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Nitrated Fatty Acids Formation and Actions Bruce Freeman, PhD Department of Pharmacology

Nitrated Fatty Acids Formation and Actions Bruce Freeman, PhD Department of Pharmacology University of Pittsburgh School of Medicine. Objectives of Course Lecture and Supplied Materials. Discuss the biological formation and structural characterisitics of nitrated fatty acid derivatives

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Nitrated Fatty Acids Formation and Actions Bruce Freeman, PhD Department of Pharmacology

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  1. Nitrated Fatty Acids Formation and Actions Bruce Freeman, PhD Department of Pharmacology University of Pittsburgh School of Medicine Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 1

  2. Objectives of Course Lecture and Supplied Materials • Discuss the biological formation and structural characterisitics of nitrated fatty acid derivatives • Teach how to synthesize, purify, quantify and handle nitrated fatty acids • Convey present knowledge about the cell signaling actions of nitrated fatty acids Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 2

  3. Fatty Acids Fatty acids have common names, IUPAC names and acronyms Linoleic Acid Octadeca-9,12-dienoic acid 18:2 LA O HO Arachidonic Acid Eicosatetraenoic Acid Dodeca-5,8,11,14-tetraenoic acid 20:4 AA O O H Myristic Acid (14:0) Linolenic Acid (18:3) Palmitic Acid (16:0) Arachidic Acid (20:0) Stearic Acid (18:0) Docosahexanoic acid (22:6) Oleic Acid (18:1) Endogenous fatty acids have the cis-stereochemical orientation Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 3

  4. LNO2 Formation A Crucial Experiment • NO manifests both pro-oxidant and antioxidant actions • Reactions of NO-derived species with oxidizing unsaturated fatty acids yields NO2 derivatives J Biol Chem 269:28066-28075, 1994 Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 4

  5. Species Compartment Fraction Concentration (nM) - _ + Total NO Plasma 205 21 2 _ + Total 7.2 1.1 RSNO Plasma _ + 0.7 0.3 Total 3-Nitro-tyrosine Plasma Total Hb-NO Blood < 50 < 50 Hb-SNO Blood Total _ + 79 35 Free Plasma LNO 2 _ + 550 275 Esterified _ + 630 240 Total _ + 619 52 Free Plasma OANO 2 _ + 302 369 Esterified _ + 921 421 Total _ + 17 Free 50 Packed red cells LNO 2 _ + Esterified 199 121 _ + 249 104 Total _ + 11 Free 59 Packed red cells ONO 2 _ + Esterified 155 65 _ + 214 76 Total _ + Total 477 128* LNO Blood 2 _ + Total 639 366* OANO Blood 2 Nitrated Fatty Acids – Most Abundant Bioactive Oxides of Nitrogen in the Vascular Compartment PNAS 101:11577-11582, 2004 J Biol Chem, 2005 Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 5

  6. Plasma Urine -(OH)NO2 -(OH)NO2 -NO2 -NO2 16:1 18:1 Base Peak Intensity 18:2 18:3 20:4 20:5 5 10 15 5 5 10 15 10 15 5 10 15 Time (min) Time (min) All Unsaturated Fatty Acid Species Display Nitrated Derivatives In Healthy Human Plasma and Urine J Biol Chem, in press 2005 Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 6

  7. Condition NO2-oleic acid (nM) Control 0.12 * NO2-, pH 3.0 0.76 * MPO/H2O2/ NO2- 1.45 * ONOO- 3.31 MPO- 50 nM, H2O2- 600 µM, NO2-- 100 µM, ONOO-- 1.5 mM Linoleic acid nitration results can be found in: O’Donnell et al. Chem Res Tox 12: 83-92, 1999. * p< 0.05 Inflammatory Conditions Induce Oleic Acid NitrationFatty Acid Micellar Emulsion J Biol Chem, in press 2005 Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 7

  8. Condition NO2-oleic acid NO2-linoleic acid Plasma (nM) Control 38 29 * * LPS 323 120 Lung (nmol/g) Control 0.017 0.006 * * LPS 0.066 0.030 Heart (nmol/g) Control 0.017 0.014 * * LPS 0.060 0.044 Kidney (nmol/g) Control 0.030 0.009 * * LPS 0.215 0.069 * p< 0.05, 3-4 animals Inflammatory Conditions Induce Fatty Acid NitrationIntraperitoneal Injection of LPS in Mice Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 8

  9. Inflammatory Conditions Induce Fatty Acid NitrationAcute Respiratory Distress Syndrome Patients Condition NO2-oleic acid NO2-linoleic acid Matched plasma (nmol/mg prot) - 3.0 ±0.7 Pulmonary edema fluid - 45 ± 6* * p< 0.05 Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 9

  10. Mechanisms of Fatty Acid Nitration Hydrogen (Nitronium Ion) NO2+ Abstraction • Dietary sources H H • Acidic nitration O N 2 • Enzymatic? O N O 2 2 O N H O O N O 2 2 Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 10

  11. Why Were Nitrated Unsaturated Fatty Acids (Nitroalkenes) Not Identified and Studied Sooner? • First identified in 1912 (Zelinsky), 1991 (Finlayson-Pitts) • To observe clinically-occurring nitrated fatty acids, required development of sensitive MS capabilities • Decay rapidly with traditional lipid extraction and FA derivatization techniques used for GC-MS • NO2 group leaves peptides as m/z = 45 in positive ion mode, leaves NO2-FA adducts as m/z = 47 • NO2-FA adducts of proteins “lost” with typical thiol-based protein reduction and electrophoresis strategies • NO2-FA adducts of proteins are retained on column with typical acetonitrile gradients used for peptide separation Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 11

  12. Synthesis of Nitrated Linoleic Acid PNAS 101:11577-11582, 2004 Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 12

  13. Detection/Characterization of LNO2 Method Results Comments Structural data from standard 1. Very low sensitivity 2. Derivatization GC MS (EI) 1. Good sensitivity 2. Extensive sample preparation 3. Sample degradation In vivo detection of LNO2 GC MS (NICI) • Low sensitivity • MS3 and above, not realistic 3-D Ion Trap ESI MS/MS Structural data from standards → Unable to characterize/quantitate in vivo samples Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 13

  14. Q1 Q2 Q3 Collision Chamber Detector Source Triple Quadrupole MS • MS– Scan specific mass range in either Q1 or Q3 • Product Ion Analysis– Set Q1 for specific mass; Q3 for product range • Precursor Ion Analysis – Set Q3 for a specific product mass; Q1 for precursor range • Multiple Reaction Monitoring (MRM) – Set Q1 for a specific precursor; set Q3 for a specific product Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 14

  15. HPLC Elution Profile for LNO2 Regioisomers Peak 2 Peak 1 Synthetic LNO2 Red Cell Plasma Peak 2 Peak 2 Peak 2 Peak 1 Peak 1 Peak 1 Relative Intensity 10 12 14 10 12 14 10 12 14 Time (min) Time (min) Time (min) MRM transition: 324 [M-H]- / 277 [M-HNO2]- Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 15 PNAS 101:11577-11582, 2004

  16. Product Ion Analysis of LNO2 Regioisomers Peak 1 PNAS 101:11577-11582, 2004 Peak 1 Peak 2 NO2 O 277 196 -O 10 12 14 Time (min) Standard Red Blood Cell Plasma 277 277 277 x 7 x 5 x 10 171 196 171 244 306 157 233 Relative Intensity 233 324 244 196 157 233 171 293 293 157 293 244 196 324 306 306 150 200 250 300 150 200 250 300 150 200 250 300 m/z m/z m/z Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 16

  17. Product Ion Analysis of LNO2 Regioisomers Peak 2 PNAS 101:11577-11582, 2004 Peak 2 Peak 1 O NO2 277 -O 228 10 12 14 Time (min) Red Blood Cell Standard Plasma 277 277 277 x 5 x 19 x 7 324 233 244 228 228 168 233 Relative Intensity 233 168 168 244 244 228 324 306 324 306 306 150 200 250 300 150 200 250 300 150 200 250 300 m/z m/z m/z Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 17

  18. 279 Synthetic 326 OA-NO 2 308 246 197 168 Relative Intensity 2X 9 10 295 264 Synthetic 344 308 [C]OA-NO 13 326 2 Relative Intensity 9 10 120 160 200 240 280 320 time (min) m/z 5X 326 246 279 197 Plasma OA-NO 308 168 2 Relative Intensity 5X 9 10 326 246 279 Red Cell 197 OA-NO 2 308 168 Relative Intensity 9 10 120 160 200 240 280 320 time (min) m/z Nitrated Oleic Acid (OA-NO2) is Also Present in Human Blood J Biol Chem, in press 2005 Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 18

  19. MS-Based Quantitation of NO2-FA • Include [13C]-fatty acid and [15N]-nitrite during sample processing and analysis to detect artifactual fatty acid nitration • Evaluate ranges of pH and adventitious NO2- • Evaluate impact of oxidized lipid derivatives • Use [13C]LNO2 and [13C]NO2-OA as internal standards to correct for sample preparation loss • Internal standard curve linear over 5-orders of magnitude; limit of quantitation = 300 amol on column Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 19

  20. Quantitative Analysis of LNO2 in Clinical Samples 10 324/277 (LNO2) y = 0.969x + 0.002 r = 0.998 1 LNO2 peak area IS peak area 0.1 342/295 (13C-LNO2) 0.01 0.001 2 0.001 0.01 0.1 10 1 LNO peak area/IS peak area 3 5 6 1 2 4 7 [LNO2]/[IS] 0.05 0.5 5 50 500 PNAS 101:11577-11582, 2004 ng/ml LNO 2 LOQ ~ 300 amol on column Standard curve linear over 5-orders of magnitude Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 20

  21. NO 2 Complex Lipid Nitro-Fatty Acid Derivatives Membrane and Lipoprotein Reservoirs? NO2 NO2 NO2 Palmitate LNO2 O O P O N + O - NO 2 NO 2 NO 2 PLA2 “Stable” LNO2 Storage Linoleic acid represents ~10% of net fatty acids in cells, with ~0.1-1.0% of net linoleate nitrated ~10-100,000 molecules LNO2/cell NO2 Cell Signaling Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 21

  22. Lipase-Treated Complex Biological Lipids Release Nitrated Fatty Acids OA-NO2 LNO2 + Lipase Released Fatty Acid (nM) - Lipase Time (hr) Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 22

  23. 16:0 16:0 20:4 18:2 O O O P O O P O N + N + O - O - Precursor scan m/z 184—molecular species of phosphatidylcholine Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 23

  24. Precursor Analysis of Nitrated Phosphatidylcholine (m/z 184) Palmitate LNO2 O O P O N + O - Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 24

  25. EPI Analysis of Nitrated PC M[-CH3] Palmitate LNO2 Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 25

  26. Nitroalkene Derivatives Redox-derived fatty acid derivatives that display unique signaling reactivities • Acidic hydrogen on C-N bond of nitroalkene capable of acid-base chemistry (Nef reaction), leading to NO release and cGMP-dependent signaling • In “native” form, function as high affinity ligands for the nuclear receptor family PPAR • C-N bond undergoes Michael addition reactions with nucleophiles (Cys, His) Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 26

  27. Agilent Array Analysisof Cell Gene Expression Responses to LNO2 Human Monocytes, Endothelium • Transcription and initiation factors • Adhesion molecules, cytokines • Enzymes, receptors HO-1, PPAR-linked genes Nitrolinoleic acid regulates 5504 genes Up 2376 genes Down 3128 genes Linoleic acid regulates 14 genes – 5 up, 9 down Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 27

  28. NO Release From Nitroalkenes Via Modified Nef Reaction JBC 280:19289-19297, 2005 Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 28

  29. * * Nitrolinoleate Mediates NO-Like Vascular Signaling 100 % Reversal Relaxation 100 50 80 0 60 (-) Endothelium LNO2 + OxyHb LNO2 LNO2 + ODQ (+ ) Endothelium % Relaxation 40 L-NAME (30 µM) 20 Linoleate 6 0 cGMP (pmol/mg cell protein) Methanol 4 0.01 0.1 10 1 2 LNO2 (µM) 0 0 0.3 1 SNP (10 µM) LNO2 (µM) Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 29 PNAS 99:15941-46, 2002

  30. Nitroalkenes are Hydrophobically Stabilized Octyl-thio-b-glucopyranoside CMC=2.8 mg/ml Octyl-b-glucopyranoside CMC=7.3 mg/ml oxyMb EPR ) r h / M µ ( ] O N [ Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 30 JBC 280:19289-19297, 2005

  31. O O NO2 NO2 HO HO Hydrophobic Stabilization of LNO2 Inflammation PLA2 NO2 NO2 NO2 NO2 NO2 Kd Esterification k K PPARg activation Kd k NO + 2o reaction products K NO2 NO2 NO2 NO2 Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 31 JBC 280:19289-19297, 2005

  32. PPAR u u PPAR-Regulated Gene Expression Cytoplasm Degradation Nucleus PPAR Ligands Co-repressor P Coactivator 9-cis-RA Target genes RXR AGGTCA (N) AGGTCA PPRE Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 32

  33. Nitroalkenes Induce PPARg-Dependent Cell Responses Monocytes • Induce CD36 expression • Inhibit TNF, IL-1, MCP expression, NFkB • Inhibit vascular adhesion Adipocytes • Induction of adipogenesis • Induce PPARg and aP2 expression • Increased glucose uptake Pulmonary Endothelium • Inhibition of TNF-induced VCAM-1 expression Alveolar Epithelium • Lipid metabolism, apoptosis, redox signaling Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 33

  34. Nuclear receptor LBDs (ligand binding domain) Gal 4 DBD (DNA binding domain) Full length PPARs Reporter Constructs for AnalyzingPPAR Ligand Activity Luciferase activity Luciferase 4 X Gal4 Normalized to GFP CV-1 cells Luciferase activity Luciferase Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 34 3 X PPRE

  35. 7 PPARg PPARa 5 PPARd Relative Luciferase Activity * 3 * * 1 10 0 0.01 0.1 1 LNO2 Concentration (µM) - - - - - + + + + + - - - - - + + + + + RXRa MR PR AR GR Other nuclear receptors LNO2 is a Specific PPAR Agonist 8 * 6 * Relative Luciferase Activity 4 Relative Luciferase Activity * Relative Luciferase Activity 2 0 Control - - - - + + + + LNO2 - - - - + + + + g a d Vector PPARs Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 35 PNAS 102:2340-2345, 2005

  36. NO2-OA is a More Potent PPARg Ligand HO-1 expression Inhibition of VCAM-1 expression Adipocyte differentiation, glc uptake PPARg 3 9 100 NO2-OA 2.5 80 PPARa 6 60 Relative Luciferase Activity 2 Remaining nitrated fatty acid (%) Relative Luciferase Activity 40 3 PPARd LNO2 1.5 20 Nitrated lipid Remaining (%) Relative Luciferase Activity Relative Luciferase Activity 0 0 1 0 20 40 60 80 100 120 Meth NO2-OA (1 µM) LNO2 (3 µM) 0 0.2 0.4 0.6 0.8 1 Time (min) Concentration (NO2-OA) NO2-OA > LNO2 “potency” may be due to stability Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 36 PNAS 102:2340-2345, 2005

  37. Nitroalkenes Undergo Reversible Michael Addition Reactions with Nucleophiles - Thiols GSH 306.3 100 H H LNO2 R ' O - R ' O + N R S - + N 75 324.2 - R S H O - H O LNO2-GSH Relative Abundance 50 H + H H H 631.3 - R ' O 25 - R ' O + N + N R S R S - H O - H O 0 200 300 400 500 600 m/z Nitroalkylation Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 37 Submitted

  38. 327 327 327 327 44 26 32 38 Mass (103 m/z) Protein Nitroalkylation Purified GAPDH GAPDH + NO2-OA GAPDH NO2-OA 100 mM GSH 2-D Ion Trap Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 38 MALDI-TOF-MS Submitted

  39. NO and Fatty Acid Signaling Converge NO terminates lipid radicals, inhibiting propagation reactions and preserving lipophilic antioxidants NO-derived species either stimulate or inhibit lipid and lipoprotein oxidation, depending on basal oxidative and inflammatory conditions NO is consumed by reactions of eicosanoid biosynthesis, gene expression and catalytic activity of eicosanoid biosynthetic enzymes regulated by NO Nitrated fatty acids transduce NO and NO2- signaling formed by reactions of NO and NO2-derived species can be misidentified as other NOx derivatives signal via receptor-dependent mechanisms and unique chemical reactivities Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 39

  40. Eugene Chen, PhD – MSM Bruce Branchaud, PhD - UO Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 40

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