Metabolomic analyses of plasma reveals new insights into asphyxia and resuscitation in pigs

Abstract

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; R(2).adj = 0.58, q-value<0.001) and of glycine to BCAA (Gly/BCAA; R(2).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 R(2)adjust = 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% O(2) 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.

Figure 1. Experimental study design.

After one hour of stabilization, 27 piglets were randomized to hypoxia and reoxygenation (HR). Hypoxemia (start of asphyxia, SA) was achieved by ventilation with a gas mixture of 8% O₂ in N₂ until either the mean arterial blood pressure decreased to 20 mmHg or the base excess (BE) reached −20 mM (end of asphyxia, EA). Before the start of resuscitation, the hypoxic piglets were block-randomized for resuscitation with 21% or 100% oxygen for 15 min and then ventilation with room air for 45 min (Groups 1 (n = 8) and 2 (n = 8)) or for receiving 100% oxygen for 60 min (Group 3 (n = 11)). Control animals were handled as the other groups without exposure to hypoxia or hyperoxia. Blood samples for metabolomic analyses were taken at the start of hypoxia (SA), the end of hypoxia (EA) and the end of reoxygenation (ER).

Figure 2. Distribution of the metabolites classes.

The distribution of the corrected p value according to the metabolites classes from the two linear models describing alterations between control and treated animals during asphyxia (left boxplot) and changes in the plasma metabolome during reoxygenation (right boxplot). The number of significant changes at q-value<0.01 is appearing below each boxplot, alongside with the total number of metabolites for a given class in brackets. A detailed description of each group of metabolites is given in Table S1. Figure 3b) Concentration changes of metabolites for treated animals during asphyxia (EA/SA, blue) and during reoxygenation (ER/EA, orange). Levels of significance are as follows: *, q<10-2, **, q<10-4 and *** q<10-6.

Figure 3. Visualization of individual metabolite concentration ratios.

The heat-map is a graphical representation of the true metabolite concentration changes in a two-dimensional, rectangular and colored grid. Metabolites are given on the y axis (i.e., row), animal profiles are given on the x axis (i.e., columns) and each “pixel” represents a metabolite change (in log basis 2 scale) between two consecutive time points. With regard to the design, each animal (as labeled by G/g) is thus represented twice: one cell for each change corresponding to the hypoxia (i.e., EA/SA) and to the resuscitation (i.e., ER/EA) steps. For a more comprehensive visualization, metabolite changes are centered around a common value, and both rows and columns are reordered so that meaningful characteristics of the data can be uncovered without any a priori knowledge about the experimental design. First, concentration changes for each metabolite (row) are centered around the average change found in the sham animals (i.e., control animals labeled by g). Following the left corner diagram, red (resp. blue)-colored cells indicate a higher (resp. a lower) change in concentration than the average change observed for the control animals. Cells for which changes are greater than 3 (c.a. 2∧3 = 8 fold change) and lower than −3 (c.a. 1/8) are coded in the darkest red and blue. Column reordering proceeds by placing observations (i.e., EA/SA or ER/EA from one animal) with the most similar profiles, using hierarchical clustering agglomeration in which the leaves represent individual observations and in which the height of the nodes reflects the dissimilarity between the two clusters of observation. Animal labels are given at the bottom, whereas a square color-coded according to its treatment group is at the top of the heat-map. Three clusters, corresponding to the profiles from the asphyxiated animals (left) followed by the sham animals (center) and profiles associated to resuscitation (right), are clearly observed. However, unsupervised clustering cannot efficiently group animals according to their reoxygenation protocol (right). Finally, metabolites (i.e., rows) are grouped according to their intensity patterns, with their partitions displayed on the left side. The upper part of the heat-map comprises metabolites that are increased during asphyxia and decreased during resuscitation, whereas the lower part regroups compounds with the opposite behavior.

Figure 4. Correlation of metabolite concentration with the duration of hypoxia.

The time-course of plasma lactate levels (in mM) of all individual animals before, during and after asphyxia in a normalized discrete timescale (animals are sampled at 15/30 minutes intervals during asphyxia and resuscitation/reoxygenation) (a). Lactate concentration (mM) does not correlate with the duration of hypoxia (in minutes) (b). The fitted values of the PLS models built on the full set (+) or reduced (*) set of metabolites and ratios of metabolites are plotted against the actual duration of asphyxia (in minutes) Combinations of metabolites by PLS modeling provided a better representation of the duration of asphyxia (c). n = 8–11. The 30 most contributing features are sorted according to the variable importance on the projection (VIP) scores; VIP are given as median and 20/80 quantiles from 30 resampling steps (d).

Figure 5. Concentration ratios of metabolites due to resuscitation protocols.

An explicit summary of concentration ratios for the set of metabolites found significant at a q value<0.01 due to resuscitation protocols (n = 8–11). Bars correspond to the paired changes during reoxygenation (to the left–decline; to the right–increase). Bars in: green: resuscitation with 21%O₂; blue: 100% O₂ for 15 min and 45 min of 21% O₂; and red: prolonged hyperoxia, 100% for 60 minutes. Levels of significance are coded as follows: *, q<10⁻², **, q<10⁻⁴.

Figure 6. Visualizations into biochemical pathways.

Summary and visualizations into biochemical pathways: a) Effect of asphyxia on Krebs cycle intermediates; Fumarate, succinate and α-ketoglutarate are strongly increased after asphyxia, as well as lactate and alanine. These increases can be seen as the result of mitochondrial dysfunction and the subsequent disturbance of the Krebs cycle, since the Krebs cycle and mitochondrial function is coupled. b) Effect of different resuscitation protocols on these intermediates. Reoxygenation with 100% oxygen for either duration (g2/3) resulted in a significant delay in the decrease in fumarate, succinate and α-ketoglutarate compared to normoxic resuscitation (g1). The significantly lower levels of these Krebs cycle intermediates in the 21% FiO₂ resuscitation group indicates better mitochondrial recovery.