usefulness as a biomarker has been known for decades. To date, the available methods for GSH determination require laborious sample processing and the use of sophisticated laboratory equipment. To the best of our knowledge, no tools suitable for point-of-care (POC) sensing have been reported. Surface-enhanced Raman spectroscopy (SERS), performed in a microvolume capillary measurement cell, is proposed in this study as a robust approach for the quantification of GSH in human whole blood samples. The use of a silver colloid allowed a highly selective signal enhancement for GSH providing analytical enhancement factors of 3 to 4 orders of magnitude. A highly accurate determination of GSH in whole blood samples with recoveries ranging from 99 to 107% and relative standard deviations less than or equal to 18% were achieved by signal normalization with the intensity of an isotopically labeled internal standard. GSH concentrations were retrieved within 4 min using small-volume blood samples (2 μL). The developed procedure was applied to the analysis of blood of 20 healthy adults and 36 newborns, obtaining comparable results between literature and those found by SERS and a reference method. The characteristics of this novel tool are suitable for its implementation in a portable optical sensor device enabling POC testing of oxidative stress levels in newborns.