Plasma Metabolomics Implicates Modified Transfer RNAs and Altered Bioenergetics in the Outcomes of Pulmonary Arterial Hypertension

Abstract

Background: Pulmonary arterial hypertension (PAH) is a heterogeneous disorder with high mortality.

Methods: We conducted a comprehensive study of plasma metabolites using ultraperformance liquid chromatography mass spectrometry to identify patients at high risk of early death, to identify patients who respond well to treatment, and to provide novel molecular insights into disease pathogenesis.

Results: Fifty-three circulating metabolites distinguished well-phenotyped patients with idiopathic or heritable PAH (n=365) from healthy control subjects (n=121) after correction for multiple testing (P<7.3e-5) and confounding factors, including drug therapy, and renal and hepatic impairment. A subset of 20 of 53 metabolites also discriminated patients with PAH from disease control subjects (symptomatic patients without pulmonary hypertension, n=139). Sixty-two metabolites were prognostic in PAH, with 36 of 62 independent of established prognostic markers. Increased levels of tRNA-specific modified nucleosides (N2,N2-dimethylguanosine, N1-methylinosine), tricarboxylic acid cycle intermediates (malate, fumarate), glutamate, fatty acid acylcarnitines, tryptophan, and polyamine metabolites and decreased levels of steroids, sphingomyelins, and phosphatidylcholines distinguished patients from control subjects. The largest differences correlated with increased risk of death, and correction of several metabolites over time was associated with a better outcome. Patients who responded to calcium channel blocker therapy had metabolic profiles similar to those of healthy control subjects.

Conclusions: Metabolic profiles in PAH are strongly related to survival and should be considered part of the deep phenotypic characterization of this disease. Our results support the investigation of targeted therapeutic strategies that seek to address the alterations in translational regulation and energy metabolism that characterize these patients.

Keywords: hypertension, pulmonary; metabolism; metabolome; metabolomics; pulmonary circulation.

Figure 1.

Analysis flow chart. Summary of analytic workflow showing numbers of metabolites that distinguish patients with pulmonary arterial hypertension (PAH) from control subjects and/or are prognostic in PAH. NT-proBNP indicates N-terminal brain natriuretic peptide; and RDW, red cell distribution width.

Figure 2.

Metabolites that discriminate patients with pulmonary arterial hypertension pulmonary arterial hypertension (PAH) from control subjects. A, Average metabolite levels in PAH and control subjects for 20 metabolites found to significantly distinguish patients with PAH from both healthy and disease control subjects independently of potential confounders. Values plotted are z scores calculated from the mean and standard deviation of all healthy volunteers in the study. Negative values indicate metabolites at lower levels in patients vs healthy control subjects, and positive values indicate higher levels of metabolites in patients. For the discovery analysis, only data from patients with PAH who were 19 to 70 years of age are plotted; for the validation analysis, all patients data are shown. B, Network analysis of the same 20 metabolites on the basis of second-order correlations. Line thickness indicates strength of correlations (all P<0.0001). *Probable metabolite identity but unconfirmed (see Methods). DHE indicates docosahexaenoyl; DHEA-S, dehydroisoandrosterone sulfate; DPE, docosapentaenoyl; EPE, eicosapentaenoyl; GPC, glycerophosphocholine; and SM, sphingomyelin.

Figure 3.

Discriminant analysis models on the basis of low numbers of metabolites distinguish patients with pulmonary arterial hypertension (PAH) from control subjects. A and C, Dot plots showing individual subjects’ model scores for healthy control subjects (HC), patients with PAH, vasoresponders, and disease control subjects (DC) in discovery and validation analyses. Metabolites were selected by logistic regression of PAH-HC (A) and PAH-DC (C) comparisons. B and D, Receiver-operating characteristic curves showing the performance of models in distinguishing PAH from HC (B) and DC (D) subjects. AUC indicates area under the curve; and CI, confidence interval.

Figure 4.

Prognostic metabolites independent of established risk factors. A, Hazard ratios after correction for creatinine and diuretic use of 36 metabolites that were prognostic in patients with pulmonary arterial hypertension independently of red cell distribution width, N-terminal brain natriuretic peptide, and 6-minute walk distance. Hazard ratios indicate the risk of a change in each metabolite of 1 SD for ease of comparison. Patients of all ages were included in both the discovery and validation survival analyses. B, Network analysis of the same 36 metabolites on the basis of second-order correlations. Line thickness indicates strength of correlations (all P<0.0001). Red lines indicate negative correlations. *Probable metabolite identity but unconfirmed (see Methods). DHE indicates docosahexaenoyl; DHEA-S, dehydroisoandrosterone sulfate; DPE, docosapentaenoyl; EPE, eicosapentaenoyl; GPC, glycerophosphocholine; GPE, glycerophosphoethanolamine; Met, Cys, SAM and Taur, methionine, cysteine, S-adenosylmethionine and taurine; and SM, sphingomyelin.

Figure 5.

Hierarchical clustering of 19 discriminating and prognostic metabolites in patients with pulmonary arterial hypertension (PAH). A, Venn diagram shows overlap between metabolites that discriminate patients with PAH from healthy control subjects in all 3 cohorts from logistic regression between patients with PAH and healthy control subjects and prognostic metabolites in the discovery and first validation cohorts. B, Clustering of the 19 overlapping metabolites from A is shown between healthy control subjects (HC; n=58), PAH survivors (n=110, alive at 3 years after sample), and nonsurvivors (n=24) in the discovery analysis. Red indicates metabolite levels that are increased (and blue levels that are decreased) in patients with PAH vs control subjects. *Probable metabolite identity but unconfirmed (see Methods). ‡Metabolites also distinguish PAH from disease control subjects. DHEA-S indicates dehydroisoandrosterone sulphate; EPE, eicosapentaenoyl; GPC, glycerophosphocholine; and SM, sphingomyelin.

Figure 6.

Analysis of serial samples. A, Receiver-operating characteristic analysis of changes in metabolite levels and survival status at last follow-up. B and D, Changes in individual patient metabolite levels grouped by survival status at last follow-up. C, Kaplan-Meier analysis illustrating survival over time in patients with pulmonary arterial hypertension divided into groups according to the changes in N-acetyl-methionine levels between serial samples.

Figure 7.

Circulating angiogenin levels. A, Plasma angiogenin levels determined by ELISA in healthy control subjects and patients with PAH. B, Scatterplot of plasma N2,N2-dimethylguanosine vs plasma angiogenin in control subjects and patients with pulmonary arterial hypertension (PAH). Statistics shown are from the Spearman rank test.