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

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Use of Metabolomics to Trend Recovery and Therapy After Injury in Critically Ill Trauma Patients 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. © 2016 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 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 © 2016 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 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). © 2016 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 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). © 2016 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 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 © 2016 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 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 © 2016 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 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
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