Download Slide 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
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
Fatty Acids
Fatty acids have common names, IUPAC names and acronyms
Linoleic Acid
Octadeca-9,12-dienoic acid
18:2 LA
O
HO
O
OH
Myristic Acid (14:0)
Palmitic Acid (16:0)
Stearic Acid (18:0)
Oleic Acid (18:1)
Endogenous
fatty
Sunrise
Free Radical School
Arachidonic Acid
Eicosatetraenoic Acid
Dodeca-5,8,11,14-tetraenoic acid
20:4
AA
Linolenic Acid (18:3)
Arachidic Acid (20:0)
Docosahexanoic acid (22:6)
acids have
the2005
cis-stereochemical
orientation
SFRBM
Austin,Tx
Bruce Freeman 3
A Crucial Experiment
LNO2 Formation
• 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
Nitrated Fatty Acids – Most Abundant Bioactive
Oxides of Nitrogen in the Vascular Compartment
Species
Compartment
NO2
RSNO
3-Nitro-tyrosine
Hb-NO
Hb-SNO
Plasma
Plasma
Plasma
Blood
Blood
LNO2
Plasma
OANO
2
Plasma
LNO2
Packed red cells
ONO2
Packed red cells
Fraction
Total
Total
Total
Total
Total
Concentration (nM)
_ 21
205 +
_ 1.1
7.2 +
_ 0.3
0.7 +
< 50
< 50
Free
Esterified
Total
Free
Esterified
Total
+ 35
79 _
_ 275
550 +
_ 240
630 +
619 +_ 52
_ 369
302 +
_ 421
921 +
Free
Esterified
Total
Free
Esterified
Total
+ 17
50 _
199 +_ 121
249 +_ 104
59 _+ 11
155 +_ 65
214 +_ 76
LNO2
Blood
Total
OANO 2
Blood
Total
_ 128*
477 +
_ 366*
639 +
PNAS 101:11577-11582, 2004
J Biol Chem, 2005
Sunrise Free Radical School
SFRBM 2005 Austin,Tx
Bruce Freeman 5
All Unsaturated Fatty Acid Species Display Nitrated
Derivatives In Healthy Human Plasma and Urine
Base Peak Intensity
Plasma
-(OH)NO2
-NO2
Urine
-(OH)NO2
-NO2
16:1
18:1
18:2
18:3
20:4
20:5
5
Sunrise Free Radical School
10 15 5 10 15 5
Time (min)
10 15 5 10 15
Time (min)
SFRBM 2005 Austin,Tx
J Biol Chem, in press 2005
Bruce Freeman 6
Inflammatory Conditions Induce Oleic Acid Nitration
Fatty Acid Micellar Emulsion
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
J Biol Chem, in press 2005
Sunrise Free Radical School
SFRBM 2005 Austin,Tx
Bruce Freeman 7
Inflammatory Conditions Induce Fatty Acid Nitration
Intraperitoneal Injection of LPS in Mice
Condition
NO2-oleic acid
NO2-linoleic acid
Plasma (nM)
Control
38
29
LPS
323 *
120 *
Control
0.017
0.006
LPS
0.066*
0.030 *
0.017
0.060 *
0.014
0.044 *
0.030
0.215 *
0.009
0.069 *
Lung (nmol/g)
Heart (nmol/g)
Control
LPS
Kidney (nmol/g)
Control
LPS
* p< 0.05, 3-4 animals
Sunrise Free Radical School
SFRBM 2005 Austin,Tx
Bruce Freeman 8
Inflammatory Conditions Induce Fatty Acid Nitration
Acute 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
Mechanisms of Fatty Acid Nitration
Hydrogen
Abstraction
NO2+ (Nitronium Ion)
H
• Dietary sources
• Acidic nitration
• Enzymatic?
H
O2N
O2
NO2
O2N
HOO
NO2
Sunrise Free Radical School
SFRBM 2005 Austin,Tx
Bruce Freeman 10
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
Synthesis of Nitrated Linoleic Acid
PNAS 101:11577-11582, 2004
Sunrise Free Radical School
SFRBM 2005 Austin,Tx
Bruce Freeman 12
Detection/Characterization of LNO2
Method
GC MS (EI)
GC MS (NICI)
3-D Ion Trap
ESI MS/MS
Results
Comments
Structural data
from standard
1. Very low sensitivity
2. Derivatization
In vivo detection
of LNO2
1. Good sensitivity
2. Extensive sample
preparation
3. Sample degradation
Structural data from
standards
1. Low sensitivity
2. MS3 and above,
not realistic
→ Unable to characterize/quantitate in vivo samples
Sunrise Free Radical School
SFRBM 2005 Austin,Tx
Bruce Freeman 13
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
Q1
Source
Sunrise Free Radical School
Q2
Collision
Chamber
SFRBM 2005 Austin,Tx
Q3
Detector
Bruce Freeman 14
HPLC Elution Profile for LNO2 Regioisomers
O
NO2
Peak 2
-O
O
O2N
Peak 1
-O
Synthetic LNO2
Peak 1
Peak 2
Red Cell
Peak 1
Peak 2
Plasma
Peak 1
Peak 2
Relative Intensity
10
12
14
Time (min)
10
12
14
Time (min)
10
12
14
Time (min)
MRM transition: 324 [M-H]- / 277 [M-HNO2]Sunrise Free Radical School
SFRBM 2005 Austin,Tx
PNAS
101:11577-11582,
Bruce
Freeman 2004
15
Product Ion Analysis of LNO2
Regioisomers Peak 1
PNAS 101:11577-11582, 2004
Peak 1
NO2
O
Peak 2
277
196
-O
10
12
14
Time (min)
Standard
Red Blood Cell
Relative Intensity
277
277
171196
244
233
293
306
150
277
x7
171
x 10
157
Plasma
200 250
m/z
Sunrise Free Radical School
300
157
x5
157
196 233
244 293
324
306
150 200 250 300
m/z
SFRBM 2005 Austin,Tx
171
233
244
196
306
324
293
150 200 250 300
m/z
Bruce Freeman 16
Product Ion Analysis of LNO2
Regioisomers Peak 2
PNAS 101:11577-11582, 2004
Peak 2
Peak 1
O
277 NO2
-O
228
10
12
14
Time (min)
Red Blood Cell
Relative Intensity
Standard
277
x 19
233 244
168
150
228
200
x7
250
m/z
Sunrise Free Radical School
277
277
x5
228
168
324
306
300
Plasma
233
228 244
168
324
306
150 200 250 300
m/z
SFRBM 2005 Austin,Tx
233
244
324
306
150 200 250 300
m/z
Bruce Freeman 17
Nitrated Oleic Acid (OA-NO2) is Also Present in
Human Blood
279
Synthetic
OA-NO2
326
246
168197
9
10
308
2X
295
Relative
264 Intensity
Synthetic
13
[C]OA-NO
2
308344
326
9
10
time (min)
120 160 200240 280320
Relative
Intensity
m/z
5X
197
Plasma
OA-NO2
168
9
10
Red Cell
OA-NO2
10
time (min)
Sunrise Free Radical School
308
5X
326
Relative
246 Intensity
197
168
9
326
246
279
279
308
120 160 200 240 280
320
Relative
Intensity
m/z
J Biol Chem, in press 2005
SFRBM 2005 Austin,Tx
Bruce Freeman 18
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
Quantitative Analysis of LNO2 in
Clinical Samples
342/295 (13C-LNO2)
1
2
3
4
5
6
7
LNO2 peak area
IS peak area
10
324/277 (LNO2)
1
y = 0.969x + 0.002
r = 0.998
0.1
0.01
20.001
0.001 0.01 0.1
10
1
LNO peak
area/IS
peak
area
[LNO2]/[IS]
PNAS 101:11577-11582, 2004
0.05
0.5
5
50
ng/ml LNO2
500
LOQ ~ 300 amol on column
Standard curve linear over 5-orders of magnitude
Sunrise Free Radical School
SFRBM 2005 Austin,Tx
Bruce Freeman 20
Complex Lipid Nitro-Fatty Acid Derivatives
Membrane and Lipoprotein Reservoirs?
NO2
NO2
NO2
LNO2
NO2
NO2
NO2
Palmitate
O
O P O
O-
N+
NO2
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
O
Cell Signaling
Sunrise Free Radical School
-O
SFRBM 2005 Austin,Tx
NO22
NO
Bruce Freeman 21
Released Fatty Acid (nM)
Lipase-Treated Complex Biological Lipids
Release Nitrated Fatty Acids
1600
OA-NO2
LNO2
1200
+ Lipase
800
400
- Lipase
0
0
2
4
6
8
Time (hr)
Sunrise Free Radical School
SFRBM 2005 Austin,Tx
Bruce Freeman 22
Precursor scan m/z 184—molecular
species of phosphatidylcholine
+Prec (184.20): Exp 1, 10.761 to 29.474 min from Sample 1 (PC Mix) of Chromatography Test.wif...
3.2e5
3.0e5
2.8e5
18:2
16:0
O
O P O
O-
Max. 3.3e5 cps.
758.8
N+
782.8
16:0
20:4
2.6e5
O
O P O
O-
2.4e5
In te n s ity , c p s
2.2e5
N+
2.0e5
786.8
760.8
1.8e5
1.6e5
1.4e5
703.8
1.2e5
1.0e5
8.0e4
813.8
780.8
788.8 815.8
6.0e4
4.0e4
Sunrise
756.8
816.8
734.8
762.8
731.8
810.8
705.8
2.0e4
735.8 744.8
806.8
675.8
802.8
770.8
691.8 695.6 720.8
604.8 622.8 636.8 650.6
600
620
640
660
680
700
720
740
760
780
800
820
m/z, amu
Free Radical School
SFRBM
2005 Austin,Tx
841.8 846.8
840
875.0 899.0
860
880
900
Bruce Freeman 23
Precursor Analysis of Nitrated
Phosphatidylcholine (m/z 184)
+Prec (184.20): 2.178 to 7.555 min from Sample 1 (PC + nitrated PC) of Precursor 184.wiff (Turb...
Max. 2.9e5 cps.
803.8
1.00e4
9500.00
Palmitate
9000.00
LNO2
8500.00
8000.00
7500.00
O
O P O
O-
N+
In te n s ity , c p s
7000.00
6500.00
6000.00
5500.00
5000.00
804.8
4500.00
4000.00
3500.00
3000.00
821.8
2500.00
814.8
2000.00
1500.00
1000.00
500.00
0.00
772.8
770.8
773.8
790.8
788.8
774.8
798.8
805.8
812.8
775.8
780.8 786.8 789.8
770
775
Sunrise Free Radical
School780
791.8
799.8
794.6
785
790
801.8
807.8
811.8
795
800
805
810
SFRBM
2005
Austin,Tx
m/z, amu
815.8
818.6
815
819.8 822.8
817.6
820
828.8
824.8
830.8
827.8
825 Bruce
830Freeman
24
-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.
787.5
7.0e4
6.5e4
6.0e4
EPI Analysis
of Nitrated PC
M[-CH3]
5.5e4
In te n s ity , c p s
5.0e4
4.5e4
4.0e4
Palmitate
3.5e4
255.2
3.0e4
2.5e4
2.0e4
480.3
LNO2
1.5e4
1.0e4
741.5
277.4
5000.0
0.0
100
150
200
Sunrise Free Radical School
250
306.2
324.2
300
350
462.2
400
450
m/z, amu
549.3
500
SFRBM 2005 Austin,Tx
550
644.4 658.6
600
650
701.4 757.5 769.4
700
750
800
Bruce Freeman 25
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
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 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
NO Release From Nitroalkenes
Via Modified Nef Reaction
JBC 280:19289-19297, 2005
Sunrise Free Radical School
SFRBM 2005 Austin,Tx
Bruce Freeman 28
% Relaxation
100
80
60
(-) Endothelium
40
(+ ) Endothelium
L-NAME (30 µM)
20
Linoleate
0
Methanol
0.01
0.1
1
LNO2 (µM)
10
cGMP (pmol/mg cell protein)
% Reversal Relaxation
Nitrolinoleate Mediates NO-Like Vascular Signaling
*
100
*
50
0
LNO2 LNO2 LNO2
+
+
ODQ OxyHb
6
4
2
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
Nitroalkenes are Hydrophobically Stabilized
Octyl-thio-b-glucopyranoside
Octyl-b-glucopyranoside
CMC=2.8 mg/ml
CMC=7.3 mg/ml
oxyMb
[ N O ] ( µ M / h r)
EPR
Sunrise Free Radical School
SFRBM 2005 Austin,Tx
Bruce Freeman 30
JBC 280:19289-19297, 2005
Hydrophobic Stabilization of LNO2
NO2
Inflammation O
PLA2
HO
NO2
NO2
NO2
NO2
NO2
Kd
k
Esterification
K
O
NO2
Kd
HO
PPARg activation
k
NO + 2o reaction products
K
NO2
NO2
NO2
Sunrise Free Radical School
NO2
SFRBM 2005 Austin,Tx
Bruce Freeman 31
JBC 280:19289-19297, 2005
PPAR-Regulated Gene Expression
Cytoplasm
u
PPAR
Ligands
u
Degradation
Nucleus
Co-repressor
P
Coactivator
PPAR
RXR
9-cis-RA
Target genes
AGGTCA (N) AGGTCA
PPRE
Sunrise Free Radical School
SFRBM 2005 Austin,Tx
Bruce Freeman 32
Nitroalkenes Induce PPARgDependent 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
Reporter Constructs for Analyzing
PPAR Ligand Activity
Nuclear receptor LBDs (ligand binding domain)
Gal 4 DBD (DNA binding domain)
Luciferase activity
4 X Gal4
Luciferase
Normalized to GFP
CV-1 cells
Full length PPARs
Luciferase activity
Sunrise Free
Radical School
3 X PPRE
Luciferase
SFRBM 2005 Austin,Tx
Bruce Freeman 34
LNO2 is a Specific PPAR Agonist
Relative Luciferase Activity
8
Relative Luciferase Activity
*
6
*
4
7
PPARg
PPARa
PPARd
5
*
3
* *
1
Relative
Activity
10
0
0.01Luciferase
0.1
1
*
Relative Luciferase
Activity
LNO Concentration
(µM)
2
2
0
Control
LNO2
+
-
-
- +
- +
Vector
g
Sunrise Free Radical School
PNAS 102:2340-2345, 2005
+
+
-
- +
+
-
- +
+
-
- +
+
-
- +
+
-
- +
+
-
- +
+
-
- +
a
d AR GR MR PR RXRa
PPARs
nuclear receptors
SFRBM Other
2005 Austin,Tx
Bruce Freeman 35
NO2-OA is a More Potent PPARg Ligand
2.5
PPARa
2
PPARd
1.5
9
6
3
Remaining nitrated fatty acid (%)
PPARg
3
Relative Luciferase Activity
Relative Luciferase Activity
HO-1 expression
Inhibition of VCAM-1 expression
Adipocyte differentiation, glc uptake
100
80
NO2-OA
60
40
LNO2
20
Nitrated
lipid Remaining
Relative LuciferaseRelative
ActivityLuciferase
Activity
0
0
1
0
0.2 0.4
0.6 0.8
1
Concentration (NO2-OA)
Meth NO2-OA LNO2
(1 µM) (3 µM)
0 20 40 60 80 100 120
Time (min)
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
Nitroalkenes Undergo Reversible Michael
Addition Reactions with Nucleophiles - Thiols
H
100
R'
N
RS-
+
O
O-
H
R'
N+
RS H
O
O-
-
H+
H
H
R'
N
RS H
+
O-
O-
H
R'
N
RS H
+
O-
O-
Relative Abundance
H
LNO2
75
324.2
LNO2-GSH
50
631.3
25
0 200
300
400
500
600
m/z
Nitroalkylation
Sunrise Free Radical School
GSH
306.3
SFRBM 2005 Austin,Tx
Bruce Freeman 37
Submitted
Protein Nitroalkylation
327
Purified
GAPDH
327
327
GAPDH
+
NO2-OA
327
GAPDH
NO2-OA
100 mM GSH
26
44
32
38
Mass (103 m/z)
Sunrise Free Radical School
MALDI-TOF-MS SFRBM 2005 Austin,Tx
2-D Ion Trap
Bruce Freeman 38
Submitted
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
Eugene Chen, PhD – MSM
Bruce Branchaud, PhD - UO
Sunrise Free Radical School
SFRBM 2005 Austin,Tx
Bruce Freeman 40