Sunrise Free Radical School SFRBM 2005 Austin,Tx Bruce Freeman 1

1
Nitrated Fatty Acids Formation and Actions Bruce
Freeman, PhD Department of Pharmacology University
of Pittsburgh School of Medicine
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

3
Fatty Acids
Fatty acids have common names, IUPAC names and
acronyms
Linoleic Acid Octadeca-9,12-dienoic acid 182 LA
O
HO
Arachidonic Acid Eicosatetraenoic
Acid Dodeca-5,8,11,14-tetraenoic acid 204 AA
O
O
H
Myristic Acid (140) Linolenic Acid (183)
Palmitic Acid (160) Arachidic Acid (200)
Stearic Acid (180) Docosahexanoic acid
(226) Oleic Acid (181)
Endogenous fatty acids have the
cis-stereochemical orientation
4
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 26928066-28075, 1994
5
Nitrated Fatty Acids Most Abundant
Bioactive Oxides of Nitrogen in the Vascular
Compartment
PNAS 10111577-11582, 2004 J Biol Chem, 2005
6
All Unsaturated Fatty Acid Species Display
Nitrated Derivatives In Healthy Human Plasma and
Urine
J Biol Chem, in press 2005
7
Inflammatory Conditions Induce Oleic Acid
Nitration Fatty Acid Micellar Emulsion
J Biol Chem, in press 2005
8
Inflammatory Conditions Induce Fatty Acid
Nitration Intraperitoneal Injection of LPS in
Mice
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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
plt 0.05
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
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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

12
Synthesis of Nitrated Linoleic Acid
PNAS 10111577-11582, 2004
13
Detection/Characterization of LNO2
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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

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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-
PNAS 10111577-11582, 2004
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Product Ion Analysis of LNO2 Regioisomers Peak 1
PNAS 10111577-11582, 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
157
306
233
Relative Intensity
233
324
196
244
233
157
171
157
293
293
293
244
196
324
306
306
150
200
250
300
150
200
250
300
150
200
250
300
m/z
m/z
m/z
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Product Ion Analysis of LNO2 Regioisomers Peak 2
PNAS 10111577-11582, 2004
Peak 2
Peak 1
O
NO2
277
-O
228
10
12
14
Time (min)
Red Blood Cell
Standard
Plasma
277
277
277
x 19
x 5
x 7
324
233
244
228
228
168
233
Relative Intensity
233
168
168
244
228
244
324
306
324
306
306
150
200
250
300
150
200
250
300
150
200
250
300
m/z
m/z
m/z
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Nitrated Oleic Acid (OA-NO2) is Also Present in
Human Blood
J Biol Chem, in press 2005
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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 13CLNO2 and 13CNO2-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

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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
10
1
0.1
0.01
0.001
LNO peak area/IS peak area
1
2
3
4
5
6
7
LNO2/IS
500
50
5
0.5
0.05
PNAS 10111577-11582, 2004
ng/ml LNO
2
LOQ 300 amol on column Standard curve linear
over 5-orders of magnitude
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Complex Lipid Nitro-Fatty Acid Derivatives Membran
e 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
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Lipase-Treated Complex Biological Lipids Release
Nitrated Fatty Acids
OA-NO2
LNO2
Lipase
Released Fatty Acid (nM)
- Lipase
Time (hr)
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Precursor scan m/z 184molecular species of
phosphatidylcholine
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Precursor Analysis of Nitrated Phosphatidylcholine
(m/z 184)
Palmitate
LNO2
O
O
P
O
N

O
-
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EPI Analysis of Nitrated PC
M-CH3
Palmitate
LNO2
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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)

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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
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NO Release From Nitroalkenes Via Modified Nef
Reaction
JBC 28019289-19297, 2005
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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
10
0.1
0.01
1
2
LNO2 (µM)
0
0
0.3
1
SNP (10 µM)
LNO2 (µM)
PNAS 9915941-46, 2002
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Nitroalkenes are Hydrophobically
Stabilized Octyl-thio-b-glucopyranoside
CMC2.8 mg/ml Octyl-b-glucopyranoside CMC7.3
mg/ml
oxyMb
EPR
)
r
h
/
M
µ
(


O
N

JBC 28019289-19297, 2005
31
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
JBC 28019289-19297, 2005
32
PPAR-Regulated Gene Expression
Cytoplasm
Degradation
Nucleus
PPAR Ligands
Co-repressor
P
Coactivator
9-cis-RA
Target genes
RXR
AGGTCA (N) AGGTCA
PPRE
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

34
Reporter Constructs for AnalyzingPPAR Ligand
Activity
Luciferase activity
Luciferase
4 X Gal4
Normalized to GFP CV-1 cells
Luciferase activity
Luciferase
3 X PPRE
35
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
PNAS 1022340-2345, 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
NO2-OA (1 µM)
LNO2 (3 µM)
Meth
0
0.2
0.4
0.6
0.8
1
Time (min)
Concentration (NO2-OA)
NO2-OA gt LNO2 potency may be due to stability
PNAS 1022340-2345, 2005
37
Nitroalkenes Undergo Reversible Michael Addition
Reactions with Nucleophiles - Thiols
GSH
306.3
100
H
H
LNO2
O
R
'
-
O
R
'

N
R
S
-

N
75
324.2
-
R
S
O
H
-
O
H
LNO2-GSH
Relative Abundance
50
H

H
H
H
631.3
-
-
O
R
'
25
O
R
'

N

N
R
S
R
S
-
H
O
-
H
O
0
200
300
400
500
600
m/z
Nitroalkylation
Submitted
38
Protein Nitroalkylation
Purified GAPDH
GAPDH NO2-OA
GAPDH NO2-OA 100 mM GSH
2-D Ion Trap
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

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Eugene Chen, PhD MSM Bruce Branchaud, PhD - UO