RESEARCH ARTICLE
New Biochemical Insights into the Mechanisms of Pulmonary Arterial Hypertension in Humans Renata Bujak1,2☯, Jesús Mateo3,6☯, Isabel Blanco4,6, José Luis Izquierdo-García3,6, Danuta Dudzik1, Michał J. Markuszewski2, Victor Ivo Peinado4,6, Martín Laclaustra3,5, Joan Albert Barberá4,6, Coral Barbas1, Jesús Ruiz-Cabello3,6,7*
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1 Centre of Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Universidad CEU San Pablo, Campus Monteprincipe, Boadilla del Monte, Madrid, Spain, 2 Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk, Al. Gen. J. Hallera 107, 80–416, Gdańsk, Poland, 3 Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain, 4 Hospital Clinic Barcelona-IDIBAPS, Barcelona, Spain, 5 CIBERESP, Preventive Medicine and Public Health Department, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain, 6 Ciber de Enfermedades Respiratorias (CIBERES), Madrid, Spain, 7 Universidad Complutense de Madrid, Madrid, Spain ☯ These authors contributed equally to this work. *
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OPEN ACCESS Citation: Bujak R, Mateo J, Blanco I, IzquierdoGarcía JL, Dudzik D, Markuszewski MJ, et al. (2016) New Biochemical Insights into the Mechanisms of Pulmonary Arterial Hypertension in Humans. PLoS ONE 11(8): e0160505. doi:10.1371/journal. pone.0160505 Editor: Daniel Monleon, Instituto de Investigación Sanitaria INCLIVA, SPAIN Received: February 15, 2016 Accepted: July 20, 2016 Published: August 3, 2016 Copyright: © 2016 Bujak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by the Polish National Science Center (2014/13/N/NZ7/04231), the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2014-58920R), by the Fondo de Investigación Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional (FEDER) (PI14-01427), and by the qualitypromoting subsidy from the Ministry of Science and Higher Education of Poland, Leading National Research Centre (KNOW programme 2012-2017).
Abstract Diagnosis of pulmonary arterial hypertension (PAH) is difficult due to the lack of specific clinical symptoms and biomarkers, especially at early stages. We compared plasma metabolic fingerprints of PAH patients (n = 20) with matched healthy volunteers (n = 20) using, for the first time, untargeted multiplatform metabolomics approach consisting of high-performance liquid and gas chromatography coupled with mass spectrometry. Multivariate statistical analyses were performed to select metabolites that contribute most to groups’ classification (21 from liquid in both ionization modes and 9 from gas chromatography-mass spectrometry). We found metabolites related to energy imbalance, such as glycolysis-derived metabolites, as well as metabolites involved in fatty acid, lipid and amino acid metabolism. We observed statistically significant changes in threitol and aminomalonic acid in PAH patients, which could provide new biochemical insights into the pathogenesis of the disease. The results were externally validated on independent case and control cohorts, confirming up to 16 metabolites as statistically significant in the validation study. Multiplatform metabolomics, followed by multivariate chemometric data analysis has a huge potential for explaining pathogenesis of PAH and for searching potential and new more specific and less invasive markers of the disease.
Introduction Pulmonary arterial hypertension (PAH) is a heterogeneous disease with multifactorial pathophysiology. PAH is currently classified into various clinical phenotypes, but they all share their severity and progressiveness as common features. The lack of specific clinical symptoms, especially at early stages hinders the diagnosis [1]. PAH, when not diagnosed, can lead to right
PLOS ONE | DOI:10.1371/journal.pone.0160505 August 3, 2016
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The CNIC is supported by the Spanish Ministry of Economy and Competitiveness and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
ventricle failure and consequently to premature death. Additionally, pathomechanisms of PAH remain still not fully understood. Knowledge regarding pathological hallmarks of PAH, such as cell proliferation, apoptosis resistance, vascular remodelling, vasoconstriction and increased angiogenesis, derives mainly from experimental animal models [2,3]. For this reason, new, sensitive and specific markers of PAH in humans are needed to better understand the pathological processes of the disease and consequently improve current diagnosis and treatment. Metabolomics focuses on qualitative and quantitative analysis of low-molecular-weight compounds (metabolites) in various biological samples (plasma/serum, urine, saliva, tissue, exhaled breath) to understand the complex and dynamic responses of living systems to diverse stimuli, such as pathological processes, drug treatments, genetic variability or environmental factors [4,5]. The metabolome, as a final consequence of derangements in genome and proteome is considered to be a link in the genotype-to-phenotype gap. In the area of PAH research, we consider that the application of metabolomics can be a potential tool for understanding its pathogenesis and to find new diagnostic markers. Only a few reports can be found in the literature suggesting the role of metabolic alterations, such as: excessive cellular glucose uptake, glycolytic metabolism, high-density lipoprotein cholesterol and insulin resistance in PAH pathogenesis [6]. One of the most recent studies employed a metabolomics approach to determine metabolic profiles of lung tissue derived from patients with severe PAH [7]. Obviously, analysis of tissue samples provides detection of site-specific metabolite alterations that might be characteristic for disease stage. However, its application in diagnosis and clinical practice is limited due to its invasiveness, while plasma analysis could provide diagnostic markers more readily available to clinicians. In order to search for potential markers of pathological conditions occurring in PAH, untargeted multiplatform metabolomics, using high-performance liquid and gas chromatography coupled with mass spectrometry (LC-MS and GC-MS), was applied to plasma samples of PAH patients and healthy controls, providing data on the plasma metabolic fingerprint of PAH.
Materials and Methods Study design and samples This case-control study included 20 patients with confirmed PAH derived from Hospital Clinic in Barcelona and 20 healthy controls. Plasma samples were collected at fasting condition at the same time of the day into EDTA tubes and frozen at -80°C for aproximately 6 months, until metabolomic analysis. The studied groups were matched according to age (p = 0.96), body mass index (p = 0.87) and sex (p = 0.62). Independent recruitment of other additional 20 patients and 12 controls processed in a separated batch and not used in the main analyses allowed external validation. The investigation was carried out in accordance with approval of The ethical committee of clinical investigations in Barcelona (CEIC, the approval number CIF-G-08431173) and the informed consent was signed by each participant of the study. The detailed characteristics of study and validation cohorts are described in S1 and S2 Tables. Plasma was separated from fasting blood samples for metabolic fingerprinting. Metabolomics included liquid chromatography-mass spectrometry (LC-MS) in positive and negative modes and gas chromatography-mass spectrometry (GC-MS).
Plasma metabolic fingerprinting with HPLC-ESI-QTOF-MS and GC-EI-Q-MS Sample preparation for LC-MS included deproteinization, centrifugation and filtration. Quality control samples (QCs) were prepared as aliquots of a pool of equal volumes from all samples
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included in the study, using the same preparation procedures. Samples were analysed by an HPLC system connected to a Q-TOF LC-MS detector. Randomized samples were analysed in two separate runs (for positive and negative modes). Sample preparation for GC-MS involved deproteinization, centrifugation, supernatant’s evaporation and two-step derivatization employing methoxymation and silylation. GC−MS analysis was performed with a gas chromatograph interfaced to an inert quadrupole analyser.
Data extraction and treatment For LC-MS, raw datasets were extracted by the Molecular Feature Extraction tool in the MassHunter Qualitative Analysis software (Agilent Technologies). For GC-MS, deconvolution and data processing were performed with Automated Mass Spectrometry Deconvolution and Identification System (www.amdis.net). Due to retention time shifts during both LC−MS and GC-MS sequence runs, samples were multi-aligned using Mass Profiler Professional (Agilent Technologies). Subsequently, data filtration regarding quality assurance criteria (QA) and frequency in at least one of the compared groups (i.e., in 90% of samples) was applied.
Statistical analysis and metabolite identification Principal component analysis (PCA) was applied to check quality of the analysis, trends in the datasets and detect potential outliers. Then, multivariate statistics were used to select compounds with statistically significant differences between the groups. To select metabolites contributing the most into groups’ discrimination, orthogonal partial least squares discriminant analysis (OPLS-DA) was applied. Statistically significant metabolites were selected based on the Jack-knife confidence interval (p
