SUPPLEMENTAL MATERIAL Supplemental Methods Metabolite Profiling For measurement of amino acids, biogenic amines, and other polar plasma metabolites we used hydrophobic interaction chromatography (HILIC) with 150 x 2.1 mm Atlantis HILIC columns (Waters; Milford, MA) as previously described.1 Briefly, formic acid, ammonium acetate, LC/MS grade solvents, and valine-d8 were obtained from Sigma-Aldrich (St. Louis, MO), with the remainder of isotopicallylabeled analytical standards obtained from Cambridge Isotope Labs, Inc (Andover, MA). Plasma samples (10 μL) were prepared for LC/MS analyses via protein precipitation with the addition of 9 volumes of 74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards for valine-d8 and phenylalanine-d8. The samples were centrifuged (10 min, 10,000 rpm, 4ºC). After centrifugation, the supernatants were injected directly, followed by HILIC with the same mobile phases (mobile phase A: 10 mM ammonium formate and 0.1% formic acid, v/v; mobile phase B: acetonitrile with 0.1% formic acid, v/v). Multiplexing was used to enable the measurement of 105 metabolite transitions divided between the 2 LC systems, and each sample was injected once on each. Each column was eluted isocratically with 5% mobile phase A (over 1 minute), followed by a linear gradient to 60% mobile phase A (over 10 minutes). MS analyses were performed using electrospray ionization and multiple reaction monitoring scans in the positive ion mode. Declustering potentials and collision energies were optimized for each metabolite by infusion of reference standards prior to sample analyses. The dwell time for each transition was 30 msec, the ion spray voltage was 4.5 kV, and the source temperature was 425ºC. Analyses of polar metabolites in the negative ionization mode were conducted using a 5500 QTRAP triple quadrupole mass spectrometer (AB SCIEX; Foster City, CA) coupled to an ACQUITY UPLC (Waters; Milford, MA). Plasma samples (30µL) were prepared via protein precipitation with the addition of four volumes of 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA). The samples were centrifuged (10 min, 9,000 x g, 4°C), and the supernatants were injected directly onto a 150 x 2.0 mm Luna NH2 column (Phenomenex; Torrance, CA) that was eluted at a flow rate of 400µL/min with initial conditions of 10% mobile phase A (20 mM ammonium acetate and 20 mM ammonium hydroxide in water) and 90% mobile phase B (10 mM ammonium hydroxide in 75:25 v/v acetonitrile/methanol) followed by a 10 min linear gradient to 100% mobile phase A. MS analyses were carried out using electrospray ionization and selective multiple reaction monitoring scans in the negative ion mode. Declustering potentials and collision energies were optimized for each metabolite by infusion of reference standards before sample analyses. The ion spray voltage was -4.5 kV and the source temperature was 500°C. Internal standard peak areas were monitored for quality control and individual samples with peak areas differing from the group mean by more than 2 standard deviations were re-analyzed. MultiQuant software (Version 1.1; AB SCIEX; Foster City, CA) was used for automated LC-MS peak integration and metabolite peaks were manually reviewed for quality of integration and compared to reference standards to confirm identification. Using sample preparation and MS replicates of human samples, we have previously documented coefficients of variation (CV) for the metabolites in the platform: 54% of metabolites have CV ≤10% and 74% have CV ≤20%.2 Animal Model Studies All animals had access to standard mouse chow and water, ad libitum, during the experiment. Each mouse was weighed weekly and housed in groups of 4 mice per cage. Quantitative Real Time Polymerase Chain Reaction Total RNA from mouse lungs were extracted and purified via the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. The RNA concentration was determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). cDNA was synthesized in a 20-µL reaction mixture with 1µg of total RNA using the RT2 First-Strand Kit (Qiagen). Quantitative RT-PCR was performed using a 7500 FAST Real-Time PCR System (Applied Biosystems) with the RT2 SYBR® Green ROX qPCR mix (Qiagen). ). The IDO1 primers (Forward TGGCGTATGTGTGGAACCG and Reverse CTCGCAGTAGGGAACAGCAA) were purchased through Invitrogen . All data were normalized to 18SrRNA and quantitative measures obtained using the D-D-CT method. Supplemental Results: Supplemental Tables arginine ornithine citrulline SDMA ADMA Arg/OnC anthranilic acid kynurenic acid kynurenine quinolinate allantoin xanthosine inosine hypoxanthine xanthine urate aconitate isocitrate citrate malate succinate/methylmalonate fumarate tryptophan 3-hydroxyanthranilic acid serotonin 5-HIAA 5-hydroxytryptophan orotate creatinine cAMP aminoisobutyric acid VMA cystathionine cystathionine VMA aminoisobutyric acid cAMP creatinine orotate 5-hydroxytryptophan 5-HIAA serotonin 3-hydroxyanthranilic acid tryptophan fumarate succinate/methylmalonate malate citrate isocitrate aconitate urate xanthine hypoxanthine inosine xanthosine allantoin quinolinate kynurenine kynurenic acid anthranilic acid Arg/OnC ADMA SDMA citrulline ornithine arginine Supplemental Table 1. Pearson correlation coefficients between levels of metabolites in pathways related to invasive indices of RV-PV function. 1.00 0.26 1.00 0.18 0.50 1.00 -0.17 0.38 0.55 -0.03 0.54 0.39 0.60 1.00 0.68 -0.46 -0.46 -0.55 -0.44 1.00 -0.19 0.32 0.32 0.55 0.39 -0.45 1.00 -0.13 0.30 0.49 0.61 0.45 -0.44 0.69 1.00 -0.27 0.33 0.34 0.57 0.50 -0.53 0.54 0.54 1.00 -0.32 0.30 0.35 0.63 0.51 -0.56 0.55 0.56 0.83 1.00 -0.28 0.02 0.19 0.32 0.00 -0.33 0.25 0.34 0.16 0.29 1.00 -0.34 0.29 0.41 0.75 0.51 -0.59 0.48 0.57 0.51 0.63 0.44 1.00 -0.13 0.28 0.34 0.54 0.45 -0.38 0.31 0.42 0.43 0.45 0.37 0.77 1.00 -0.07 0.29 0.19 0.36 0.39 -0.28 0.27 0.31 0.31 0.31 0.30 0.41 0.50 1.00 -0.27 0.21 0.29 0.60 0.38 -0.45 0.41 0.53 0.51 0.55 0.40 0.79 0.82 0.49 1.00 -0.27 0.05 0.01 0.21 0.01 -0.27 0.29 0.16 0.33 0.31 0.31 0.03 0.00 0.33 0.12 1.00 -0.26 0.07 0.10 0.34 0.08 -0.31 0.34 0.32 0.22 0.32 0.48 0.28 0.16 0.27 0.27 0.52 1.00 -0.32 0.24 0.17 0.48 0.17 -0.47 0.48 0.44 0.51 0.49 0.44 0.37 0.25 0.44 0.42 0.61 0.70 1.00 -0.26 0.00 0.18 0.32 0.10 -0.30 0.26 0.33 0.22 0.18 0.33 0.18 0.13 0.24 0.16 0.45 0.67 0.51 1.00 -0.44 0.09 0.21 0.44 0.20 -0.51 0.38 0.44 0.29 0.35 0.47 0.41 0.25 0.42 0.35 0.37 0.64 0.55 0.60 1.00 -0.25 0.04 0.16 0.20 0.05 -0.30 0.46 0.27 0.23 0.18 0.37 0.13 0.09 0.33 0.16 0.50 0.51 0.50 0.48 0.63 -0.21 0.01 -0.07 0.05 -0.03 -0.16 0.08 0.07 -0.02 -0.01 0.02 0.03 -0.11 0.28 0.01 0.39 0.23 0.18 0.14 0.24 0.09 1.00 0.18 -0.18 -0.12 -0.23 -0.04 0.30 -0.05 -0.18 0.03 -0.18 -0.35 -0.32 -0.19 -0.05 -0.13 0.08 -0.24 -0.22 -0.12 -0.20 -0.02 0.11 1.00 -0.06 0.19 0.18 0.27 0.38 -0.21 0.37 0.31 0.47 0.56 -0.06 0.34 0.23 0.32 0.31 0.09 -0.06 0.13 -0.22 0.04 0.01 0.19 0.26 1.00 0.10 -0.32 -0.02 -0.10 -0.09 0.25 0.04 0.14 -0.07 -0.16 0.04 -0.18 -0.04 0.16 0.02 0.14 0.03 0.00 0.13 0.12 0.33 -0.18 0.21 -0.15 1.00 -0.07 0.24 0.43 0.33 0.14 -0.33 0.28 0.34 0.17 0.08 0.05 0.19 0.18 0.15 0.12 0.19 0.07 0.28 0.24 0.22 0.19 0.08 -0.02 0.01 0.02 1.00 -0.01 -0.04 -0.04 -0.12 0.01 0.02 0.00 0.13 -0.03 -0.07 0.08 0.03 0.09 0.29 0.06 -0.07 -0.01 -0.05 -0.12 0.11 0.24 -0.20 -0.03 0.06 0.92 -0.14 1.00 -0.25 0.12 0.38 0.50 0.27 -0.42 0.31 0.49 0.34 0.34 0.51 0.72 0.76 0.40 0.84 0.06 0.32 0.36 0.29 0.38 0.23 -0.07 -0.24 0.06 0.19 0.25 0.13 1.00 -0.19 0.05 0.46 0.63 0.29 -0.35 0.44 0.60 0.38 0.43 0.54 0.52 0.39 0.34 0.52 0.35 0.33 0.48 0.37 0.43 0.22 0.05 -0.03 0.23 0.15 0.32 0.05 0.54 1.00 -0.18 0.22 0.29 0.32 0.20 -0.37 0.39 0.33 0.38 0.41 0.19 0.36 0.20 0.45 0.21 0.42 0.18 0.48 0.30 0.25 0.33 0.20 -0.20 0.24 -0.09 0.38 0.12 0.20 0.37 1.00 -0.33 0.35 0.45 0.67 0.49 -0.63 0.48 0.36 0.51 0.61 0.19 0.72 0.53 0.29 0.50 0.04 0.17 0.32 0.24 0.37 0.14 0.08 -0.15 0.40 -0.29 0.19 -0.20 0.37 0.37 0.33 1.00 -0.22 0.15 0.39 0.47 0.09 -0.40 0.31 0.33 0.33 0.32 0.45 0.40 0.29 0.43 0.29 0.40 0.40 0.58 0.49 0.48 0.37 0.18 -0.23 -0.17 0.11 0.36 0.09 0.42 0.43 0.48 0.33 1.00 -0.36 0.40 0.06 0.42 0.25 -0.55 0.39 0.34 0.48 0.52 0.52 0.46 0.37 0.27 0.46 0.34 0.29 0.48 0.07 0.30 0.25 0.10 -0.27 0.21 -0.17 -0.04 -0.08 0.33 0.29 0.11 0.40 0.25 1.00 1.00 1.00 Supplemental Table 2. Relationships between IDO-TM scores and co-morbidities as well as medication exposures IDO-TMs P value β-coefficients BMI -0.026 0.83 Hypertension 0.080 0.52 Diabetes 0.151 0.21 Smoking 0.040 0.74 Beta-blocker 0.127 0.29 ACE I/ARB -0.009 0.94 CCB -0.084 0.48 Diuretic 0.193 0.11 Statin 0.166 0.17 ASA 0.184 0.18 Coumadin 0.146 0.23 Supplemental References 1. 2. Lewis GD, Farrell L, Wood MJ, Martinovic M, Arany Z, Rowe GC, Souza A, Cheng S, McCabe EL, Yang E, Shi X, Deo R, Roth FP, Asnani A, Rhee EP, Systrom DM, Semigran MJ, Vasan RS, Carr SA, Wang TJ, Sabatine MS, Clish CB, Gerszten RE. Metabolic signatures of exercise in human plasma. Science translational medicine.2:33ra37. Wang TJ, Larson MG, Vasan RS, Cheng S, Rhee EP, McCabe E, Lewis GD, Fox CS, Jacques PF, Fernandez C, O'Donnell CJ, Carr SA, Mootha VK, Florez JC, Souza A, Melander O, Clish CB, Gerszten RE. Metabolite profiles and the risk of developing diabetes. Nature medicine. 2011;17:448-453. Supplemental Figure 50 Subject 1 Subject 2 40 Mean PA Pressure (mmHg) Slope 4.9mmHg/L/min 30 20 Slope 1.4 mmHg/L/min 10 0 0.0 5.0 10.0 15.0 Cardiac Output (L/min) Supplemental Figure Legend : Representative changes in mean pulmonary arterial pressure relative to changes in cardiac output (PQ) during exercise in two subjects. Although neither patient has resting pulmonary hypertension, serial measurements of pulmonary arterial pressure and cardiac output during exercise demonstrate a much steeper PQ in subject 1 compared to subject 2. These distinct responses to exercise illustrate the advantage of integrating multi-point assessment of pressure flow relationships during exercise to resting hemodynamic measurements in assessing pulmonary vascular function.