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Reisdorph Lab Assays

Reisdorph Lab Assays. Director: Nichole.Reisdorph@UCDenver.edu Targeted Assays: Mike.L.Armstrong@UCDenver.edu Jon.Manke@UCDenver.edu. Targeted Assays.

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Reisdorph Lab Assays

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  1. Reisdorph Lab Assays Director: Nichole.Reisdorph@UCDenver.edu Targeted Assays: Mike.L.Armstrong@UCDenver.edu Jon.Manke@UCDenver.edu

  2. Targeted Assays • Targeted analysis can be performed on tissues, cultured cells, cell culture media, biofluids (urine, plasma, serum, CSF, BAL, sputum, etc.) • Deliverables: • Report includes concentration values for all targeted compounds • Calibration curves and QC data available upon request • Please inquire about required sample quantities for each assay if not listed.

  3. Targeted Acylcarnitine Analysis • Assay offers absolute quantitation for 8 acylcarnitines • Targeted relative quant for 40 additional compounds • Requires a minimum 25 µL plasma aliquot • 20 Samples can be analyzed per day

  4. AcylcarnitinesAbsolute Quantitation • Quantified using isotopically labeled internal standard

  5. AcylcarnitinesTargeted Relative Quantitation • Targeted multiple reaction monitoring (MRM) assay. • Quantified relative to similar labeled internal standard and known aclycarnitine product ions

  6. Targeted Amino Acid Analysis • Uses HILIC chromatography (Hydrophilic Interaction LIquidChromatography) • Can analyze 40 samples per day • Requires 25-50 µl Plasma or serum.

  7. Lipid mediator and oxylipin pathways DHA*, DPA*, EPA* Linoleic Acid* Arachidonic Acid* Alpha-linolenic Acid* Membrane PLs containing DHA, EPA Membrane PLs containing Arachidonic Acid Phospholipase A2 Phospholipase A2 Phospholipase A2 HETEs DPA 12 or 15 LOX Arachidonic Acid n-6 PUFA C20:4 Eicosapentaenoic acid (EPA) n-3 PUFA C20:5 Docosahexaenoic acid (DHA) n-3 PUFA C22:6 Lipoxygenase (LOX) Lipoxygenase (LOX) 12 or 15 LOX Cyclooxygenase (COX1,2) PGD,E,F3, LTA-D5 Prostaglandin H2 (PGH2) HPETE 17(S)-HDHA 14(S)-HDHA HODEs Lipoxin A/B PGE2 PGD2 ^PGF2 7-R-Maresin 1, 7-S-Maresin 1 LTA4 10(S),17(S)-DiHDoHE (Protectin D1) PGI2 TXA2 LTB4 LTC4, LTD4, LTE4 6-keto-PGF1a Resolvin D1, Resolvin D2 H2O2/O2- Arachidonic Acid 8-Iso-PGF2a and other Isos

  8. Targeted Lipid Mediator and Oxylipin Assay Target compounds and associated pathways • Required sample quantities • 100ul of plasma or serum • 20-200mg of tissue (dependent on tissue type, please inquire) • 100-200ul of urine • For other sample matrices, please inquire for required quantities

  9. EndocannabinoidsLipid based neurotransmitters that bind to cannabinoid receptors • Required sample quantities • 50ul of plasma or serum • Please inquire about required tissue quantities or other sample matrices • Endocannabinoids targeted • 2-Arachidonoyl Glycerol (2-AG) • 2-Linoleoyl Glycerol (2-LG) • ArachidonoylEthanolamide (AEA) • ArachidonoylGlycine (NAGly) • DocosahexaenoylEthanolamide (DHEa) • DocosatetraenoylEthanolamide (DEA) • EicosapentaenoylEthanolamide (EPEa) • LinoleoylEthanolamide (LEA) • OleoylEthanolamide (OEA) • PalmitoleoylEthanolamide (POEA) • PalmitoylEthanolamide (PEA) • StearoylEthanolamide (SEA) 2-AG AEA NAGly

  10. Targeted nucleoside/nucleotide/cofactor assay • Required sample quantities • 1E6 cultured cells preferred • Please inquire about tissue or biofluid quantities NaM Cyclic AMP Adenosine ADP Adenosine monophosphate ATP

  11. Targeted Bile Acid Assay 1 2 • Required sample quantities • 50ul of serum or plasma • 100mg of liver • 20mg Feces 3 4 5 6 7 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 2 17

  12. Lipid identification and quantitation • Estimated quantitation of lipids using Avanti SPLASH lipid spike • Estimated concentrations of the following lipid classes • Phosphatidylcholines (PC) • Phosphatidylethanolamines (PE) • Phosphatidylserines (PS) • Phosphatidylglycerol (PG) • Phosphatidic acid (PA) • Lysophosphatidylcholine (LPC) • Lysophosphatidylethanolamine (LPA) • Cholesterol esters • Monoglycerides (MG) • Diglycerides (DG) • Triglycerides (TG) • Sphingomyelins (SM) • Cholesterol Liquid/Liquid extraction Cultured cells, biofluids or tissue HPLC reverse phase separation High resolution time-of-flight mass spectrometry Targeted data extraction using Lipidmaps database MS/MS confirmation Estimated concentration

  13. Assays Currently Under Development (Not Validated)

  14. Fatty Acid Methyl Esters (FAMEs) by GC/MS 8% HCL In MeOH Fatty Acid Methyl Ester Esterified lipids Glycerol Acid hydrolysis and esterification of free fatty acids

  15. Targeted TCA Assay (Not Validated) Fructose C6H12O6 MW: 180.16 Pyruvate C3H4O3 MW: 88.06 Acetyl CoA C23H38N7O17P3S MW: 809.57 Glucose C6H12O6 MW: 180.16 Glucose-6-phosphate C6H13O9P MW: 260.14 Malate C4H6O5 MW: 134.09 Alpha-ketoglutarate C5H6O5 MW: 146.10 Lactic Acid C3H6O3 MW: 90.06 Isocitrate C6H8O7 MW: 192.12 Succinicate C4H6O4 MW: 118.09 Fumarate C4H4O4 MW: 116.07 Oxaloacetate C4H4O5 MW: 132.07 Citrate C6H8O7 MW: 192.12

  16. Citations • Acylcarnitines • A.K. Ghoshal, T. Guo, N. Soukhova, S.J. Soldin, Rapid measurement of plasma acylcarnitines by liquid chromatography-tandem mass spectrometry without derivatization, ClinChimActa, 358 (2005) 104-112. • Amino acids • M. Armstrong, K. Jonscher, N.A. Reisdorph, Analysis of 25 underivatized amino acids in human plasma using ion-pairing reversed-phase liquid chromatography/time-of-flight mass spectrometry, Rapid Commun Mass Spectrom, 21 (2007) 2717-2726. • M.S. Choi, S.U. Rehman, I.S. Kim, H.J. Park, M.Y. Song, H.H. Yoo, Development of a mixed-mode chromatography with tandem mass spectrometry method for the quantitative analysis of 23 underivatized amino acids in human serum, J Pharmaceut Biomed, 145 (2017) 52-58. • Lipid mediators/Oxylipins • M. Armstrong, A.H. Liu, R. Harbeck, R. Reisdorph, N. Rabinovitch, N. Reisdorph, Leukotriene-E4 in human urine: Comparison of on-line purification and liquid chromatography-tandem mass spectrometry to affinity purification followed by enzyme immunoassay, J Chromatogr B AnalytTechnol Biomed Life Sci, 877 (2009) 3169-3174. • D. Balgoma, J. Larsson, J. Rokach, J.A. Lawson, K. Daham, B. Dahlen, S.E. Dahlen, C.E. Wheelock, Quantification of lipid mediator metabolites in human urine from asthma patients by electrospray ionization mass spectrometry: controlling matrix effects, Anal Chem, 85 (2013) 7866-7874. • D. Balgoma, M. Yang, M. Sjodin, S. Snowden, R. Karimi, B. Levanen, H. Merikallio, R. Kaarteenaho, L. Palmberg, K. Larsson, D.J. Erle, S.E. Dahlen, B. Dahlen, C.M. Skold, A.M. Wheelock, C.E. Wheelock, Linoleic acid-derived lipid mediators increase in a female-dominated subphenotype of COPD, EurRespir J, 47 (2016) 1645-1656. • Endocannabinoids • S. Gouveia-Figueira, M.L. Nording, Validation of a tandem mass spectrometry method using combined extraction of 37 oxylipins and 14 endocannabinoid-related compounds including prostamides from biological matrices, Prostaglandins Other Lipid Mediat, 121 (2015) 110-121. • Nucleotides/Nucleosides/cofactors • C.R. Martens, B.A. Denman, M.R. Mazzo, M.L. Armstrong, N. Reisdorph, M.B. McQueen, M. Chonchol, D.R. Seals, Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD(+) in healthy middle-aged and older adults, Nat Commun, 9 (2018) 1286. • Bile acids • M.H. Sarafian, M.R. Lewis, A. Pechlivanis, S. Ralphs, M.J. McPhail, V.C. Patel, M.E. Dumas, E. Holmes, J.K. Nicholson, Bile acid profiling and quantification in biofluids using ultra-performance liquid chromatography tandem mass spectrometry, Anal Chem, 87 (2015) 9662-9670. • Lipid quantitation • P.T. Ivanova, S.B. Milne, M.O. Byrne, Y. Xiang, H.A. Brown, Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry, Methods Enzymol, 432 (2007) 21-57.

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