REVIEW ARTICLE
Hippokratia 2017, 21(3 ):115-123
Efstathiou N1, Theodoridis G2, Sarafidis K1
11st Department of Neonatology, School of Medicine, 2School of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece
Abstract
Background: Hypoxic-ischemic encephalopathy (HIE), a serious complication of perinatal asphyxia, is commonly associated with an unfavorable outcome. In-depth research is important not only for the interpretation of the underlying biological alternations but may also provide the basis for the development of novel diagnostic and therapeutic tools. The application of metabolomics in perinatal asphyxia/HIE is a relatively new approach.
Methods: We performed a narrative, non-systematic review in the literature of metabolomic studies involving newborn animals and humans exposed to hypoxia-ischemia or developing perinatal asphyxia/HIE.
Results: Fifteen animal studies, nine studies in human neonates, and two review articles were evaluated. Changes in the metabolomic profile of newborn animals exposed to hypoxia-ischemia and of asphyxiated neonates with HIE are presented in relation to the underlying pathophysiology. The clinical relevance of these findings is further discussed in a comprehensible to the bedside clinician manner.
Conclusions: Metabolomics may provide an explanation for the various metabolic alternations occurring in perinatal asphyxia/HIE, elucidate the biological background of the applied therapeutic interventions and promote the development of novel diagnostic-prognostic biomarkers of the disease. HIPPOKRATIA 2017, 21(3): 115-123.
Key words: Neonatal care, perinatal asphyxia, metabolic pathways, therapeutic hypothermia, biomarker
Corresponding author: Kosmas Sarafidis, Associate Professor in Neonatology, 1st Department of Neonatology, Aristotle University of Thessaloniki, Hippokrateion General Hospital, 49 Kostantinoupoleos Str, 54642, Thessaloniki, Greece, tel: +302310892426, fax: +302310992787, e-mail: saraf@med.auth.gr
Introduction
Hypoxic-ischemic encephalopathy
Hypoxic-ischemic encephalopathy (HIE) following perinatal asphyxia remains an issue of great clinical importance. The majority of the nearly one million deaths that occur every year consequent to perinatal asphyxia worldwide happen in low- and middle-resource countries1. Nevertheless, 1-5 term neonates per 1,000 live births will develop HIE even in countries with advanced medical systems2,3. Moreover, despite progress in perinatal and neonatal care, depending on the severity of the brain injury, a significant number of the affected neonates will die or suffer neurological sequels4,5.
HIE is mainly caused by energy failure of the brain due to an acute perinatal event. However, experimental and clinical observations have demonstrated that the development of HIE is rather an evolving process. The initial hypoxic and/or ischemic insult switches metabolism to anaerobic energy production, and with prolonged oxygen deprivation “primary energy failure” is established characterized by depletion of high-energy metabolites [adenosine triphosphate (ATP)]. This leads to depolarization of the mitochondrial membranes, intracellular calcium accumulation, cytotoxic edema, extracellular accumulation of excitatory amino acids, and finally death of some neuronal cells6,7. After resuscitation, oxidative metabolism returns gradually (in 30-60 min) to baseline whereas many neurons recover, at least partially. However, after a latent period of around six hours, high-energy metabolites decrease again, owing to the deterioration of the mitochondrial function, and give rise to the so-called “secondary energy failure”. Oxidative stress, accumulation of toxic metabolites and delayed apoptosis are additional features of this phase. There is a “tertiary phase” of repair and reorganization that may last for months, but the precise mechanisms involved in this phase are not clear6,7.
Therapeutic hypothermia (TH) initiating during the first six hours is nowadays considered the standard of care for the management of asphyxiated neonates with moderate-severe HIE, as it significantly alters prognosis, improving survival and neurodevelopmental outcome4. On the other hand, TH is not a panacea and has endogenous limitations, as, for example, its applicability only during the first six hours after birth7. This fact renders the need for more research mandatory in order to elucidate underlying biological mechanisms of perinatal asphyxia/HIE. Potentially, this research could aid in the development of future pharmaceutical interventions for neuroprotection and also of reliable disease biomarkers. Metabolomics, the younger “-omics” discipline, could be the scientific answer to these issues.
Metabolomics
Metabolomics involves the development and application of unbiased/global analysis of biological samples (e.g., animal/human biofluids, tissue or cell extracts) with the aim to discover biomarkers related to a certain condition such as disease, stress or environmental factors. Analytical methods utilized in metabolomics research aim at the characterization of the entire metabolic content of the samples under study and then at the relation of their concentration patterns to properties of the samples8. Therefore, metabolomics involves multidisciplinary research driven by scientists with different expertise, for example, life and medical sciences, analytical chemistry, statistics, biochemistry, nutrition, and agricultural or environmental sciences.
Holistic methods apply a hypothesis-free approach and aim at the identification and determination of non-anticipated markers. In this approach, data leads the analysis. The markers found should subsequently be validated and linked within biochemical pathways. Metabolomics approaches can be combined with data from other -omics fields such as genomics, proteomics, and transcriptomics in order to reach a systems biology perspective to integrate different systems to provide descriptive and predictive models.
Thus far, biomarker discovery for disease and drug efficacy represent the major fields of metabolomics research and development9. In particular, the search for early biomarkers of disease (diagnostic markers)10, biomarkers of drug efficacy (pharmacometabonomics)11, biomarkers of disease progression (prognostic markers) or drug toxicity (safety assessment)12 have attracted major efforts from the research community.
With regard to instrumental methods, holistic analysis necessitates the use of information-rich spectroscopy techniques such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS)13,14. Liquid phase separations, in particular, High-Performance Liquid Chromatography (HPLC) and Ultra High-Performance Liquid Chromatography [U(H)PLC], are the key analytical platforms. These are used in the pharmaceutical industry for the determination of small molecules in an extensive range of applications. When combined with mass spectrometry (MS), they provide the essential technology in the quest for wide metabolome coverage for biomarker discovery13,14. Due to the versatility offered, HPLC/U(H)PLC is the most powerful tool for the analysis of a multitude of molecules from different groups, molecules that have different molecular properties and exist in the same sample in different concentration ranges. As a result, LC-MS dominates the field of metabolomics analysis.
Methods
Given the dramatic consequences of perinatal asphyxia/HIE and the potential of metabolomics in the exploration of diseases and biomarker discovery, this review aims to make metabolomics comprehensible to the bedside clinician presenting the main results of the pertinent metabolomic studies regarding pathophysiology and current clinical practice. Therefore, we searched the Medline (PubMed) for metabolomic articles in English related to perinatal asphyxia/HIE, hypoxia-ischemia, and neonatal resuscitation published from 2000 to 2017. The following keywords were used in advanced search: (metabolom*) AND (neonat* OR perinatal OR newborn) AND (asphy* OR hypox* OR ischem* OR ischaem* OR anoxia OR encephalopathy OR brain injury). Moreover, our search was expanded to most recent neonatal resuscitation guidelines and therapeutic hypothermia (TH) in neonates with HIE to support our statements.
We proceeded in discussing metabolic derangements and their biological interpretation in perinatal asphyxia/HIE, and translate metabolomic investigations to clinical practice focusing on oxygen concentration during neonatal resuscitation, therapeutic hypothermia, as well as outcome prognosis. Finally, we investigated the role of metabolomics in the discovery of novel HIE biomarkers. To answer these topics, a narrative but comprehensive review is presented.
Results
From the 102 articles retrieved, only a number of them, either experimental or human (summarized in Table 1 and Table 2, respectively), were specifically referred to metabolomics in relation to perinatal asphyxia/HIE, neonatal hypoxia-ischemia, and resuscitation protocols. We also retrieved two review articles explicitly focusing on metabolomics and asphyxia/HIE15,16 and the most recent guideline article on neonatal resuscitation from the International Liaison Committee on Resuscitation (ILCOR)17. Duplicate and irrelevant articles were not included. However, evaluations of methodological quality were not used to exclude studies from this review. The flow chart of the recovered and analyzed studies from the PubMed is shown in Figure 1.
Figure 1: Flow chart of the studies retrieved from Medline (PubMed) that were finally analyzed.
Discussion
A. Metabolomics in perinatal asphyxia-HIE: possible clinical relevance
Metabolomics, as a holistic approach, has significant advantages over the traditional measurement of individual metabolites, allowing the simultaneous evaluation of various metabolic pathways in large-scale events such as hypoxia-ischemia following perinatal asphyxia. Metabolomics may not only markedly improve our understanding of the pathophysiological-biochemical alternations occurring in perinatal asphyxia-HIE but also provide evidence for important clinical questions.
Metabolic derangements and biological interpretation
Recent metabolomic studies in animal models and neonates have confirmed the involvement of known pathways and revealed the contribution of other heretofore unknown pathways in the development of perinatal asphyxia/HIE.
As expected, alternations in energy metabolites that indicate a shift towards anaerobic metabolism (increased lactate) and disturbance of the Kreb’s cycle were the most prominent findings of the relevant metabolomic studies. Due to energy depletion (ATP)18 and dysfunction of the respiratory chain, lactate is formed and accumulated19-21, as are the Kreb’s cycle intermediates (citrate, alpha keto-glutarate, succinate, and fumarate)19,22-26. As suggested by data from infants with severe HIE, increased succinate levels may play a particular role in the development of encephalopathy after asphyxia, possibly through the hypoxia-inducible factor-1α24. Previous animal studies have already documented increasedlactate levels immediately after oxygen deprivation27. Moreover, lactate measured within one hour after birth has been used as an early predictor of HIE development after intrapartum asphyxia28. It is noteworthy that even 24-48 hours after resuscitation, neonates suffering asphyxia seem to have their own metabolic fingerprint compared to controls, suggesting ongoing metabolic alternations21, seemingly due to delayed recovery of the Kreb’s cycle19.
Urine NMR analysis in term asphyxiated newborns also showed reduced acetate21, a precursor of acetyl-coenzyme A, which has a central role in the metabolism of carbohydrates and fats by entering the Kreb’s cycle. Interestingly, results from animal studies imply that hypothermia achieves its neuroprotective effects, through the coordinated suppression of acetyl-CoA content, which in turn down-regulates the production of acetylcholine in specific regions of the brain29.
Increased glucose was one of the significant changes observed in the urine metabolic fingerprint of asphyxiated neonates20,21. Nevertheless, circulating (and probably urine) glucose levels may not reflect those in the brain. As a matter of fact, rats exposed to hypoxia were found to have elevated plasma glucose in spite of a profound decrease in brain glucose. Increased brain demands for glucose exceeding the supply from the periphery was speculated to explain this paradox27. Previous experimental data had shown that glucose supplementation reduces perinatal hypoxic-ischemic brain injury30. Results of a retrospective study in term infants with HIE, none of which received TH, indicated that early hypoglycemia occurring during the first six hours of life was associated with an adverse neurodevelopmental outcome at 24 months of age, irrespective of the grade of HIE. Nevertheless, this unfavorable outcome was not observed in hypoglycemia occurring after the sixth hour of life as well as in hyperglycemia31. Currently, although it is generally considered that normoglycemia should be maintained in asphyxiated neonates, no specific glucose targets are recommended for the post-resuscitation care of the neonates17.
Hypoxanthineis a purine derivative and for many years has been considered as a hallmark indicator of asphyxia32. When metabolized by xanthine oxidase, it generates oxygen radicals that are highly destructive to the tissues, including the brain. In recent metabolomic studies involving animal models of severe neonatal asphyxia or hypoxia, plasma hypoxanthineconcentrations were significantly increased33-35. In the latter investigations, levels of other products of purines’ catabolism (e.g., inosine, uric acid, etc.) and pyrimidine were also found to be increased33. Allopurinol, a xanthine-oxidase inhibitor, could potentially reduce the formation of these superoxides and, thus, brain damage in HIE. However, the available data are not sufficient to determine whether allopurinol has clinically important benefits for newborn infants with HIE36. A European Union-funded project (the ALBINO trial) will further evaluate the efficacy and safety of allopurinol administered immediately after birth to near-term and term infants with HIE in addition to TH37.
Metabolomic studies suggest that lipids play a crucial role in perinatal asphyxia-HIE. According to experimental and clinical studies, choline is one of the most significantly increased analytes during postnatal hypoxia/aspyxia24,33-34. Choline and its metabolites are very important for the structural integrity and signaling of cell membranes (phospholipids), neurotransmission (acetylcholine synthesis), lipid transport (lipoproteins), and methyl-group metabolism (homocysteine reduction)38. On the contrary, as shown in animal studies, arachidonic acid is characteristically elevated in asphyxia-HIE, possibly reflecting cell disruption22,39. This fatty acid is a component of the cell membrane found abundantly in the brain and is a precursor of biologically important metabolites such as prostacyclins, thromboxanes, and leukotrienes. Moreover, activation of cytosolic phospholipases following hypoxia-ischemia increases eicosanoid release and brings about inflammation, which represents a major mechanism of brain injury in HIE7. Carnitine, the transporter of the fatty acids across the inner mitochondrial membrane for the β-oxidation, is also altered in asphyxia. Plasma metabolomic analysis in studies involving asphyxia-resuscitation or hypoxia in newborn pigs revealed lower free carnitine and higher long-chain acylcarnitines after hypoxia (owing to the hypoxia-induced incomplete β-oxidation)19,33. In a recent study, we were also able to confirm elevated urine acylcarnitines levels in the HIE compared to controls26. Other derangements in metabolites include ketone bodies24 and amino acids19,23, both of which may serve as alternative energy sources. Moreover, ketogenesis was proposed as playing an important role in preventing neurological injury during perinatal asphyxia as acetone and 3-hydroxybutyrate were found increased two-fold in asphyxiated infants and decreased by two-fold in severe HIE24.
Inositolis a precursor for phosphorylated compounds that are involved in signal transduction40 widely distributed in human tissues and cells. Its most widely occurring stereoisomer, myo-inositol, has been documented to increase in the blood following perinatal asphyxia or HIE in animals22 and humans24,26. A similar elevation of myo-inositol has been observed in the cerebrospinal fluid of fetal sheep suffering from hypoxia. In this case, brain injury was attributed to osmolytic cell changes causing cell edema41.
Finally, perturbations of the amino acids seem also to be the case in hypoxia and HIE. Nevertheless, in some animal19 and humans studies42,43, a significant increase in the levels of several amino acids was noted, whereas others in humans showed elevated or reduced amino acids26,44. Among the latter metabolites, glutamine merits specific consideration, as is a crucial link between carbon metabolism of carbohydrates and proteins, and also plays an essential role in the growth of several cells. Most importantly, glutamine is related to glutamate45, the major excitatory neurotransmitter in perinatal brain injury46. Previous studies documented significantly elevated glutamine levels in the plasma and brain of newborn animals following hypoxia33,47.
B. Translation of experimental data to clinical practice
Oxygen concentration and neonatal resuscitation
A clinically important point in neonatology is how to optimize resuscitation to prevent morbidity and mortality. Cardinal clinical studies have shown that neonates resuscitated with ambient air (21 % oxygen) had a faster recovery as indicated by the significantly higher heart rate at 90 seconds after birth and Apgar score at five minutes compared to those resuscitated with 100 % oxygen48. Ensuing metabolomic investigations in asphyxiated piglets resuscitated with 21 % versus 100 % oxygen provided a biological explanation of the findings of the aforementioned clinical studies, documenting not only metabolic variations with different oxygen concentrations49 but also an earlier recovery of the mitochondrial function (decline of the Krebs cycle intermediates) with 21 % oxygen19. As derived from another metabolomic study in animals, resuscitation with oxygen at 21 % seems to be associated with optimal cellular function and maintenance whereas lower (18 %) and higher oxygen concentrations with carbohydrate metabolism (increased glucose and lactate) and free radical scavenging, respectively20. Progress in analytical techniques allowed the identification of novel lipid peroxidation biomarkers (isoprostanoids) related to hypoxia and reoxygenation in neonates with HIE using very small volumes of plasma50 that is clinically challenging. Altogether, these data corroborate recent resuscitation guidelines in term neonates, in which initiation of resuscitation with room air is suggested17. Interestingly, an animal study evaluating six different neonatal cardiopulmonary resuscitation protocols with the parallel use of plasma and urine metabolomics, although confirmed the presence of severe metabolic alterations in hypoxia-induced cardiac arrest up to four hours after recovery of the circulation, yetprovided no evidence for a differential metabolic response to the various resuscitation protocols or in terms of survival34.
Therapeutic hypothermia
The theoretical background of TH involves the protection of the nervous system by lowering body temperature and, thus, the metabolic rate of the brain. With a series of pioneer studies, Liu et al using the NMR analysis of rodent brain tissue documented the important role of metabolomics for the evaluation of different hypothermia strategies on the outcome18,25,51. In any case, though, early recognition of neonates with HIE is crucial as the efficacy of TH is time-dependent. HIE-affected, full-term neonates could be discriminated from non-affected ones on the basis of umbilical cord serum metabolome. Nevertheless, only separation of the severe HIE cases could be accurately made in the latter studies24,42. Clinical parameters such as the combination of umbilical arterial lactate and neonatal resuscitation level were reported to accurately identify at birth neonates that may benefit from neuroprotective therapies as TH, that is, those with moderate-severe HIE52. Major cooling trials4 and subsequent guidelines6,7,17 excluded babies with mild encephalopathy from receiving therapeutic hypothermia, as prognosis in these cases is generally considered “good”. The issue, however, is precisely the neonates with mild HIE. These infants may subsequently develop moderate-severe HIE that has escaped initial diagnosis with the existing clinical tools (e.g., the Thompson Score)53 and, in general, require a high level of expertise. Additionally, emerging evidence indicates that mild HIE may not be as safe as assumed. As it is proven in animal studies, cerebrospinal fluid levels of lactic acid, alanine, phenylalanine, lysine, tyrosine, branched chain amino acids, and hypoxanthine were increased even in cases with mild hypoxia compared to control41. These findings are in agreement with recent clinical data reporting a larger than expected proportion of infants with mild encephalopathy and abnormal outcomes54. It is reasonable, then, that the existing clinical practice not to cool neonates with mild encephalopathy is considered a matter of controversy55. Recent metabolomic studies indicate potential biomarkers that, if measured at birth, could distinguish neonates recovering from perinatal asphyxia from those developing HIE, helping thus to direct treatment43.
Prognosis of outcome
In one of the earliest works in the field of metabolomics, using Gas Chromatography-MS (GC-MS), Chu et al showed that the urine metabolic profile of the asphyxiated neonates with a good neurodevelopmental outcome is different from that of the neonates who develop HIE or die. In Chu’s study, eight organic acids involved in distinct biochemical pathways were significantly associated with neurodevelopmental handicap having high sensitivity and specificity56. Similarly, preclinical studies documented an association between specific metabolites (arachidonic acid, butanoic acid, citric acid, fumaric acid, lactate, malate, propanoic acid, and succinic acid) and early or long-term neurodevelopmental outcome39. Moreover, in cooled asphyxiated neonates, the urine metabolic profiles of those who died after seven days of life were closely comparable to each other and significantly different from those of survivors57. Overall, metabolomic studies performed so far on neonatal asphyxia/HIE, although limited in number, support the possibility of developing an HIE biomarker in the future using metabolomics.
C. Metabolomics and novel HIE biomarkers
A unique metabolomic “fingerprint” might be used in the future for the development of diagnostic and prognostic biomarkers, thus individualizing treatment and allowing more accurate prediction of important clinical outcomes. Currently, our urgent need to develop HIE biomarkers is mainly based on the following scientific gaps: a) our inability to differentiate mild HIE from perinatal depression without encephalopathy or to identify those neonates who are at risk of progressing to moderate-severe HIE; b) our inability to predict response, for instance to TH, whenever it is applied, and possibly add or apply other neuroprotective interventions; and c) the need to prognosticate short- and long-term outcomes related to the severity of the disease, coexisting perinatal conditions and management.
So far, only a few metabolites have been proved to be robust enough, diagnostically, in neonatal asphyxia/HIE. Most likely, a panel of metabolites will offer higher diagnostic accuracy than a single one24,42. It is to be hoped that an easy-to-apply test will be available for clinical practice soon, contributing significantly to the improvement in neonatal care. A 1H-NMR-derived metabolomic index based on early umbilical cord blood alterations of succinate, glycerol, 3-hydroxybutyrate, and O-phosphocholine has shown potential for the prediction of HIE severity. Although the latter metabolite index outperformed other standard biochemical markers at birth for prediction of neurodevelopmental outcome at three years, it was not found to be superior to EEG or the Sarnat score58.
Conclusions
Metabolomics is a valuable tool for the exploration of the multiple biochemical alternations observed in HIE. A better understanding of the disease, potentially, may allow the discovery of new neuroprotective interventions as well as of novel diagnostic-prognostic biomarkers, thus individualizing the management of the affected neonates. So far, relevant research is limited. Large-scale studies are needed to prove the utility of metabolomics in perinatal asphyxia/HIE, ultimately clarifying important clinical dilemmas and questions. Nevertheless, as highlighted in a previous review article by Deniham et al, issues related to the highly unpredictable nature of HIE, and thus patient recruitment, renders metabolomic research in neonatal HIE a difficult task16.
Conflict of interest
Authors declare no conflict of interest.
Acknowledgments
We acknowledge that this review was supported by the “IKY Fellowships of Excellence for postgraduate studies in Greece -SIEMENS program”.
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