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Do levels of lipid peroxidation biomarkers reflect the degree of brain injury in newborns?

The pathogenesis and progression of Hypoxic-Ischemic Encephalopathy (HIE), a major cause of severe neurological disability and mortality in the perinatal period, is shaped by the interplay of multiple processes including inflammation, oxidative stress, and excitotoxicity. We conducted a longitudinal study determining biomarkers of oxidative stress and inflammation in non-invasive urine samples of newborns with moderate/severe HIE (N=51) employing Liquid Chromatography - Mass Spectrometry. We noted that levels of several biomarkers of oxidative stress increased over time demonstrating the ongoing propagation of oxidative injury. Prostaglandins, in contrast, showed a decreasing trend in their concentration profiles over time, which probably reflects their mediation on pathogenic mechanisms, including the inflammatory response. Statistically significant differences in the levels oxidative stress of neonates with distinct brain lesion patterns, as detected with Magnetic Resonance Imaging (MRI), were observed, revealing an increase of lipid peroxidation biomarkers in newborns with cerebral lesions (MRI score 1) as compared to scores 0 and 2. Moreover, a gender-dependent study showed no statistically significant differences in biomarker concentrations between male and female infants. Our observation leads to the hypothesis that the monitoring of non-invasive lipid peroxidation biomarkers could aid diagnosis and predicting of long-term outcomes as complementary tool to standard explorations.


Introduction
Approximately 10% of newborns require assistance to initiate breathing at birth, and around 2% require resuscitation with clinical and/or biochemical signs of perinatal asphyxia with ca. 20% of these infants developing hypoxic ischemic encephalopathy (HIE), a major cause of severe neurological disability and perinatal mortality. Strong evidence indicates that the pathogenesis and progress ion of HIE is shaped by the interplay of multiple processes including inflammation, oxidative stress, and excitotoxicity which finally lead to brain injury. After a hypoxic-ischemic insult, brain cells initially undergo impaired oxidative metabolism, followed by reperfusion injury that leads to the death of neuronal cells. Neuronal death is triggered by a cascade of events which include reactive oxygen species (ROS). The generation of ROS is involved in physiological reactions during the fetalto-neonatal transition, and it has been demonstrated that ROS generation up-regulates the expression of specific genes whose end-products are beneficial for postnatal adaptation. However, triggered by hypoxia and ischemia, ROS production rapidly increases and overwhelms antioxidant defenses and ultimately lead to the development of specific conditions. Hence, the beneficial and toxic effects of O 2 administration for newborn therapy need to balance to prevent cellular and tissue damage. Both, hypoxia and reoxygenation, generate excessive amounts of ROS and subsequently, oxidative stress.
Several studies illustrate molecular and metabolomics changes associated with asphyxia, duration of hypoxia, and resuscitation with different oxygen concentrations. At high concentrations, ROS modify or degenerate a range of macromolecules, such as lipids, proteins, and polysaccharides and can damage DNA and RNA and lead to a cascading inflammatory response. The neonatal brain with a high concentration of polyunsaturated fatty acids (PUFAs), high rate of oxygen consumption, low concentration of antioxidants, and high levels of iron is especially vulnerable to excessive amounts of ROS. Inflammation is considered as an important contributor in the pathophysiology of cerebral HI injury. The initial inflammatory response to HI produces a secondary neuronal injury followed by an anti-inflammatory response toward resolution of inflammation.
The state-of-the-art reference method for the early characterization of structural damage of the grey matter (i.e., the basal ganglia, the thalamus, and the cortex) and to a lesser extent infarction in white matter, is magnetic resonance imaging (MRI) acquired within [5][6][7] This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. days after birth. The prognostic ability of MRI obtained in the neonatal period to determine neurodevelopmental outcomes in childhood has been demonstrated (8).
Currently, literature providing evidence of the relationship between oxidative stress biomarkers and the degree of brain damage is scant and based on the comparison of HIE versus healthy control infants and determinations in blood samples. In preterm infants, a reference range for lipid peroxidation byproducts in urine samples was established and preterm babies who later developed bronchopulmonary dysplasia (BPD), a disease associated to oxidative stress, showed significantly higher urinary elimination of isofurans in analytical determinations performed during the first days after birth (5).
Access to an early and accurate, biomarker-guided diagnosis of neonatal HIE could be a powerful ally to reduce neonatal mortality, morbidity, and degree of disability, through a personalized adjustment of treatment options. In the present study, we monitored urinary oxidative biomarkers levels in a cohort of term newborns with moderate or severe HIE before, during, and after TH. We compared oxidative stress levels between neonates with different degrees of brain injury vs. normal MRI outcomes following a standardized scoring system (8) and carried out a gender-dependent study of the detected oxidative stress biomarkers.

Results
A sub-cohort of 51 newborns from the HYPOTOP trial recruiting infants with moderate or severe HIE qualifying for TH were included in this study. Relevant clinical and demographic characteristics are summarized in Table 1. A total of 22 patients (43%) had normal (score 0) and 29 (57%) pathologic (scores 1, 2 or 3) MRI outcomes. There was no statistically significant difference among both groups (i.e., normal and pathologic MRI outcomes) regarding gestational age (GA), gender, and biometric characteristics (Table 1). From this population, 24 (47%) patients were males and 27 (53%) females and there was no statistically significant difference between recorded clinical parameters between genders.
Serial collection of urine samples enabled the longitudinal assessment of the evolution of urinary oxidative stress biomarkers before and during cooling and upon rewarming. Out of measured lipid peroxidation compounds, five resolved isoprostanoids and six total This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
intensities of relative UPLC-MS/MS signals characteristic of lipid peroxidation products (i.e., total parameters) were detected (i.e., levels above the Limit of Quantification, LOQ) in 50% of samples. Two out of five measured prostaglandins (PGs) were detected, in addition to one biomarker of oxidative damage to DNA and one out of four biomarkers of oxidative damage to proteins. Median values and IQR of each determined parameter, as well as the detection frequency are shown in Table 2. No significant trend in the levels of 5(RS)-5-F 2t -IsoP, 15(RS)-15-F 2t -IsoP, 2,3-dinor-15-F 2t -IsoP, 4(RS)-4-F 4t -NeuroP as well as total IsoPs, IsoFs, NeuroPs, NeuroFs and dihomo-IsoFs was observed during the study period.
Variable Importance in Projection (VIP) scores vs. regression vector ( Figure 2B, bottom) were used to measure the influence of each metabolite on the PLSDA models. Several determined biomarkers showed characteristic patterns in samples from newborns belonging to the MRI score 1 group, i.e., levels of 14(RS)-14-F 4t -NeuroP, total IsoPs, 15(RS)-This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Finally, levels of oxidative stress biomarkers in male vs. female infants were assessed in the search of sex-specific associations. However, no statistically significant differences were found (data not shown).

Discussion
This is the first study to examine the relationship between a large panel of oxidative stress and inflammatory biomarkers and brain injury in infants with moderate/severe HIE. Our data demonstrate that specific indicators for oxidative stress mediated damage and inflammation can be longitudinally monitored in newborns with HIE, thanks to the use of non-invasively collected urine samples. The results ( Figure 2A) reflect an increase of a specific NeuroP isomer (i.e., 14(RS)-14-F 4t -NeuroP) as well as total dihomo-IsoPs over the study period revealing protracted oxidative stress that persisted during TH and the rewarming period. NeuroPs are lipid mediators generated by non-enzymatic lipid peroxidation of docosahexaenoic acid (DHA), a polyunsaturated fatty acid highly abundant in brain tissue and have been suggested as markers of oxidative damage to brain tissue, urine, plasma, or cerebrospinal fluid (9). Dihomo-IsoPs, derived from radical attack on adrenic acid (AdA), are considered biomarkers of cerebral white matter injury. Likewise, increasing levels of biomarkers of damage to proteins (mTyr/Phe) and DNA (8OhdG/2dG) were observed. The spontaneous oxidation of Phe in presence of hydroxyl free radicals produces m-Tyr and o-Tyr that are stable molecules, and their concentrations are useful markers of oxidative protein damage. Protein oxidation has been associated with neuronal dysfunction in vivo and may be involved in the pathology of neurodegenerative disorders (1). 8OHdG, produced by the oxidation of the nucleotide 2dG, is excreted in urine and is commonly used as biomarker of oxidative damage to DNA (7). Oxidative stress is strongly related to acute brain damage in children, and 8-OHdG is a useful marker of brain damage Simultaneously we studied inflammatory biomarkers in HIE. Decreasing levels of biomarkers of PGs (i.e., 6-keto-PGF 1α and PGF 2α ) were observed. PGs are produced by the enzymatic peroxidation of AA, and they play a key role in the inflammatory response.
Inflammation is a response to injury, and it is an intrinsically beneficial event that leads to This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. removal of offending factors and restoration of tissue structure and physiological function.
PGs, particularly prostaglandins E and F 2 , lead to cerebral vasodilation and their concentrations increase in response to cerebral ischemia (4). More specifically, 6-keto-PGF 1α , a stable metabolite of prostacyclin also known as prostaglandin I 2 (PGI 2 ), is a vasodilative and is associated with brain perfusion and cerebral infarction. It is probably the most important PG due to its effect on sensory neurons (2). The decrease of PGs with time observed in the present study might reflect a progressive return to normal levels after the hypoxic insult that occurred before/during delivery.
Access to biomarkers that early and accurately diagnose oxidative stress and response could potentially aid the diagnosis and improve prognosis of newborns with HIE. In this regard, we compared between infants with pathologic and normal MRI outcomes by using standardized scores discerning between injury patterns. Our data provides evidence of oxidative stress biomarker changes in urine samples of newborns with HIE depending on their lesion type. Specifically, biomarker patterns in urine samples from newborns with MRI score 1 were significantly different from scores 0 and 2, while no difference between the latter sub-groups were found. Infants belonging to the sub-groups with MRI score 0 and 2 presented lower values of DHA-derived NeuroPs (14(RS)-14-F 4t -NeuroP), AA-derived IsoPs (total IsoPs and 15(RS)-15-F 2t -IsoP), and AdA-derived dihomo-IsoPs (total dihomo-IsoPs) in comparison to infants with MRI score 1. Thus, lipid peroxidation biomarker profiles linked to oxidative damage of different PUFAs, which are all highly abundant in the brain, change according to specific injury patterns (i.e., MRI score 1).
Previous studies established male sex as a risk factor for unfavorable outcome of HIE during the perinatal period regardless of lesion types. Some authors found notable differences between microglia and neuroimmune signaling in the male and female brain and suggested that these differences play a relevant role . However, in this study we did not observe consistent and significant alterations between male and female infants associated to oxidative stress levels.
In summary, this is the first study presenting detailed insights on the evolution of a broad panel of oxidative stress and inflammation biomarkers in newborns infants with moderate/severe HIE undergoing TH. We identified a set of biomarkers with constantly increasing concentrations that demonstrate the propagation of oxidative injury through the whole study period including the secondary phase of HIE. PGs, in contrast, showed a decreasing tendency which reflects inflammatory response. We found statistically significant differences in newborns with different patterns of pathologic vs. normal HIE outcomes, but not between male and female infants.

Innovation
Oxidative stress and inflammation are known to be deeply involved in the pathogenesis of cerebral lesions in newborns after a hypoxic-ischemic insult. However, the determination of specific related biomarkers for diagnosis and prediction of long-term outcomes as a complementary tool to standard evaluations is not yet exploited in newborn critical care.
Our findings demonstrate the feasibility and potential usefulness of a continuous monitoring of biomarkers in non-invasive biofluids from newborn infants with HIE undergoing therapeutic hypothermia as oxidative stress and inflammation biomarkers may provide clinically relevant additional information on the patient's status and progression.

Notes
Study approval and population Receiver operating characteristic (ROC) curves were based on PLSDA models. Significance of models was tested by permutation testing (500 permutations). Group comparisons of demographic and clinical characteristics were carried out by using the Student's t-test for unequal variances, Chi square test or Wilcoxon ranksum test for parametric, categorical and non-parametric variables, respectively. P-values<0.05 were considered as statistically significant. This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.       This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.