Longitudinal perturbations of plasma nuclear magnetic resonance profiles in neonatal encephalopathy.

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.

Resuscitators' opinions on using a respiratory function monitor during neonatal resuscitation.

AIM: The aim of this study was to assess the resuscitators' opinions of the usefulness and clinical value of using a respiratory function monitor (RFM) when resuscitating extremely preterm infants with positive pressure ventilation. METHODS: The link to an online survey was sent to 106 resuscitators from six countries who were involved in a multicentre trial that compared the percentage of inflations within a predefined target range with and without the RFM. The resuscitators were asked to assess the usefulness and clinical value of the RFM. The survey was online for 4 months after the trial ended in May 2019. RESULTS: The survey was completed by 74 (70%) resuscitators of which 99% considered the RFM to be helpful during neonatal resuscitation and 92% indicated that it influenced their decision-making. The majority (76%) indicated that using the RFM improved their practice and made resuscitation more effective, even when the RFM was not available. Inadequate training was the key issue that limited the effectiveness of the RFM: 45% felt insufficiently trained, and 78% felt more training in using and interpreting the RFM would have been beneficial. CONCLUSION: Resuscitators considered the RFM to be helpful to guide neonatal resuscitation, but sufficient training was required to achieve the maximum benefit.

Noninvasive monitoring of evolving urinary metabolic patterns in neonatal encephalopathy.

BACKGROUND: Infants with moderate and severe neonatal encephalopathy (NE) frequently suffer from long-term adverse outcomes. We hypothesize that the urinary metabolome of newborns with NE reflects the evolution of injury patterns observed with magnetic resonance imaging (MRI). METHODS: Eligible patients were newborn infants with perinatal asphyxia evolving to NE and qualifying for therapeutic hypothermia (TH) included in the HYPOTOP trial. MRI was employed for characterizing brain injury. Urine samples of 55 infants were collected before, during, and after TH. Metabolic profiles of samples were recorded employing three complementary mass spectrometry-based assays, and the alteration of detected metabolic features between groups was assessed. RESULTS: The longitudinal assessment revealed significant perturbations of the urinary metabolome. After 24 h of TH, a stable disease pattern evolved characterized by the alterations of 4-8% of metabolic features related to lipid metabolism, metabolism of cofactors and vitamins, glycan biosynthesis and metabolism, amino acid metabolism, and nucleotide metabolism. Characteristic metabolomic fingerprints were observed for different MRI injury patterns. CONCLUSIONS: This study shows the potential of urinary metabolic profiles for the noninvasive monitoring of brain injury of infants with NE during TH. IMPACT: A comprehensive approach for the study of the urinary metabolome was employed involving a semi-targeted capillary electrophoresis-time-of-flight mass spectrometry (TOFMS) assay, an untargeted ultra-performance liquid chromatography (UPLC)-quadrupole TOFMS assay, and a targeted UPLC-tandem MS-based method for the quantification of amino acids. The longitudinal study of the urinary metabolome identified dynamic metabolic changes between birth and until 96 h after the initiation of TH. The identification of altered metabolic pathways in newborns with pathologic MRI outcomes might offer the possibility of developing noninvasive monitoring approaches for personalized adjustment of the treatment and for supporting early outcome prediction.

A multi-centre randomised controlled trial of respiratory function monitoring during stabilisation of very preterm infants at birth.

AIM: To determine whether the use of a respiratory function monitor (RFM) during PPV of extremely preterm infants at birth, compared with no RFM, leads to an increase in percentage of inflations with an expiratory tidal volume (Vte) within a predefined target range. METHODS: Unmasked, randomised clinical trial conducted October 2013 - May 2019 in 7 neonatal intensive care units in 6 countries. Very preterm infants (24-27 weeks of gestation) receiving PPV at birth were randomised to have a RFM screen visible or not. The primary outcome was the median proportion of inflations during manual PPV (face mask or intubated) within the target range (Vte 4-8 mL/kg). There were 42 other prespecified monitor measurements and clinical outcomes. RESULTS: Among 288 infants randomised (median (IQR) gestational age 26+2 (25+3-27+1) weeks), a total number of 51,352 inflations were analysed. The median (IQR) percentage of inflations within the target range in the RFM visible group was 30.0 (18.0-42.2)% vs 30.2 (14.8-43.1)% in the RFM non-visible group (p = 0.721). There were no differences in other respiratory function measurements, oxygen saturation, heart rate or FiO2. There were no differences in clinical outcomes, except for the incidence of intraventricular haemorrhage (all grades) and/or cystic periventricular leukomalacia (visible RFM: 26.7% vs non-visible RFM: 39.0%; RR 0.71 (0.68-0.97); p = 0.028). CONCLUSION: In very preterm infants receiving PPV at birth, the use of a RFM, compared to no RFM as guidance for tidal volume delivery, did not increase the percentage of inflations in a predefined target range. TRIAL REGISTRATION: Dutch Trial Register NTR4104, clinicaltrials.gov NCT03256578.

Metabolic Phenotypes of Hypoxic-Ischemic Encephalopathy with Normal vs. Pathologic Magnetic Resonance Imaging Outcomes.

Hypoxic-Ischemic Encephalopathy (HIE) is one of the most relevant contributors to neurological disability in term infants. We hypothesized that clinical outcomes of newborns with (HIE) can be associated with changes at plasma metabolic level enabling the detection of brain injury. Plasma samples of a cohort of 55 asphyxiated infants who evolved to moderate/severe HIE were collected between birth and completion of therapeutic hypothermia (TH). Samples were analyzed employing a quantitative gas chromatography-mass spectrometry method for the determination of lactate and pyruvate and an untargeted liquid chromatography-time-of-flight mass spectrometry method for metabolic fingerprinting. Brain injury was assessed employing magnetic resonance imaging (MRI). A critical assessment of the usefulness of lactate, pyruvate, and pyruvate/lactate for outcome prediction was carried out. Besides, metabolic fingerprinting identified a dynamic perturbation of eleven metabolic pathways, including amino acid and purine metabolism, and the steroid hormone biosynthesis, in newborns with pathologic MRI outcomes. Although data suggest the usefulness of lactate and pyruvate monitoring during 72 h for discerning outcomes, only the steroid hormone biosynthesis pathway was significantly altered in early plasma samples (i.e., before the initiation of TH). This study highlights pathways that might potentially be targeted for biomarker discovery or adjuvant therapies to be combined with TH.

Does Pasteurized Donor Human Milk Efficiently Protect Preterm Infants Against Oxidative Stress?

Pasteurized donor human milk (DHM) is the preferred alternative for infant nutrition when own mother's milk (OMM) is unavailable. Whether DHM is an efficient means for protecting preterm infants from oxidative stress remains unknown. We quantified a panel of oxidative stress biomarkers in urine samples from preterm infants (≤32 weeks of gestation and a birth weight ≤1500 g) receiving ≥80% of feeding volume as either DHM or OMM. The noninvasive in vivo assessment of oxidative stress showed no statistically significant difference between both groups at the time when full enteral nutrition (150 mL/kg body weight) was achieved and until hospital discharge. In addition, the changes of urinary biomarker levels with time were assessed. This is the first longitudinal study on oxidative stress levels in preterm infants fed with DHM in comparison with OMM. There is no statistically significant difference in urinary oxidative stress levels of preterm infants from both groups indicating that despite the effects of pasteurization, DHM is a valid alternative when OMM is not available. Based on the results, we raise the hypothesis that pasteurized DHM protects preterm infants from oxidative stress as good as OMM, and consequently, its use could prevent oxidative stress-related diseases. Antioxid. Redox Signal. 31, 791-799.

Topiramate plus Cooling for Hypoxic-Ischemic Encephalopathy: A Randomized, Controlled, Multicenter, Double-Blinded Trial.

BACKGROUND AND OBJECTIVES: Therapeutic interventions to improve the efficacy of whole-body cooling for hypoxic-ischemic encephalopathy (HIE) are desirable. Topiramate has been effective in reducing brain damage in experimental studies. However, in the clinical setting information is limited to a small number of feasibility trials. We launched a randomized controlled double-blinded topiramate/placebo multicenter trial with the primary objective being to reduce the antiepileptic activity in cooled neonates with HIE and assess if brain damage would be reduced as a consequence. STUDY DESIGN: Neonates were randomly assigned to topiramate or placebo at the initiation of hypothermia. Topiramate was administered via a nasogastric tube. Brain electric activity was continuously monitored. Topiramate pharmacokinetics, energy-related and Krebs' cycle intermediates, and lipid peroxidation biomarkers were determined using liquid chromatography-mass spectrometry and MRI for assessing brain damage. RESULTS: Out of 180 eligible patients 110 were randomized, 57 (51.8%) to topiramate and 53 (48.2%) to placebo. No differences in the perinatal or postnatal variables were found. The topiramate group exhibited less seizure burden in the first 24 h of hypothermia (topiramate, n = 14 [25.9%] vs. placebo, n = 22 [42%]); needed less additional medication, and had lower mortality (topiramate, n = 5 [9.2%] vs. placebo, n = 10 [19.2%]); however, these results did not achieve statistical significance. Topiramate achieved a therapeutic range in 37.5 and 75.5% of the patients at 24 and 48 h, respectively. A significant association between serum topiramate levels and seizure activity (p < 0.016) was established. No differences for oxidative stress, energy-related metabolites, or MRI were found. CONCLUSIONS: Topiramate reduced seizures in patients achieving therapeutic levels in the first hours after treatment initiation; however, they represented only a part of the study population. Our results warrant further studies with higher loading and maintenance dosing of topiramate.

The Oxygen Load Supplied during Delivery Room Stabilization of Preterm Infants Modifies the DNA Methylation Profile.

OBJECTIVES: To determine whether the amount of oxygen provided during postnatal stabilization changes the DNA methylome in preterm infants. STUDY DESIGN: This prospective, observational study included 32 preterm infants ≤32 weeks of gestation who received oxygen in the delivery room. Patients were monitored using a respiratory function monitor to determine the amount of oxygen received upon stabilization. Blood samples were processed for comparison of DNA methylation before and after resuscitation using a DNA methylation high-resolution microarray Infinium Human DNA methylation EPIC 850K BeadChip. RESULTS: The median amount oxygen provided to preterm infants during stabilization was 644 mLO2/kg. Male sex and vaginal delivery were associated with increased oxygen needs. There were 2626 differentially methylated CpGs representing 1567 genes that showed an association with oxygen load selected and, of these, 85% were hypomethylated. We found that oxygen loads of >500 mLO2/kg changed the methylation pattern of the selected CpGs. Genes associated with these CpGs were "enriched" in KEGG pathways involved in cell cycle progression, DNA repair, and oxidative stress. CONCLUSIONS: The oxygen load provided upon resuscitation modified the DNA methylome. Differential methylation may lead to altered expression of genes related to cell cycle progression, oxidative stress, and DNA repair. The reversibility of these early epigenetic changes is unknown but merits further study.

Preterm Gut Microbiome Depending on Feeding Type: Significance of Donor Human Milk.

Preterm microbial colonization is affected by gestational age, antibiotic treatment, type of birth, but also by type of feeding. Breast milk has been acknowledged as the gold standard for human nutrition. In preterm infants breast milk has been associated with improved growth and cognitive development and a reduced risk of necrotizing enterocolitis and late onset sepsis. In the absence of their mother's own milk (MOM), pasteurized donor human milk (DHM) could be the best available alternative due to its similarity to the former. However, little is known about the effect of DHM upon preterm microbiota and potential biological implications. Our objective was to determine the impact of DHM upon preterm gut microbiota admitted in a referral neonatal intensive care unit (NICU). A prospective observational cohort study in NICU of 69 neonates <32 weeks of gestation and with a birth weight ≤1,500 g was conducted. Neonates were classified in three groups according to feeding practices consisting in their MOM, DHM, or formula. Fecal samples were collected when full enteral feeding (defined as ≥150 cc/kg/day) was achieved. Gut microbiota composition was analyzed by 16S rRNA gene sequencing. Despite the higher variability, no differences in microbial diversity and richness were found, although feeding type significantly influenced the preterm microbiota composition and predictive functional profiles. Preterm infants fed MOM showed a significant greater presence of Bifidobacteriaceae and lower of Staphylococcaceae, Clostridiaceae, and Pasteurellaceae compared to preterm fed DHM. Formula fed microbial profile was different to those observed in preterm fed MOM. Remarkably, preterm infants fed DHM showed closer microbial profiles to preterm fed their MOM. Inferred metagenomic analyses showed higher presence of Bifidobacterium genus in mother's milk group was related to enrichment in the Glycan biosynthesis and metabolism pathway that was not identified in the DHM or in the formula fed groups. In conclusion, DHM favors an intestinal microbiome more similar to MOM than formula despite the differences between MOM and DHM. This may have potential beneficial long-term effects on intestinal functionality, immune system, and metabolic activities.

Influence of Sex on Gestational Complications, Fetal-to-Neonatal Transition, and Postnatal Adaptation.

Fetal sex is associated with striking differences during in utero development, fetal-to-neonatal transition, and postnatal morbidity and mortality. Male sex fetuses are apparently protected while in utero resulting in a higher secondary sex rate for males than for females. However, during fetal-to-neonatal transition and thereafter in the newborn period, female exhibits a greater degree of maturation that translates into a better capacity to stabilize, less incidence of prematurity and prematurity-associated morbidities, and better long-term outcomes. The present review addresses the influence of sex during gestation and postnatal adaptation that includes the establishment of an adult-type circulation, the initiation of breathing, endurance when confronted with perinatal hypoxia ischemia, and a gender-related different response to drugs. The intrinsic mechanisms explaining these differences in the perinatal period remain elusive and further experimental and clinical research are therefore stringently needed if an individual oriented therapy is to be developed.

Oxygen needs during resuscitation and surfactant to achieve stabilisation were independent risks factors for pulmonary interstitial emphysema in preterm infants.

AIM: Pulmonary interstitial emphysema is a severe complication of mechanical ventilation in preterm infants that leads to air leakage and, or, chronic lung disease. We determined the associated risk factors. METHODS: This was a retrospective case-control study from 2005 to 2014 at a regional referral centre in Valencia, Spain. The cases were 54 preterm infants up to 30 weeks' gestation and, or, born weighing less than 1500 g, who were diagnosed with pulmonary interstitial emphysema (PIE). The 54 controls were preterm infants without PIE matched by gestational age. Univariate analysis and multivariate analysis were performed to assess the independent predicting factors. RESULTS: Infants with PIE had been resuscitated with higher mean fractional inspired oxygen concentration (FiO2 ) (p = 0.008), had received higher peak mean positive end expiratory pressure (p = 0.00) and higher mean airway pressure (p = 0.026) 24 hours before diagnosis. PIE patients also received more surfactant (p = 0.00) and had higher mortality (p = 0.034). A Cox regression model identified that independent risk factors were the total amount of surfactant administered and the mean FiO2 during the 24 hours before diagnosis. CONCLUSION: Independent risk factors for pulmonary interstitial emphysema in preterm infants were higher oxygen during resuscitation and a higher need for surfactant and ventilatory pressures before diagnosis.

Congenital Critical Heart Defect Screening in a Health Area of the Community of Valencia (Spain): A Prospective Observational Study.

Despite the progress in the fetal echocardiographic detection of congenital critical heart defects and neonatal physical examination, a significant number of newborn infants are discharged and readmitted to the hospital in severe condition due to cardiac failure or collapse. The aim of this study was to assess the incidence of undetected critical congenital heart disease (CCHD) by a pulse oximetry-screening program in the maternity wards of hospitals with Perinatal Services in a specific geographic area. This is a prospective observational study performed in in the health area corresponding to the city of Valencia. Eligible infants were consecutively admitted newborn infants in the maternities of the participating hospitals with negative fetal echocardiography after normal physical examination in the delivery room. All patients were screened following a specific pulse oximetry protocol before discharge. A total of 8856 newborn infants were screened. A total of three babies presented with severe congenital cardiac malformation and two babies presented with early onset sepsis. Sensitivity was 100% and specificity was 99.97%, with a positive predictive value of 60% and negative predictive value of 100%. Pulse oximetry screening programs in the early neonatal period constitute a valuable tool to avoid inadvertent hospital discharge of severe cardiac malformations and the subsequent life-threatening complications derived.

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 ß-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 ß-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.

Abdominal Near-Infrared Spectroscopy Detects Low Mesenteric Perfusion Early in Preterm Infants with Hemodynamic Significant Ductus Arteriosus.

BACKGROUND: The therapeutic decision to close patent ductus arteriosus in preterm infants entails great uncertainty. Near-infrared spectroscopy is a noninvasive bedside technique used to monitor mixed regional oxygen saturation. We hypothesized that near-infrared spectroscopy could identify preterm infants at risk of mesenteric hypoperfusion associated with hemodynamically significant ductus arteriosus. METHODS: This is a prospective observational study including consecutively admitted preterm infants with a gestational age <32 weeks. Mesenteric regional oxygenation was blindly monitored using an INVOS 5100 device. The presence of a hemodynamically significant patent ductus arteriosus was routinely confirmed by echocardiography/Doppler. Statistical analysis including Bland-Altman plots was performed to assess near-infrared spectroscopy intraobserver repeatability. RESULTS: A total of 72 preterm infants were enrolled. The daily mean regional oxygen saturation for preterm infants was determined both in mesenteric and cerebral regions and plotted against time. We identified a differential temporary baseline. Hemodynamic significant ductus arteriosus was associated with lower blood pressures and lower regional splanchnic oxygenation. There was a significant relationship between reversal diastolic flow in the descending aorta and the regional oxygen saturation, which remained significant after controlling for ductal size and nil per os. CONCLUSIONS: The simultaneous monitoring of splanchnic near-infrared spectroscopy and echocardiography could identify low mesenteric perfusion in the presence of hemodynamic ductus arteriosus.

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.

Novel biomarkers in amniotic fluid for early assessment of intraamniotic infection.

Intra-amniotic infection/inflammation (IAI) is associated with preterm birth, short and long-term adverse clinical outcomes and oxidative stress. The diagnosis of IAI is based on histological and clinical findings; however, often these results are unspecific. Therefore, efforts have been directed towards validating reliable methods for patients lacking overt clinical symptoms. In this study, amniotic fluid (AF) samples were prospectively collected from 23 women grouped into two categories (with or without IAI) following clinical, microbiological and histological criteria. AFs were analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the determination of the following biomarkers: oxidized and nitrated tyrosines (Tyr), 8-hydroxy-2'-deoxyguanosine (8OHdG), oxidized glutathione (GSSG) and glutathione sulfonamide (GSA). 3-NO2-Tyrosine (3NO2-Tyr) and GSSG concentrations in AF were not identified as significantly relevant biomarkers in the presence of IAI. However, inflammatory biomarkers such as GSA (p=0.002) and 3-Chloro-Tyrosine [3Cl-Tyr (p=0.049)], and oxidative stress biomarker 8OHdG (p=0.021) were significantly increased in AF with IAI as compared to normal controls. Biomarkers of inflammation and oxidative stress determined in AF samples could represent a new approach towards an early diagnosis of IAI and subsequent chorioamnionitis in the clinical setting.

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.