Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 1 Nitrated Fatty Acids Formation and Actions Bruce Freeman, PhD Department of Pharmacology University of Pittsburgh School of Medicine
Jan 12, 2016
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 1
Nitrated Fatty AcidsFormation and Actions
Bruce Freeman, PhDDepartment of Pharmacology
University of Pittsburgh School of Medicine
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 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 3
Fatty AcidsFatty acids have common names, IUPAC names and acronyms
Linoleic AcidOctadeca-9,12-dienoic acid18:2 LA
Arachidonic AcidEicosatetraenoic AcidDodeca-5,8,11,14-tetraenoic acid20:4AA
O
HO
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)
OH
O
Endogenous fatty acids have the cis-stereochemical orientation
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 4
• NO manifests both pro-oxidant and antioxidant actions
• Reactions of NO-derived species with oxidizing unsaturated fatty acids yields NO2 derivatives
LNO2 Formation
J Biol Chem 269:28066-28075, 1994
A Crucial Experiment
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 5
Species Compartment Concentration (nM)
NO-2 Plasma
RSNO
Plasma
LNO2 Plasma
Free
Esterified
Total
Hb-NO Blood < 50
Hb-SNO
Blood
< 50
LNO2
Packed red cells Free Esterified
Total
LNO2 Blood
205 +_ 217.2 +_ 1.1
79 +_ 35
550 +_ 275 630 +_ 240
50 + 17_ 199 +_ 121
477 +_ 128*Total
TotalTotal
TotalTotal
Fraction
3-Nitro-tyrosine Plasma 0.7 +_ 0.3 Total
249 +_ 104
OANO2
Plasma
Free Esterified
Total
619 +_ 52
302 +_ 369 921 +_ 421
ONO2
Packed red cells Free
Esterified Total
59 + 11_ 155 +_ 65 214 +_ 76
OANO2
Blood
639 +_ 366*
Total
Nitrated Fatty Acids – Most Abundant BioactiveOxides 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 6
16:1
18:1
18:2
18:3
20:4
20:5
-NO2-(OH)NO2
Time (min)5 10 15 5 10 15
Plasma Urine
Time (min)
Bas
e P
eak
Inte
nsi
ty
5 10 15 5 10 15
-NO2-(OH)NO2
All Unsaturated Fatty Acid Species Display NitratedDerivatives In Healthy Human Plasma and Urine
J Biol Chem, in press 2005
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 7
Inflammatory Conditions Induce Oleic Acid Nitration Fatty Acid Micellar Emulsion
Control
NO2-, pH 3.0
MPO/H2O2/ NO2-
ONOO-
0.12
0.76
1.45
3.31
Condition NO2-oleic acid (nM)
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
*
*
*
J Biol Chem, in press 2005
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 8
Inflammatory Conditions Induce Fatty Acid Nitration Intraperitoneal Injection of LPS in Mice
* *
* *
* p< 0.05, 3-4 animals
*
* *
*
Condition NO2-oleic acid NO2-linoleic acid
Heart (nmol/g)
Control
LPS
Lung (nmol/g)
Control
LPS
Plasma (nM)
Control
LPS
Kidney (nmol/g)
Control
LPS
38
323
0.017
0.066
0.017
0.060
0.030
0.215
29
120
0.006
0.030
0.014
0.044
0.009
0.069
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 9
Inflammatory Conditions Induce Fatty Acid Nitration Acute Respiratory Distress Syndrome Patients
* p< 0.05
Condition NO2-oleic acid NO2-linoleic acid
Matched plasma (nmol/mg prot) - 3.0 ± 0.7
Pulmonary edema fluid - 45 ± 6*
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 10
Mechanisms of Fatty Acid Nitration
• Acidic nitration• Enzymatic?
• Dietary sources
NO2+
NO2O2N
O2HOO
H H
O2N
NO2
(Nitronium Ion)Hydrogen
Abstraction
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 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 12
Synthesis of Nitrated Linoleic Acid
PNAS 101:11577-11582, 2004
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 13
Detection/Characterization of LNO2
Method Results Comments
GC MS (EI)Structural data from standard
1. Very low sensitivity2. Derivatization
GC MS (NICI)In vivo detection
of LNO2
1. Good sensitivity2. Extensive sample
preparation3. Sample degradation
3-D Ion Trap ESI MS/MS
Structural data from standards
1. Low sensitivity2. MS3 and above,
not realistic
→ Unable to characterize/quantitate in vivo samples
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 14
Triple Quadrupole MS
Q1 Q2 Q3
CollisionChamberSource Detector
• 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 15
10
Peak 1 Peak 2
12 14
Time (min)10
Peak 1 Peak 2
12 14Time (min)
10
Peak 1 Peak 2
12 14Time (min)
Synthetic LNO2 Red Cell Plasma
HPLC Elution Profile for LNO2 Regioisomers
MRM transition: 324 [M-H]- / 277 [M-HNO2]-
-O
O NO2
-O
O O2NPeak 1
Peak 2
PNAS 101:11577-11582, 2004
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 16
157
171
196 233244
277
293
306324
x 7
150 200 250 300m/z
x 10
150 200 250 300m/z
Rel
ativ
e In
tens
ity
196171
277
244
293
306
233157157
171196
233244
277
293
306324
x 5
150 200 250 300m/z
277196
Standard Red Blood Cell Plasma
O
-O
NO2
10
Peak 1 Peak 2
12 14
Time (min)
Product Ion Analysis of LNO2
Regioisomers Peak 1PNAS 101:11577-11582, 2004
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 17
x 19
150 200 250 300
m/z
Rel
ativ
e In
tens
ity
277
244233
168
306324
228 168
x 7
228233
244
277
306
324
150 200 250 300
m/z
168
228
233
244
277
306
324x 5
m/z
150 200 250 300
277
228
Standard Red Blood Cell Plasma
O
-O
NO2
10
Peak 1Peak 2
12 14
Time (min)
Product Ion Analysis of LNO2
Regioisomers Peak 2 PNAS 101:11577-11582, 2004
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 18
PlasmaOA-NO2
Red CellOA-NO2
279
326
308
246
168
197
5X
279
326
308
246
168
197
5X
120 160 200240 280320m/z
SyntheticOA-NO2
279
326
308246168197
Synthetic[C]OA-NO13
2
120 160 200240 280320m/z
295
344
326308
2642X
9 10
9 10
9 10
9 10
time (min)
time (min)J Biol Chem, in press 2005
Nitrated Oleic Acid (OA-NO2) is Also Present in Human Blood
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 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 20
1 2 3 4 5 6 7
324/277 (LNO2)
342/295 (13C-LNO2)
1010.10.010.001
5005050.50.05
10
1
0.1
0.01
0.001
[LNO2]/[IS]
ng/ml LNO2
y = 0.969x + 0.002r = 0.998
LOQ ~ 300 amol on columnStandard curve linear over 5-orders of magnitude
Quantitative Analysis of LNO2 in Clinical Samples
LNO
2 p
eak
are
aIS
pea
k a
rea
PNAS 101:11577-11582, 2004
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 21
Complex Lipid Nitro-Fatty Acid DerivativesMembrane and Lipoprotein Reservoirs?
“Stable” LNO2 StoragePLA2
-O
O NO2
O PO
OO -
N +LNO2
Palmitate
Cell SignalingNO2
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
NO 2
NO2
NO2NO2
NO2
NO2 NO2
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 22
0 2 4 6 8
0
400
800
1200
1600 OA-NO2
LNO2
+ Lipase
- Lipase
Time (hr)
Rel
ease
d F
atty
Aci
d (
nM
)
Lipase-Treated Complex Biological Lipids Release Nitrated Fatty Acids
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 23
+Prec (184.20): Exp 1, 10.761 to 29.474 min from Sample 1 (PC Mix) of Chromatography Test.wif... Max. 3.3e5 cps.
600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900m/z, amu
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
Inte
ns
ity, c
ps
758.8
782.8
786.8760.8
703.8
813.8
780.8788.8 815.8
756.8 816.8734.8762.8731.8 810.8705.8
735.8 806.8744.8675.8 802.8770.8 841.8720.8 846.8691.8 875.0 899.0650.6604.8 636.8622.8 695.6
Precursor scan m/z 184—molecular species of phosphatidylcholine
O PO
OO -
N +18:2
16:0
O PO
OO -
N +20:4
16:0
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 24
+Prec (184.20): 2.178 to 7.555 min from Sample 1 (PC + nitrated PC) of Precursor 184.wiff (Turb... Max. 2.9e5 cps.
770 775 780 785 790 795 800 805 810 815 820 825 830m/z, amu
0.00
500.00
1000.00
1500.00
2000.00
2500.00
3000.00
3500.00
4000.00
4500.00
5000.00
5500.00
6000.00
6500.00
7000.00
7500.00
8000.00
8500.00
9000.00
9500.00
1.00e4
Inte
ns
ity, c
ps
803.8
804.8
821.8
814.8
790.8788.8 798.8774.8 805.8 822.8819.8772.8 815.8812.8830.8791.8789.8 799.8775.8 828.8786.8780.8 801.8773.8 817.6807.8 818.6794.6 811.8770.8 824.8 827.8
Precursor Analysis of Nitrated Phosphatidylcholine (m/z 184)
O PO
OO -
N +LNO2
Palmitate
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 25
-EPI (787.50) CE (-45): 0.227 to 0.692 min from Sample 8 (EPI of 787) of nitrated PC.wiff (Turbo ... Max. 7.2e4 cps.
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800m/z, amu
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e4
Inte
ns
ity, c
ps
787.5
255.2
480.3
741.5306.2
277.4 324.2549.3 658.6462.2 644.4 701.4 769.4757.5
EPI Analysis of Nitrated PC
Palmitate
LNO2
M[-CH3]
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 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 27
Agilent Array Analysis of Cell Gene Expression Responses to LNO2
Human Monocytes, Endothelium
• Transcription and initiation factors
• Adhesion molecules, cytokines
• Enzymes, receptors
HO-1, PPAR-linked genesNitrolinoleic acid regulates 5504 genes
Up 2376 genesDown 3128 genes
Linoleic acid regulates 14 genes – 5 up, 9 down
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 28
NO Release From NitroalkenesVia Modified Nef Reaction
JBC 280:19289-19297, 2005
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 29
100
50
0
LNO2 LNO2
+ODQ
LNO2
+OxyHb
*
*
1010.10.01
0
20
40
60
80
100
LNO2 (µM)
(-) Endothelium
(+ ) Endothelium
L-NAME (30 µM)
Linoleate
Methanol
% R
elax
atio
n
% R
ever
sal R
elax
atio
n
Nitrolinoleate Mediates NO-Like Vascular Signaling
PNAS 99:15941-46, 2002
0 0.3 1 SNP (10 µM)0
2
4
6
cGM
P (
pm
ol/
mg
cel
l p
rote
in)
LNO2 (µM)
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 30
Nitroalkenes are Hydrophobically StabilizedOctyl-thio--glucopyranoside CMC=2.8 mg/mlOctyl--glucopyranoside CMC=7.3 mg/ml
[NO
] (µ
M/h
r)
EPR oxyMb
JBC 280:19289-19297, 2005
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 31
NO + 2o reaction products
K
k
EsterificationK
InflammationPLA2
PPAR activation
NO2
HO
O NO2
Kd
Kdk
HO
O NO2
NO2NO2
NO2
NO2
NO2
NO2
NO2
NO2
Hydrophobic Stabilization of LNO2
JBC 280:19289-19297, 2005
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 32
Nucleus
Cytoplasm
AGGTCA (N) AGGTCA
Target genesRXR
PPRE
PPAR
Coactivator
Co-repressor
Degradation
PPARLigands
P
u u
9-cis-RA
PPAR-Regulated Gene Expression
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 33
Nitroalkenes Induce PPAR-Dependent Cell Responses
• Induce CD36 expression• Inhibit TNF, IL-1, MCP expression, NFB• Inhibit vascular adhesion
Monocytes
Adipocytes• Induction of adipogenesis• Induce PPAR 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 34
Reporter Constructs for AnalyzingPPAR Ligand Activity
Nuclear receptor LBDs (ligand binding domain)
Gal 4 DBD (DNA binding domain)
Luciferase Luciferase activity
4 X Gal4
Full length PPARs
Luciferase Luciferase activity
3 X PPRE
Normalized to GFPCV-1 cells
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 35
LNO2 is a Specific PPAR Agonist
0
2
4
6
8
Vector
Control
LNO2
+ - + -+ -+ -
+- +- +- +-
*
*
*
Rel
ativ
e L
uci
fera
se A
ctiv
ity
PPARs
LNO2 Concentration (µM)
1
3
5
7
0 0.01 0.1 1 10
PPAR
PPAR PPAR
*
* *
Re
lati
ve
L
uc
ife
ras
e A
cti
vit
y
AR GR MR PR RXR
+ -+ -+ -+ -+ -
+-+- +- +- +-
Other nuclear receptorsPNAS 102:2340-2345, 2005
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 36
0 20 40 60 80 100 120Time (min)
100
80
60
40
20
01
1.5
2
2.5
3
0 0.2 0.4 0.6 0.8 1
Concentration (NO2-OA)
PPAR
PPAR
PPAR
0
3
6
9
NO2-OA(1 µM)
LNO2
(3 µM)Meth
LNO2
NO2-OA
Rel
ati
ve
Lu
cife
ras
e A
ctiv
ity
Rel
ati
ve
Lu
cife
ras
e A
ctiv
ity
Rem
ain
ing
nit
rate
d f
atty
aci
d (
%)
NO2-OA > LNO2 “potency” may be due to stability
NO2-OA is a More Potent PPAR LigandHO-1 expressionInhibition of VCAM-1 expressionAdipocyte differentiation, glc uptake
PNAS 102:2340-2345, 2005
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 37
Nitroalkenes Undergo Reversible Michael Addition Reactions with Nucleophiles - Thiols
N+O
O-
R'
H
H
RS- N+O-
O-
R'
H
H
RS
N+O-
O-
R'
H
H
RS
H+
N+O-
O-
R'
H
H
RS
H
200 300 400 500 600
m/z
0
25
50
75
100
Rel
ativ
e A
bu
nd
ance
306.3
324.2
631.3
GSH
LNO2
LNO2-GSH
Submitted
Nitroalkylation
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 38
Protein Nitroalkylation
4426 32 38Mass (103 m/z)
PurifiedGAPDH
GAPDH+
NO2-OA
GAPDHNO2-OA
100M GSH
MALDI-TOF-MS
2-D Ion Trap
327
327
327
327
Submitted
Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 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
speciescan 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 40
Eugene Chen, PhD – MSMBruce Branchaud, PhD - UO