David Enot

Targeted Metabolomics for Biomarker Discovery

Metabolomics is a truly interdisciplinary field of science, which combines analytical chemistry, platform technology, mass spectrometry, and NMR spectroscopy with sophisticated data analysis. Applied to biomarker discovery, it includes aspects of pathobiochemistry, systems biology/medicine, and molecular diagnostics and requires bioinformatics and multivariate statistics. While successfully established in the screening of inborn errors in neonates, metabolomics is now widely used in the characterization and diagnostic research of an ever increasing number of diseases. In this Review we highlight important technical prerequisites as well as recent developments in metabolomics and metabolomics data analysis with special emphasis on their utility in biomarker identification and qualification, as well as targeted metabolomics by employing high-throughput mass spectrometry.

Metabolomic Analyses of Plasma Reveals New Insights into Asphyxia and Resuscitation in Pigs

Background Currently, a limited range of biochemical tests for hypoxia are in clinical use. Early diagnostic and functional biomarkers that mirror cellular metabolism and recovery during resuscitation are lacking. We hypothesized that the quantification of metabolites after hypoxia and resuscitation would enable the detection of markers of hypoxia as well as markers enabling the monitoring and evaluation of resuscitation strategies. Methods and Findings Hypoxemia of different durations was induced in newborn piglets before randomization for resuscitation with 21% or 100% oxygen for 15 min or prolonged hyperoxia. Metabolites were measured in plasma taken before and after hypoxia as well as after resuscitation. Lactate, pH and base deficit did not correlate with the duration of hypoxia. In contrast to these, we detected the ratios of alanine to branched chained amino acids (Ala/BCAA; R2.adj = 0.58, q-value<0.001) and of glycine to BCAA (Gly/BCAA; R2.adj = 0.45, q-value<0.005), which were highly correlated with the duration of hypoxia. Combinations of metabolites and ratios increased the correlation to R2adjust = 0.92. Reoxygenation with 100% oxygen delayed cellular metabolic recovery. Reoxygenation with different concentrations of oxygen reduced lactate levels to a similar extent. In contrast, metabolites of the Krebs cycle (which is directly linked to mitochondrial function) including alpha keto-glutarate, succinate and fumarate were significantly reduced at different rates depending on the resuscitation, showing a delay in recovery in the 100% reoxygenation groups. Additional metabolites showing different responses to reoxygenation include oxysterols and acylcarnitines (n = 8–11, q<0.001). Conclusions This study provides a novel strategy and set of biomarkers. It provides biochemical in vivo data that resuscitation with 100% oxygen delays cellular recovery. In addition, the oxysterol increase raises concerns about the safety of 100% O2 resuscitation. Our biomarkers can be used in a broad clinical setting for evaluation or the prediction of damage in conditions associated with low tissue oxygenation in both infancy and adulthood. These findings have to be validated in human trials.

Effect of propofol in the immature rat brain on short- and long-term neurodevelopmental outcome.

Background Propofol is commonly used as sedative in newborns and children. Recent experimental studies led to contradictory results, revealing neurodegenerative or neuroprotective properties of propofol on the developing brain. We investigated neurodevelopmental short- and long-term effects of neonatal propofol treatment. Methods 6-day-old Wistar rats (P6), randomised in two groups, received repeated intraperitoneal injections (0, 90, 180 min) of 30 mg/kg propofol or normal saline and sacrificed 6, 12 and 24 hrs following the first injection. Cortical and thalamic areas were analysed by Western blot and quantitative real-time PCR (qRT-PCR) for expression of apoptotic and neurotrophin-dependent signalling pathways. Long-term effects were assessed by Open-field and Novel-Object-Recognition at P30 and P120. Results Western blot analyses revealed a transient increase of activated caspase-3 in cortical, and a reduction of active mitogen-activated protein kinases (ERK1/2, AKT) in cortical and thalamic areas. qRT-PCR analyses showed a down-regulation of neurotrophic factors (BDNF, NGF, NT-3) in cortical and thalamic regions. Minor impairment in locomotive activity was observed in propofol treated adolescent animals at P30. Memory or anxiety were not impaired at any time point. Conclusion Exposing the neonatal rat brain to propofol induces acute neurotrophic imbalance and neuroapoptosis in a region- and time-specific manner and minor behavioural changes in adolescent animals.

Complexity and pitfalls of mass spectrometry-based targeted metabolomics in brain research.

Current quantitative metabolomic research in brain tissue is challenged by several analytical issues. To compare data of metabolite pattern, ratios of individual metabolite concentrations and composed classifiers characterizing a distinct state, standardized workup conditions, and extraction medium are crucial. Differences in physicochemical properties of individual compounds and compound classes such as polarity determine extraction yields and, thus, ratios of compounds with varying properties. Also, variations in suppressive effects related to coextracted matrix components affect standards or references and their concentration-dependent responses.The selection of a common tissue extraction protocol is an ill-posed problem because it can be regarded as a multiple objective decision depending on factors such as sample handling practicability, measurement precision, control of matrix effects, and relevance of the chemical assay. This study systematically evaluates the impact of extraction solvents and the impact of the complex brain tissue on measured metabolite levels, taking into account ionization efficiency as well as challenges encountered in the trace-level quantification of the analytes in brain matrices. In comparison with previous studies that relied on nontargeted platforms, consequently emphasizing the global behavior of the metabolomic fingerprint, here we focus on several series of metabolites spanning over extensive polarity, concentration, and molecular mass ranges.

Hepatic induction of cholesterol biosynthesis reflects a remote adaptive response to pneumococcal pneumonia

Community‐acquired pneumonia presents a spectrum of clinical phenotypes, from lobar pneumonia to septic shock, while mechanisms underlying progression are incompletely understood. In a transcriptomic and metabolomic study across tissues, we examined serotype‐specific regulation of signaling and metabolic pathways in C57BL/6 mice intratracheally instilled with either serotype 19F Streptococcus pneumoniae (S19; causing lobar pneumonia), or serotype 2 S. pneumoniae (S2; causing septic pneumococcal disease,) or vehicle (Todd‐Hewitt broth). Samples of lung, liver, and blood were collected at 6 and 24 h postinfection and subjected to microarray analysis and mass spectrometry. Results comprise a preferential induction of cholesterol biosynthesis in lobar pneumonia at low‐infection doses (105 colony forming units/mouse) leading to increased plasma cholesterol (vehicle: 1.8 ±0.12 mM, S2: 2.3±0.10 mM, S19: 2.9±0.15 mM; P>0.05, comparing S19 to vehicle and S2). This induction was pneumolysin dependent, as a pneumolysin‐deficient strain of serotype 19F failed to induce cholesterol biosynthesis (S19ΔPLY: 1.9±0.03 mM). Preincubation of pneumolysin with purified cholesterol or plasma from hypercholesterolemic mice prior to intratracheal instillation protected against lung barrier dysfunction and alveolar macrophage necrosis. Cholesterol may attenuate disease severity by neutralizing pneumolysin in the alveolar compartment and thus prevent septic disease progression.—Weber, M., Lambeck, S., Ding, N., Henken, S., Kohl, M., Deigner, H. P., Enot, D. P., Igwe, E. I., Frappart, L., Kiehntopf, M., Claus, R. A., Kamradt, T., Weih, D., Vodovotz, Y., Briles, D. E., Ogunniyi, A. D., Paton, J. C., Maus, U. A., Bauer, M. Hepatic induction of cholesterol biosynthesis reflects a remote adaptive response to pneumococcal pneumonia. FASEB J. 26, 2424‐2436 (2012). www.fasebj.org

Metabolite profiles reveal energy failure and impaired beta-oxidation in liver of mice with complex III deficiency due to a BCS1L mutation.

Background & Aims Liver is a target organ in many mitochondrial disorders, especially if the complex III assembly factor BCS1L is mutated. To reveal disease mechanism due to such mutations, we have produced a transgenic mouse model with c.232A>G mutation in Bcs1l, the causative mutation for GRACILE syndrome. The homozygous mice develop mitochondrial hepatopathy with steatosis and fibrosis after weaning. Our aim was to assess cellular mechanisms for disease onset and progression using metabolomics. Methods With mass spectrometry we analyzed metabolite patterns in liver samples obtained from homozygotes and littermate controls of three ages. As oxidative stress might be a mechanism for mitochondrial hepatopathy, we also assessed H2O2 production and expression of antioxidants. Results Homozygotes had a similar metabolic profile at 14 days of age as controls, with the exception of slightly decreased AMP. At 24 days, when hepatocytes display first histopathological signs, increases in succinate, fumarate and AMP were found associated with impaired glucose turnover and beta-oxidation. At end stage disease after 30 days, these changes were pronounced with decreased carbohydrates, high levels of acylcarnitines and amino acids, and elevated biogenic amines, especially putrescine. Signs of oxidative stress were present in end-stage disease. Conclusions The findings suggest an early Krebs cycle defect with increases of its intermediates, which might play a role in disease onset. During disease progression, carbohydrate and fatty acid metabolism deteriorate leading to a starvation-like condition. The mouse model is valuable for further investigations on mechanisms in mitochondrial hepatopathy and for interventions.

Inflammatory-induced hibernation in the fetus: priming of fetal sheep metabolism correlates with developmental brain injury.

Prenatal inflammation is considered an important factor contributing to preterm birth and neonatal mortality and morbidity. The impact of prenatal inflammation on fetal bioenergetic status and the correlation of specific metabolites to inflammatory-induced developmental brain injury are unknown. We used a global metabolomics approach to examine plasma metabolites differentially regulated by intrauterine inflammation. Preterm-equivalent sheep fetuses were randomized to i.v. bolus infusion of either saline-vehicle or LPS. Blood samples were collected at baseline 2 h, 6 h and daily up to 10 days for metabolite quantification. Animals were killed at 10 days after LPS injection, and brain injury was assessed by histopathology. We detected both acute and delayed effects of LPS on fetal metabolism, with a long-term down-regulation of fetal energy metabolism. Within the first 3 days after LPS, 121 metabolites were up-regulated or down-regulated. A transient phase (4–6 days), in which metabolite levels recovered to baseline, was followed by a second phase marked by an opposing down-regulation of energy metabolites, increased pO2 and increased markers of inflammation and ADMA. The characteristics of the metabolite response to LPS in these two phases, defined as 2 h to 2 days and at 6–9 days, respectively, were strongly correlated with white and grey matter volumes at 10 days recovery. Based on these results we propose a novel concept of inflammatory-induced hibernation of the fetus. Inflammatory priming of fetal metabolism correlated with measures of brain injury, suggesting potential for future biomarker research and the identification of therapeutic targets.

Bioinformatics for mass spectrometry-based metabolomics.

The broad view of the state of biological systems cannot be complete without the added value of integrating proteomic and genomic data with metabolite measurement. By definition, metabolomics aims at quantifying not less than the totality of small molecules present in a biofluid, tissue, organism, or any material beyond living systems. To cope with the complexity of the task, mass spectrometry (MS) is the most promising analytical environment to fulfill increasing appetite for more accurate and larger view of the metabolome while providing sufficient data generation throughput. Bioinformatics and associated disciplines naturally play a central role in bridging the gap between fast evolving technology and domain experts. Here, we describe the strategies to translate crude MS information into features characteristics of metabolites, and resources available to guide scientists along the metabolomics pipeline. A particular emphasis is put on pragmatic solutions to interpret the outcome of metabolomics experiments at the level of signal processing, statistical treatment, and biochemical understanding.

Systematic characterization of amplitude-integrated EEG signals for monitoring the preterm brain

Background:In preterm infants, the amplitude-integrated electroencephalogram (aEEG) is not established in clinical routine. The aim of this study was to derive normative data on aEEG parameters by means of longitudinal characterization and to evaluate the impact of gestational age (GA), postnatal age (PNA), postmenstrual age, sedation, and patent ductus arteriosus (PDA).Methods:Recordings from 61 infants with GA 28–31 weeks were obtained during the first 72 h, then weekly until the age of 4 wk. Infants were divided into three groups: (i) no sedation, no PDA, (ii) sedation, no PDA, and (iii) sedation, PDA. Assessed parameters included background activity, cycling, amplitude, and log ratio of the maximum/minimum amplitude.Results:GA and PNA had a significant impact within 72 h. Sedation modified aEEG, and presence of PDA was associated with reduced aEEG scores within 72 h. The log ratio of the amplitude correlated with GA but was unaffected by sedation and PDA.Conclusion:Evaluation of electrocortical background activity within the first postnatal hours and longitudinally over days and weeks is important to better understand the postnatal factors impacting cerebral function in preterm infants. There is a need to agree on definitions and a standardized reporting system in order to permit comparisons between studies and establish aEEG as a method for routine monitoring of preterm infants.

Biomarkers of Developmental Brain Injury in Preterm Infants: Reporting on Metabolomics Activities of the Neobrain Consortium

Background and aims: A novel component in the Neobrain consortium was the large scale quantification of metabolites and their implication/use for translational research. Here we report on the 1) adequacy of the metabolomics technology2) characterisation of metabolic responses to several types of injury in plasma3) identification of metabolic markers of brain injury in animal models and humans.Methods: Study comprises animal models of hypoxia-ischemia, excitotoxic brain injury and intrauterine LPS exposure in fetal sheep and a human cohort of 41 preterm infants (28-32 weeks of gestation). Metabolite panel covering 230 compounds (acylcarnitines, lipids, prostanoids, amino acids and derivatives, oxidised products of cholesterol, small organic acids,sugars) was applied in plasma. In the sheep model and infants, extend of brain damage has been assessed by MRI, aEEG and histopathology in animals.Results: Insults causing brain injury induced significant changes in the plasma metabolome in all animal models. In the sheep model we detected a significant correlation of metabolites with outcome at several time points. In the preterm infants ADMA/SDMA, specific acyl carnitines, lysoPCs and amino acids were significantly increased in infants with abnormal MRI at term, overlapping with metabolites detected in the animal models.Conclusions: Hypothesized at the start at the project, metabolic changes associated with perinatal brain injury is demonstrated in several animal models and translated in humans. Metabolite profiling can effectively be applied to pediatric research to complete our fragmented knowledge. It enables identification of patterns of dysregulation (biomarker discovery) together with the elucidation of their underlying mechanism (biological plausibility).