Untargeted metabolomic analysis and pathway discovery in perinatal asphyxia and hypoxic-ischaemic encephalopathy

Abstract

Elucidating metabolic effects of hypoxic-ischaemic encephalopathy (HIE) may reveal early biomarkers of injury and new treatment targets. This study uses untargeted metabolomics to examine early metabolic alterations in a carefully defined neonatal population. Infants with perinatal asphyxia who were resuscitated at birth and recovered (PA group), those who developed HIE (HIE group) and healthy controls were all recruited at birth. Metabolomic analysis of cord blood was performed using direct infusion FT-ICR mass spectrometry. For each reproducibly detected metabolic feature, mean fold differences were calculated HIE vs. controls (ΔHIE) and PA vs. controls (ΔPA). Putative metabolite annotations were assigned and pathway analysis was performed. Twenty-nine putatively annotated metabolic features were significantly different in ΔPA after false discovery correction ( q < 0.05), with eight of these also significantly altered in ΔHIE. Altered putative metabolites included; melatonin, leucine, kynurenine and 3-hydroxydodecanoic acid which differentiated between infant groups (ΔPA and ΔHIE); and D-erythrose-phosphate, acetone, 3-oxotetradecanoic acid and methylglutarylcarnitine which differentiated across severity grades of HIE. Pathway analysis revealed ΔHIE was associated with a 50% and 75% perturbation of tryptophan and pyrimidine metabolism, respectively. We have identified perturbed metabolic pathways and potential biomarkers specific to PA and HIE, which measured at birth, may help direct treatment.

Trial registration: ClinicalTrials.gov NCT02019147.

Keywords: Metabolomics; biomarker; hypoxic-ischaemic encephalopathy; metabolic pathway; perinatal asphyxia.

Figures

Figure 1.

Summary of the number of mass features detected and significantly altered in nESI(+) and nESI(−), respectively. Red Venn diagrams show the distribution of putatively annotated metabolic features, of those significantly altered for each comparison groups; perinatal asphyxia vs. matched controls (ΔPA) and HIE vs.matched controls (ΔHIE).

Figure 2.

Metabolite log fold changes stratified by disease severity. Fold changes compare the difference in log transformed data in individual cases compared to their matched healthy controls in each disease group; perinatal asphyxia, mild HIE, moderate HIE and severe HIE.

Figure 3.

Pathway analysis. Altered metabolomic pathways and networks of UCB serum as a result of HIE (ΔHIE in red), PA (ΔPA in green) and those altered in both comparisons (ΔHIE and ΔPA in blue). The y-axis displays the pathway name alongside the total number of empirical formulae from that pathway detected in the analysis. The x-axis displays the % perturbation for each pathway. For example, the TCA cycle was 100% perturbed meaning the three empirical formulae were detected were all altered as a result of PA and HIE.

Figure 4.

Alterations in tryptophan metabolism. The structure is shown for the compounds identified by FT-ICR-MS. Arrows indicate an increases (↑) or decreases (↓) in metabolite fold change; ΔHIE (red) and ΔPA (blue), while metabolite IDs in bold indicate the significantly altered compounds with fold change (95% CI) included.