Use of Metabolomics to Trend Recovery and Therapy After Injury in Critically Ill Trauma Patients

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Supplementary Online Content

Parent BA, Seaton M, Sood RF, et al. Use of metabolomics to trend recovery and therapy after injury in critically ill trauma patients. JAMA Surg. Published online May 25, 2016. doi:10.1001/jamasurg.2016.0853.

eAppendix. Methods eFigure. In a Cohort of Severely Injured Blunt Trauma Patients, Mass Spectrometry (MS)–Based Plasma Glucose Correlates Well With Hospital-Based Plasma Glucose eTable 1. List of Plasma Metabolites and Biologic Pathways Which Were Assayed By Plasma Mass Spectrometry–Based Metabolomics in a Cohort of Blunt Trauma Subjects and Healthy Volunteers eTable 2. List of Statistically Significant Plasma Metabolites Which Are Associated With Exposure to Blunt Trauma eTable 3. List of Statistically Significant Plasma Metabolites in Blunt Trauma Subjects Associated With Recovery From Day 1 to Day 7 After Initial Injury, as Indicated by Mass Spectrometry–Based Metabolomics

This supplementary material has been provided by the authors to give readers additional information about their work.

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eAppendix. Methods

Sample Preparation

Plasma samples were thawed at 4 ⁰C. 250 µL methanol with 53.1 µM 13C-arginine and

50.1 µM 13C-glucose (Sigma-Aldrich, Saint Louis, MO) was added to each 50 µL plasma

aliquot, and all samples were vortexed for 2 min. Samples were kept at -20 ⁰C for 20

min, and then centrifuged at 20,800g for 20 min. Supernatants were collected and dried

for 60 min using an Eppendorf Vacufuge Drier (Happauge, NY), then each reconstituted

in 600 µL 40% Solution A and 60% Solution B, also containing 5.13 µM 13C-tyrosine

13 and 22. 6 µM C-lactate. Solution A was 30mM ammonium acetate in 85% H2O/ 15%

acetonitrile and 0.2% acetic acid. Solution B was 15% H2O/ 85% acetonitrile and 0.2%

acetic acid. Reagents for these solutions were purchased from Fisher Scientific

(Pittsburgh, PA). All samples were then filtered through Millipore PVDF filters

(Pittsburgh, PA) immediately prior to chromatography.

Chromatography Conditions

The LC system was composed of two Agilent 1260 binary pumps, an Agilent 1260 auto-

sampler and Agilent 1290 column compartment containing a column-switching valve

(Agilent Technologies, Santa Clara, CA). Each sample was injected twice, 10 µL for

analysis using negative ionization mode and 2 µL for analysis using positive ionization

mode. Both chromatographic separations were performed in HILIC mode on two parallel

Waters XBridge BEH Amide columns (150 x 2.1 mm, 2.5 µm particle size, Waters

Corporation, Milford, MA). While one column was performing the separation, the other

column was reconditioned and ready for the next injection. The flow rate was 0.300

mL/min, auto-sampler temperature was kept at 4 ⁰C, and the column compartment was

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set at 40 ⁰C, and total separation time for both ionization modes was 20 min. The

gradient conditions for both separations were identical and consisted of 10% Solvent A

from 0-2 min, a ramp to 50% during min 2-5, continued 50% Solvent A from 5-9 min, a

ramp back to 10% from 9-11 min, and then 10% Sovlent A from 11-20 min.

Mass Spectrometry (MS) Conditions

After the chromatographic separation, MS ionization and data acquisition was performed

using an AB Sciex QTrap 5500 mass spectrometer (AB Sciex, Toronto, ON, Canada)

equipped with an electrospray ionization (ESI) source. The instrument was controlled by

Analyst 1.5 software (AB Sciex, Toronto, ON, Canada). Targeted data acquisition was

performed in multiple-reaction-monitoring (MRM) mode. We monitored 121 and 80

MRM transitions in negative and positive mode, respectively (201 transitions total). The

source and collision gas was N2 (99.999% purity). The ion source conditions in negative

mode were: Curtain Gas (CUR) = 25 psi, Collision Gas (CAD) = high, Ion Spray Voltage

(IS) = - 3.8KV, Temperature (TEM) = 500 ⁰C, Ion Source Gas 1 (GS1) = 50 psi and Ion

Source Gas 2 (GS2) = 40 psi. The ion source conditions in positive mode were: CUR =

25 psi, CAD = high, IS = 3.8KV, TEM = 500 ⁰C, GS1 = 50 psi and GS2 = 40 psi.

MS Data Processing

The extracted MRM peaks were integrated using MultiQuant 2.1 software (AB Sciex,

Toronto, ON, Canada).

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eFigure. In a Cohort of Severely Injured Blunt Trauma Patients, Mass Spectrometry (MS)–Based Plasma Glucose Correlates Well With Hospital-Based Plasma Glucose (r = 0.84).

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eTable 1. List of Plasma Metabolites and Biologic Pathways Which Were Assayed By Plasma Mass Spectrometry–Based Metabolomics in a Cohort of Blunt Trauma Subjects and Healthy Volunteers. Metabolite Metabolic Pathway Acetoacetate Ketone Acetylcarnitine Fatty acid Oxidation Acetylcholine Lipids/phospholipids, ligand Aconitate TCA Cycle Adenine Nucleotide/Purine metabolism Adenosine Nucleotide/Purine metabolism Adenosine 3 phosphate (ATP) Nucleotide Adenosine diphosphate (ADP) Nucleotide/Purine metabolism Adenosine monophosphate (AMP) Nucleotide Adenylosuccinate Nucleotide/Purine metabolism Adipic Acid Caprolactam degradation Agmatine Polyamine metabolism Alanine (Ala) Amino Acid Allantoin Nucleotide Degredation Allopurinol Nucleotide metabolism (Drug) Alpha-Ketoglutaric Acid TCA Cycle Aminoisobutyrate Amino acids metabolism/Val, Leu, iso-Leu Aminolevulinate Amino acids metabolism/Gly, Thr,Ser 2-Aminoadipate Amino acids metabolism/Lys 5-Aminovalerate Amino Acid Anthranilate Amino Acid metabolism/Trp, Phe, Tyr Arachidonate Lipids/phospholipids, ligand Arginine Amino Acid Ascorbate Vitamins Ascorbic Acid (Vit. C) Vitamin, Cofactor Asparagine (Asp) Amino Acid Aspartic Acid Amino Acids Azelaic Acid Fatty Acids and Conjugates Benzoic acid Amino Acid (Phenylalanine Metabolism, others) Betaine Amino acids metabolism/Gly,Ser, Thr metabolism Biotin Vitamins Cadaverine Amino Acid Carnitine Amino acids metabolism/Lys Chenodeoxycholate Bile acid metabolism Choline Vitamins Citraconic Acid Amino Acid metabolism/Val, Leu, IL

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Citric Acid TCA Cycle Citrulline Urea Cycle Creatine Amino acids metabolism/Arg, Gly Creatinine Amino acids metabolism/Arg, Gly Cystamine (Cys) Amino acids metabolism/Cys Cystathionine Amino acids metabolism/cys Cysteine Amino Acid Cystine Amino acids metabolism Cytidine Nucleotide/Pyrimidine metabolism Cytidine 5 monophosphate (CMP) Nucleotide/Pyrimidine metabolism Cytosine (Cyt) Nucleotide Deoxycarnitine Lipids/phospholipids, ligand 2-Deoxycytidine Diphosphate Nucleotide/Pyrimidine metabolism (DCDP) Deoxycytidylic acid (DCMP) Nucleotide/pyrimidine 2-Deoxyuridine Nucleotide/Pyrimidine metabolism Deoxyuridine 3 phosphate Nucleotide/pyrimidine (DUTP) Deoxyuridylic acid (DUMP) Nucleotide/pyrimidine D-Glyceraldehyde-3-phosphate Glycolysis/Glycogenesis (D-GA3P) Dihydroxy acetone phosphate Glycolysis/phospholipid (DHAP) Dimethylglycine Vitamins Dopamine Adrenaline Metabolism D-Leucic Acid Amino Acid metabolism/Leu D-Thymidine monophosphate Nucleic Acid (DTMP) Epinephrine Amino Acid metabolism/Tyr,ligand Erythrose Sugar Folic Acid One carbon pool by folate Fructose Sugar Fructose 1 phosphate (F1P) Glycolosis Fructose 1,6 bi-phosphate Glycolysis (F16BP) Fructose 2,6 bi-phosphate Glycolosis (F26BP) Fructose 6 phosphate (F6P) Glycolosis Fumaric Acid TCA Cycle Galactose Sugars gama-Aminobutyrate Amino Acid metabolism/Ala, Glu, Asp Geranyl Pyrophosphate Ubiquinone and other terpenoid-quinone biosynthesis

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Gibberellin Diterpenoid biosynthesis Glucoronate Amino sugar and nucleotide sugar metabolism Glucosamine Amino sugar and nucleotide sugar metabolism Glucose Glycolysis/sugar Glucose 1,6 biphosphate (G16BP) Glycolysis/Glycogenesis Glucose 1-phosphate (G1P) Glycolysis Glucose 6-phosphate (G6P) Glycolosis/PPP Glutamic acid (Glu A) Amino Acid Glutamine (Glu) Amino Acid Glutaric Acid Amino Acids/lys, trp, fatty acids Glyceraldehyde Sugar Glycerate Amino Acid metabolism/Gly, Ser Glycerol-3-P Lipids/Glycerollipid Glycine (Gly) Amino Acid Glycochenodeoxycholate Bile acid metabolism Glycocholate Bile acid metabolism Guanidinoacetate Amino Acid metabolism/Gly, Ser,Thr Guanosine Nucleotide/Purine metabolism Guanosine 3,5 cyclic Nucleotide/Purine metabolism monophosphate (cGMP) Guanosine 5 diphosphate (GDP) Nucleotide/Purine metabolism Guanosine 5 triphosphate (GTP) Nucleotide/Purine metabolism Guanosine-5'-monophosphate Nucleotide/Purine metabolism (GMP) 12-HETE Lipids/phospholipids, ligand 13-HODE Lipids/phospholipids, ligand Hippuric Acid Amino Acid (Phenylalanine Metabolism Hippuric Acid Gut flora metabolism Histamine Nucleotide Histidine (His) Amino Acid Homocysteine Amino acids metabolism/Cys, Met Homogentisate Amino Acid metabolism/Tyr Homoserine Amino acids metabolism/Thr, Met, Asp Homovanillate Amino acids metabolism/Tyr,ligand 2-Hydroxyglutarate Amino-acid metabolism & Glycine/Serine/Threonine metabolism 3-Hydroxybutyric Acid (3HBA ) TCA Cycle 4-Hydroxybutyric Acid (4HBA) Lipids/phospholipids, ligand 3-Hydroxyisovaleric Acid Amino Acid 3-Hydroxykynurenine Tryptophan Cycle Hydroxyphenylpyruvate Amino acids metabolites/phe, tyr Hydroxyproline Amino acids metabolism/Pro

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5-Hydroxytryptophan Tryptophan Cycle 8-Hydroxyguanosine Nucleotide/Purine metabolism Hypoxanthine Nucleotide Inosine Nucleotide/Purine metabolism Inosine monophosphate (IMP) Nucleotide/Purine metabolism Inositol Glucose/inositol metabolism isoButyric Acid (2- Fatty acid metabolism methylpropanoic acid) iso-Leucine (iso-Leu) Amino Acid isoValeric Acid Amino Acid Kynurenate Amino Acid metabolism/Try lactate Glycolysis/TCA Lactose Sugar/Galactose Leucine (Leu) Amino Acid Linoleic Acid Fatty acid metabolism Linolenic Acid Fatty acid metabolism L-Kynurenine Tryptophan Cycle Lysine (Lys) Amino Acid Malate TCA Cycle Maleic Acid TCA Cycle Malonic Acid TCA Cycle Mannose Sugar/Galactose Metabolism Margaric Acid Fatty acid metabolism Melatonin Neuroactive ligand Methionine (Met) Amino Acid 1-Methyladenosine Nucleotide/Purine metabolism 1-Methylguanosine Nucleotide/Purine metabolism 1-Methylhistamine Amino acids metabolism/His 1/3-Methylhistidine Amino Acid (Histidine Metabolism) Methyl-OH-isobyturate Fatty acid metabolism 3-Methyl-2-Oxovaleric Acid Amino Acid Methylmalonate Vitamins MethylSuccinate Amino Acid metabolism/Isoleu Mevalonate Terpenoid backbone biosynthesis Myristic Acid Fatty acid metabolism N-Carbamoyl-B-Alanine Urea cycle N2,N2-Dimethylguanosine Nucleotide/Purine metabolism N-AcetylGlycine Amino Acid metabolism N-acetylneuraminate Sugars N-acetylputrescine Polyamine metabolism Niacinamide Vitamins

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Nicotinate (Niacin) Nicotinate and nicotinamide metabolism 3-Nitro-tyrosine Oxidative Damage Normetanephrin Amino Acid metabolism/Tyr,ligand OH-Phenylpyruvate (4- Amino Acid / Tyrosine metabolism Hydroxyphenylpyruvate) Ornithine Urea cycle Orotate Nucleotide/Pyrimidine metabolism Orotidylic acid (OMP) Nucleotide/Pyrimidine metabolism Oxalic Acid Glyoxylate and dicarboxylate metabolism Oxaloacetate TCA Cycle Oxidized glutathione Oxidative Damage 8-Oxo-2'-deoxyguanosine Nucleotide/Purine metabolism Oxypurinol Nucleotide metabolism Pantothenate Amino acids metabolism/alanine, CoA PGE Lipids/Arachidonic acid metabolism Phenylalanine Amino Acid Phenylpyruvic acid (PPA) Amino acids metabolism/Phe Phosphoenolpyruvic Acid (PEP) Glycolosis Phosphoribosyl pyrophosphate Nucleotide/Purine metabolism (PRPP) Phosphotyrosine Amino acids metabolism 2-Phosphoglyceric Acid Glycolosis 3-Phosphoglyceric Acid Glycolosis Pipecolate Amino Acid Proline (Pro) Amino Acid Propionate Nicotinate and nicotinamide metabolism Putrescine Polyamine metabolism Pyridoxal-5-P Vitamins/B6 4-Pyridoxic acid Vitamins/B6 Pyroglutamic Acid Amino acids/Glu Pyruvate Glycolysis/amino acids metabolism Quinolinate Amino Acid/ Tryptophan, bela alanine metabolism Reduced glutathione Oxidative Damage Ribose-5-P Pentose phosphate pathway Salicylurate Amino Acid metabolism/Gly Sarcosine Amino Acid Serine (Ser) Amino Acid Shikimic Acid Amino Acid metabolism/Phe, Tyr,Trp Sorbitol Sugar Spermidine Polyamine metabolism Spermine Polyamine metabolism

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Succinate TCA Cycle Sucrose Sugar Taurine (Tau) Amino acids metabolism/Sulfur metabolism Taurocholate Conjugated bile acid biosynthesis Threonine (Thr) Amino Acid Thymidine 5 phosphate (DTMP) Nucleotide/pyrimidine Trimethylamine (TMA) Gut flora metabolism / Redox Trimethylamine-N-oxide Gut flora metabolism / Redox Tryptamine Amino Acid Tryptophan (Try) Tryptophan Cycle Tyramine Amino Acid Tyrosine (Tyr) Amino Acid Uracil Nucleotide/Pyrimidine metabolism Urate Nucleotide/Purine metabolism Uridine Nucleotide/Pyrimidine metabolism Uridine diphosphate (UDP) Nucleotide/Pyrimidine metabolism Valine (Val) Amino Acid Xanthine Nucleotide Xanthosine Nucleotide/Purine metabolism Xanthurenate Amino acids metabolism Cysteinyl-Glycine (Cys-Gly) Amino acids degradation

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eTable 2. List of Statistically Significant Plasma Metabolites Which Are Associated With Exposure to Blunt Trauma.a Pathway Metabolite Changeb associated Raw BH-FDRd- with trauma P- corrected P- exposurec value value Sarcosine 6.27 <0.000 0.0008 01 Tryptophan -0.9 0.0001 0.0023 AminovalericAc -0.56 0.0001 0.0019 id Proline -0.68 0.0001 0.0023 Methionine -0.66 0.0002 0.0024 ShikimicAcid 2.29 0.0007 0.0075 Asparagine -0.84 0.0012 0.0118 Pipecolate -0.6 0.0015 0.0125 Lysine -0.63 0.0019 0.0145 Taurine -1.16 0.0019 0.0137 Amino acid Serine -0.41 0.0022 0.0152 turnover LKynurenine -0.57 0.0029 0.0163 Threonine -0.49 0.0035 0.0190 Cystine -1.64 0.0040 0.0187 Histidine -0.46 0.0067 0.0254 Glycerate -0.64 0.0099 0.0335 Alanine -0.47 0.0119 0.0393 Glycine -0.38 0.0127 0.0403 Valine -0.54 0.0001 0.0020 CitraconicAcid -0.9 0.0023 0.0148 Leucine -0.42 0.0078 0.0283 Fatty acid MargaricAcid -0.56 0.0043 0.0185 Adenylosuccinat -0.07 0.0046 0.0189 e Nucleotide Urate -0.53 0.0148 0.0444 turnover Cytidine -0.09 0.0166 0.0502 Sucrose 13.01 0.0000 0.0000 Sugars NAcetylneurami -0.83 0.0039 0.0190 nate Glyceraldehyde -0.4 0.0128 0.0395 Krebs cycle Succinate -0.15 0.0085 0.0300 Citrulline -0.61 0.0013 0.0122 Urea Cycle Ornithine -0.57 0.0038 0.0196 Allantoin -0.75 0.0041 0.0180

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Choline -0.1 0.0003 0.0044 Oxidation and Dimethylglycine -1.11 0.0028 0.0169 Vitamins Niacinamide -0.93 0.0058 0.0229 Biotin -0.76 0.0169 0.0492 a Statistical model: multiple regression, with outcome variable = metabolite; predictor variable = trauma exposure; covariates = body mass index and processing batch. b relative concentrations, determined by plasma mass spectrometry c relative to healthy volunteers d Benjamini-Hochberg false discovery rate

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eTable 3. List of Statistically Significant Plasma Metabolites in Blunt Trauma Subjects Associated With Recovery From Day 1 to Day 7 After Initial Injury, as Indicated by Mass Spectrometry–Based Metabolomics.a Pathway Metabolite Raw P- BH-FDRc- value corrected P- value Serine 0.0001 0.0024 Threonine 0.0017 0.0209 Amino acid turnover Shikimic Acid 0.0029 0.0318 Aminovaleric Acid 0.0051 0.0360 Lysine 0.0052 0.0360 Phenylalanine 0.0059 0.0367 Cadaverine 0.0080 0.0442 Iso-leucine 0.0002 0.0030 Branched Chain amino acids Leucine 0.0039 0.0357 Valine 0.0044 0.0360 Nucleotide turnover Adenylosuccinate 0.0001 0.0118 Cytidine 0.0070 0.0412 Sugars Sucrose 0.0000 0.0035 Inositol 0.0054 0.0360 Urea Cycle Ornithine 0.0009 0.0127

a Statistical model: paired t-testing for subject plasma samples from day one and day seven.

b relative concentrations, determined by plasma mass spectrometry c Benjamini-Hochberg false-discovery rate

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