Increased mortality of acute respiratory distress syndrome was associated with high levels of plasma phenylalanine

Abstract

Background: There is a dearth of drug therapies available for the treatment of acute respiratory distress syndrome (ARDS). Certain metabolites play a key role in ARDS and could serve as potential targets for developing therapies against this respiratory disorder. The present study was designed to determine such "functional metabolites" in ARDS using metabolomics and in vivo experiments in a mouse model.

Methods: Metabolomic profiles of blood plasma from 42 ARDS patients and 28 healthy controls were captured using Ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) assay. Univariate and multivariate statistical analysis were performed on metabolomic profiles from blood plasma of ARDS patients and healthy controls to screen for "functional metabolites", which were determined by variable importance in projection (VIP) scores and P value. Pathway analysis of all the metabolites was performed. The mouse model of ARDS was established to investigate the role of "functional metabolites" in the lung injury and mortality caused by the respiratory disorder.

Results: The metabolomic profiles of patients with ARDS were significantly different from healthy controls, difference was also observed between metabolomic profiles of the non-survivors and the survivors among the ARDS patient pool. Levels of Phenylalanine, D-Phenylalanine and Phenylacetylglutamine were significantly increased in non-survivors compared to the survivors of ARDS. Phenylalanine metabolism was the most notably altered pathway between the non-survivors and survivors of ARDS patients. In vivo animal experiments demonstrated that high levels of Phenylalanine might be associated with the severer lung injury and increased mortality of ARDS.

Conclusion: Increased mortality of acute respiratory distress syndrome was associated with high levels of plasma Phenylalanine.

Trial registration: Chinese Clinical Trial Registry, ChiCTR1800015930. Registered 29 April 2018, http://www.chictr.org.cn/edit.aspx?pid=25609&htm=4.

Keywords: Acute respiratory distress syndrome; Metabolites; Metabolomics; Phenylacetylglutamine; Phenylalanine; Phenylalanine metabolism.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Multivariate data analysis of the metabolites. a Principal Component Analysis (PCA) scores plots (left panels) and Partial Least Square-Discriminant Analysis (PLS-DA) scores plots (right panels) of ARDS patients vs healthy controls. Shaded areas are the 95% confidence regions of each group. b PCA and PLS-DA scores plots of the survivors vs the non-survivors of ARDS. Shaded areas are the 95% confidence regions of each group. c The cross validation test for PLS-DA model of the ARDS patients vs the healthy controls. d The cross validation test for PLS-DA model of the survivors vs non-survivors of ARDS. Deep blue represents the cross-validated R2 (also known as Q2),pink represent the sum of squares captured by the model (R2), and light blue represents the prediction accuracy. Red star indicates the best component number in building the model. In PLS-DA of the ARDS patients vs the healthy controls analysis, R2 = 0.93 and Q2 = 0.86, the survivors vs the non-survivors of ARDS analysis, R2 = 0.89 and Q2 = 0.49, respectively

Fig. 2

VIP score plot and Heatmap of metabolites. a Variable Importance in Projection (VIP) score plot of the metabolites that differed in ARDS patients vs healthy controls. b Variable Importance in Projection (VIP) score plot of the metabolites that differed in survivors vs the non-survivors of ARDS. c The heatmap showing abundance of the top 30 metabolites based on VIP scores of ARDS patients vs healthy controls. d The heatmap showing abundance of the top 30 metabolites based on VIP scores of the survivors vs the non-survivors of ARDS

Fig. 3

Screening of differentially expressed metabolites as potential mortality predictors for ARDS. a The differentially expressed metabolites in both the comparison groups, the pink circle representing the group of ARDS vs healthy controls, and the blue circle representing the group of survivors vs non-survivors. The metabolites in the cross area were those identified in both comparison groups. b The box whisker plot of Phenylalanine in the survivors (n = 28) and the non-survivors (n = 15) of ARDS and healthy controls (n = 42). c The box whisker plot of D-Phenylalanine in the survivors (n = 28) and the non-survivors (n = 15) of ARDS and healthy controls (n = 42). d The box whisker plot of phenylacetylglutamine in the survivors (n = 28) and the non-survivors (n = 15) of ARDS and healthy controls (n = 42). e ROC curve of Phenylalanine (AUC = 0.803), D-Phenylalanine (AUC = 0.785) and phenylacetylglutamine (AUC = 0.709) in predicting the mortality of ARDS. In the combined model the area under the curve (AUC) was 0.882. The data was normalized. 1-Lino = 1-Linoleoylglycerophosphocholine; 1,2,3,4-Tera = 1,2,3,4-Tetrahydro-beta-carboline-1,3-dicarboxylic acid; 2-Amino = 2-Aminoacetophenone

Fig. 4

Pathway analysis reveals the Phenylalanine pathway to be one of the most altered pathways in ARDS patients. a Pathway analysis uncovered the altered pathways in ARDS patients vs healthy controls. b Pathway analysis uncovered the altered pathways in the survivors vs non-survivors of ARDS. All matched pathways were shown according to P values from pathway enrichment analysis (y-axis) and all pathway impact values were according to pathway topology analysis (x-axis). The color and size of each circle are based on P values and pathway impact values, respectively. The deeper the red of the nod, the more significant alteration of the pathway is observed. Small P value and big pathway impact circles indicate that the pathway is greatly influenced. c The pathways that are altered between ARDS and healthy controls (pink circle) or between survivors and non-survivors (blue circle). The pathways in the cross area were identified in both groups. d Schematic diagram of metabolic pathway networks. The metabolites involved in the selected pathways (P < 0.05,impact factor > 0) altered between the survivors and the non-survivors were marked in different colors. Light blue means those metabolites are not in my data and are used as background for enrichment analysis; red (P < 0.05) and yellow (P > 0.05) means the metabolites are upregulated in the survivors deep green (P < 0.05) and light green (P > 0.05) mean that the metabolites are downregulated in survivors with different levels of significance. The metabolites in the box belong to the same pathway

Fig. 5

Phenylalanine administration increased the lung injury and mortality of ARDS. a The protocol of Phenylalanine administration and ARDS model establishment b The survival rate of ARDS mice (n = 27/group) and Sham mice (n = 5/group) treated with Phenylalanine or PBS. c The levels of Phenylalanine and tyrosine in BALF of sham mice treated with Phenylalanine or PBS (n = 4–5/group). d The protein concentration in bronchoalveolar lavage fluid (BALF) (n = 3–5 /group). e The white cell counts in BALF (n = 3–5 /group). f The hematoxylin and eosin staining of lung tissue (n = 3–5 /group) Phe = Phenylalanine. Each value represents the mean ± SEM of one of the three independent experiments. Kaplan Meier Survival analysis and comparisons were performed by log-rank test