Evolution of Energy Related Metabolites in Plasma from Newborns with Hypoxic-Ischemic Encephalopathy during Hypothermia Treatment

Abstract

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 β-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 β-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.

Figure 1

Chromatograms acquired during the injection of a plasma sample (blue line) and a spiked plasma sample (red line). Note: for lactate the 1:10 diluted sample is depicted.

Figure 2

Boxplots of metabolite concentrations in plasma samples from newborns included in the HYPOTOP trial. Note: acetoacetate, succinate, malate and α-ketoglutarate detected in 77, 95, 92 and 60% and other analytes in 100% of samples, concentrations < LLOQ were set to 1/2xLLOQ; p-values calculated employing the two-tailed Wilcoxon rank sum test for equal medians.

Figure 3

Relative changes of metabolites as a function of age of newborns with HIE enrolled in the HYPOTOP trial. Note: median values as a relative measure to median values at t0; letter size proportional to concentration levels; arrows indicate tendencies.

Figure 4

Flow diagram of the HYPOTOP trial.