Primary metabolic disorders induced by toxicants

1
Markers of oxidative stress Proteins, lipids or
DNA in the path of destruction Detection and
meaning Albert van der Vliet,
Ph.D. University of Vermont XIIth ISFRR
International Conference Free Radical School, May
6, 2004
2
Introduction
What is the role of oxidants in biology? What
oxidants do we mean? - Reactive oxygen species
comprise various metabolites with highly
variable reactive properties (O2, H2O2, OH,
1O2) - In addition, analogous families of
reactive nitrogen species (NO, N2O3, NO2,
ONOO, etc.) and reactive halogen species (HOCl,
Cl2, HOBr, NH2Cl, chloramines, bromamines,
etc.). Biological oxidants are important in
physiology and pathology - Involved in normal
cell function (e.g. growth regulation, host
defense), but also in (age-related) disease? -
Is this due to altered production of oxidants?
Oxidative modification of specific cell
targets? - How do we measure this?
3
Biological targets for oxidation
Unsaturated fatty acids, phospholipids,
lipoproteins Nucleic acids, DNA/RNA
Proteins, peptides, amino acids Sugars,
carbohydrates Other small biomolecules
(antioxidants, cofactors, etc.)
4
Lipid oxidation

• Defined as the oxidative deterioration of
  polyunsaturated fatty acids (A.L. Tappel)
• Although general peroxidation mechanisms well
  characterized, highly variable range of products,
  due to
• range of different biological lipid classes
  (phospholipids, cholesterol esters,
  triglycerides)
• variable unsaturated fatty acids (161, 182,
  204, 225) generally, the more unsaturated,
  the more oxidizable
• non-enzymatic (e.g. hydroxyl radical, metal ion
  catalyzed) and enzymatic oxidation mechanisms
  (e.g. lipoxygenases, cyclooxygenases)
• variable endproducts depending on secondary
  reactions, effects of antioxidants and
  repair/turnover

5
Radical-mediated lipid peroxidation initiation
and propagation
R
Linoleic acid (182)
Hydrogen abstraction (Initiation)
OH
R
Bisallylic carbon radical

Diene conjugation

R
Conjugated diene
O2
O
Oxygen addition
O
Peroxyl radical
R
Hydrogen abstraction (Propagation)
LH
HOO
R
Lipid hydroperoxide
Fe(II)
Fenton reaction

O
Alkoxyl radical
R
H
Fragmenation
H
H
O
LH
H
H
R
Ethane
H

H
H
Aldehyde
6
Reactive lipid oxidation products
a,b-unsaturated aldehydes
H
H
H
H
b
2
Malondialdehyde (b-hydroxy-acrolein)
2
3
1
O
a
HO
3
1
O
O
H
H
Acrolein (2,3-propenal)
H
b
Crotonaldehyde (2,3-butenal)
2
3
1
O
a
H3C
H
b
2
4-Hydroxy-2,3-nonenal (HNE)
4
9
6
8
3
a
1
5
7
O
OH
Strong electrophiles because of unsaturated
carbonyl group, react rapidly with nucleophilic
targets (e.g. GSH, protein thiols) (Esterbauer et
al., Free Rad. Biol. Med. 1991 11, 81-128
Uchida, Free Rad. Biol. Med. 2000 28, 1685-1696)

7
Reactive lipid oxidation products cyclopentenone
prostaglandins
Cyclopentenone prostaglandins of A and J series
are generated by dehydration of prostaglandins E2
and D2. Highly reactive because of unsaturated
carbonyl group which conjugates rapidly with
thiols, which may be involved in its bioactive
properties. (Milne et al., Chem. Res. Toxicol.
2004 17, 17-25)
8
Arachidonic acid oxidation products isoprostanes
Isoprostanes comprise four regioisomers with
eight stereoisomers. Relatively stable
endproducts that are currently viewed as most
reliable marker of lipid oxidation in vivo
(Lawson et al., J. Biol. Chem. 1999 274,
2444-24444)
9
Lipid oxidation

• Significance
• formation of bioactive substances, secondary
  effects on DNA/RNA or proteins
  (receptor-mediated or alkylation)
• alteration of membrane function, fluidity
• Detection of stable endproducts
• Colorometric/fluorometric methods (TBARS,
  conjugated dienes)
• HPLC, GC/LC-MS (lipid hydroperoxides, alkanes,
  isoprostanes, chlorohydrins, nitrated lipids)
• Immunochemical methods, ELISA (isoprostanes,
  MDA-dG, HNE-His, etc.)

10
Significance of lipid oxidation repair of
oxidized lipids
(I) Lipid-soluble antioxidants (vitamin E)
prevent oxidative degradation of lipids and
terminate lipid oxidation to form lipid
hydroperoxides. (II) Phospholipid hydroperoxide
can be reduced by phospholipid hydroperoxide GSH
peroxidase (PHGPX), or excised by phospholipase
A2 (PLA2 which preferentially cleaved oxidized
fatty acids) (III) Cleaved fatty acid
hydroperoxide can be reduced by GSH peroxidase
(GPX), and reduced phospholipid hydroperoxide can
be cleaved by PLA2. (IV) Lyso-phospholipid
(lacking one fatty acid chain) can be repaired by
acyltransferase
Ghirotti (1998) J. Lipid Res. 39 1529-1542
11
Oxidative damage to DNA and its detection

• Single and double strand breaks
• Comet assay (Fairbairn et al. Mutat. Res. 1995
  339, 37-59)
• Oxidative modification of DNA-bases (e.g.
  8-OH-guanine, 5-OH-uracil, etc.)
• GS/MS based methods (Dizdaroglu, Mutat. Res.
  2003 531, 109-126)
• Immunochemical methods (e.g. 8-oxodG)
• Nitrosative deamination of DNA bases
• Guanine - xanthine
• Cytosine - uracil (Burney et al. Mutat. Res.
  1999 424, 37-49)
• Formation of DNA adducts with oxidized lipids
  or proteins (e.g. dG-MDA, dT-Tyr).

12
DNA base oxidation oxidation of guanine by OH
O
N
Guan(os)ine
HN
N
O
N
H2N
O
R
N
OH
N
HN
OH
HN
OH
H
H
N
N
H2N
G8OH
N
N
H2N
R
Oxidation Ring opening
Reduction
R
O
O
H
O
N
N
HN
HN
OH
N
N
N
H2N
N
H2N
R
8-OH-guanine
R
FAPy-guanine
13
Lipid oxidation products and addition to dG
O
N
deoxy-Guanosine
HN
N
N
H2N
dR
Acrolein Crotonaldehyde Hydroxynonenal (HNE)
O
O
OH
N
N
N
N
N
N
HO
N
N
R
N H
N H
dR
dR
Acr-dG 12
-R Acr-dG3 -H Cro-dG -CH3 HNE-dG -CH(OH)-CH2-CH2
-CH2-CH2-CH3
14
DNA repair mechanisms

• Direct reversal of DNA modification
• - photolyase (only in light-exposed cells)
• - removal of methyl groups (O6-alkylguanine-DNA-a
  lkyltransferase)
• Base excision repair (small modifications not
  causing major helix disruption)
• - removal of modified base (DNA-glycosylase)
• - AP site is opened by endonuclease
• - abasic suger replaced by correct nucleotide
  (DNA polymerase)
• - resealing (DNA ligase)
• Nucleotide-excision repair (bulky lesions or
  cross-links)
• - Excision of damaged nucleotide with
  neighboring nucleotides (ATP-dependent nuclease)
• - Insertion of correct nucleotides (DNA
  polymerase)
• Recombinational repair (DNA replication)
• - sister chromatid exchange
• - double-strand-break repair (nonhomologous DNA
  end joining NHEJ)

15
Consequences of DNA oxidation
Repair can be ineffective This causes
mismatch repair and mutations, e.g. - adenine
deamination AT-GC transitions - 8OH-dG GC-TA
transversions If DNA repair is excessive -
Massive ADP-ribosylation by activation PARP -
Causes depletion of NAD and consequently ATP
(metabolic cell stress)
16
Poly(ADP-ribose) polymerase (PARP) and DNA repair
NAD
Nicotinamide
n
ADPr
DNA damage causes the activation of PARP, which
causes poly(ADP-ribosyl)ation of itself as well
as other DNA-bound proteins (transcription
factors, histones), causing their dissociation to
facilitate DNA repair. Excessive PARP activation
causes depletion of NAD and ATP.
DAmours et al. (1999) Biochem. J. 342 249-268
17
Protein oxidation

• Why is protein oxidation important?
• constitute most of the functional components in
  the cell (enzymes, ion channels, structural
  proteins) oxidative modification may have direct
  consequences
• relatively abundant in cells/tissues compared to
  e.g. lipids or DNA
• (e.g. per kg wet weight 146 g protein, 2.6 g
  DNA, 49 g total lipid)
• certain amino acids or protein functional groups
  susceptible to oxidation importance in oxidant
  signaling.
• Difficulties with proteins
• High variability 20 amino acids, thousands of
  different proteins.
• Highly variable abundance and turnover of
  proteins
• Many types of oxidative modifications oxidative
  fragmentation/crosslinking, reversible and
  irreversible oxidations of amino acid side chains

18
Protein oxidation redox signaling or oxidant
injury

• Reversible modifications (causing dysfunction of
  targets)
• Electron transfer or coordination reaction
  with transition metal ions (e.g. heme centers,
  iron-sulfur clusters) Aconitase, hemoglobin,
  cytochrome c, antioxidant enzymes (SOD,
  catalase)
• Oxidation/nitros(yl)ation of cysteine
  residues Cell signaling proteins (G-proteins,
  Ras, phosphatases, etc.), transport proteins
  (e.g. Ca2-ATPases), transcription factors (e.g.
  NF-kB), proteases (e.g. caspases)
• Methionine oxidation May represent important
  defense mechanisms against oxidative stress
• Tyrosine nitration (reversed by denitrases?)
• Irreversible modifications (destruction of
  targets)
• Oxidative protein fragmentation/crosslinking
  usually due to radical-mediated hydrogen
  abstraction in protein backbone
• Formation of protein carbonyls Oxidation of
  amino acid residues, addition reactions with
  aldehydes or sugars
• Aromatic amino acid modifications
  Hydroxylation, nitration, halogenation

19
Amino acid residues most susceptible to oxidation

• Amino acid Oxidation products
• Cysteine disulfides, cysteic acid
• Methionine methionine sulfoxide, methionine
  sulfone
• Tryptophan 2-, 4-, 5-, 6-, 7-hydroxytryptophan,
  nitrotryptophan,
• kynurenine, 3-hydroxykynurenine,
  formylkynurenine
• Phenylalanine 2,3-dihydroxyphenylalanine, 2-,
  3-, and
• 4-hydroxyphenyalanine
• Tyrosine 3,4-dihydroxyphenylalanine,
  tyrosine-tyrosine cross-linkages
• Tyr-O-Tyr, cross-linked nitrotyrosine
• Histidine 2-oxohistidine, asparagine, aspartic
  acid
• Arginine glutamic semialdehyde
• Lysine a-aminoadipic semialdehyde
• Proline 2-amino-3-ketobutyric acid
• Glutamyl Oxalic acid, pyruvic acid
• Berlett and Stadtman, 1997

20
Sulfhydryl/thiol oxidation reversible and
irreversible
21
Reversal of protein cysteine oxidation
thioredoxin and glutaredoxin
THIOREDOXIN PATHWAY
GLUTAREDOXIN PATHWAY
Prot-SS Prot1-SS-Prot2 Prot-SOH Prot-MetO
Prot-SH2 Prot1-SH Prot2-SH Prot-SH
H2O Prot-Met H2O
Prot-SSG
Prot-SH GSH
GRO-SH2
GRO-SS
Glutaredoxin
TR-SH2
TR-SS
Thioredoxin
GSSG
2 GSH
TRR-SS
TRR-SH2
Thioredoxin reductase
GR-SH2
GR-SS
GSH reductase
NADPH H
NADP
NADPH H
NADP
22
Thiol oxidation and cell signaling tyrosine
phosphorylation
Ligand/growth factor
membrane
Receptor tyrosine kinase
RTK
Y-PO4
PO4-Y
Oxidase
Y-PO4
protein
H2O2
PTP-S PTP-SOH
Protein tyrosine phosphatase
Trx
23
Reversible sulfenic acid formation in oxidant
signaling
Peroxiredoxins (Prx) are important in
detoxification of H2O2. Inactivation by
formation of sulfinic acid (-SO2H) may allow H2O2
signaling (floodgate model). An enzymatic
activity exists that reduces sulfinic acid
(-SO2H) to the corresponding thiol (-SH)
(Georgiou and Masip, Science 2003 300, 592-594).
24
Thiol oxidation by RNS formation of
S-nitrosothiols
NO
O2, H
O2
Fe(III)
N2O3
ONOOH
Fe(II)-NO
RS
RS
RS
RS
RS
RSNO2
RS
RSNO
RS
RS
RS
RSSR
25
Regulation of protein function by
S-nitros(yl)ation
Protein target Functional effect Reference Chan
nels/Transporters NMDA receptor Inhibition L
ipton, 1993 Ryanodine receptor Activation or
inhibition Xu, 1998 Eu, 2000 Na/K-ATPase
Unknown Jaffrey, 2001 Transport/storage
Serum albumin Bioactivation Stamler, 1992
Nedospasov, 2000 Metallothionin Zinc
release? Kroncke, 1994 Pearce, 2000 Protein
kinases/phosphatases JNK Inhibition So,
1998 Park, 2000 Src Activation Akhand,
1999 Creatine kinase Inhibition Arstall,
1998 Konorev, 2000 Protein phosphatases Inhib
ition Xian, 2000 Caselli, 1995 Metabolic
enzymes GAPDH Inactivation Mohr, 1996
Molina y Vedia, 1992 Methionine adenosyl
transferase Inhibition Ruiz, 1998 Perez-Mato,
1999 Thioredoxin Inhibition Dimmeler,
2002 Signaling proteins p21
ras Activation Lander, 1997 Hsp
72 Unknown Jaffrey, 2001 Proteases Tissue
plasminogen activator Activation Stamler, 1992
Caspases 1-8 Inhibition Dimmeler, 1997,
Mannick, 1999 Transcription factors
AP-1 Inhibition Nikitovic, 1998 Melino,
2000 p50 (FNF-kB) Inhibition Matthews,
1996 Marshall, 2001
26
Detection of thiol oxidation and nitros(yl)ation
Sulfenic acids (RS-OH) Indirect methods
based on unique reactivity toward nucleophiles
(dimedone, TNB, benzylamine). In general,
difficult to detect because very unstable (Poole
et al., 2004). Glutathionylation, mixed
disulfides (RS-SG, R1S-SR2) 35S-GSH
incorporation, labeling with biotinylated
glutathione ethyl ester. Immunochemical
detection with anti-GSH antibody. Can be
confirmed by reversibility with reducing agents
(DTT). Sulfinic or sulfonic acids (RS-O2H,
RS-O3H) HPLC/MALDI (Hamann et al., 2002),
immunochemical detection of hyperoxidized
cysteines (Woo et al., J. Biol. Chem. 2003 278,
47361-47364). S-nitrosothiols
(RS-NO) Chemiluminescence detection of NO after
selective chemical reduction (KI/I2 e.g.
Feelisch et al., FASEB J. 2002 16,
1590-1596) Immunochemical detection with
anti-S-nitrosocysteine antibodies (e.g. Gow et
al., J. Biol. Chem. 2002 277,
9637-9640) Indirect detection of thiols after
selective reduction (e.g. biotin switch method
Jaffrey, 2001).
27
Methionine oxidation reversible and irreversible
O
O
O
oxidation
oxidation
irreversible
reduction
O
S
O
S
S
O
H3C
H3C
H3C
Methionine
Methionine sulfoxide
Methionine sulfone
Methione oxidation is reversed by methionine
sulfoxide reductase (Msr). Two forms exist, MsrA
and MsrB that specifically reduce S- and
R-stereo- isomers of Met(O), respectively. Msr
deletion increases susceptibility to oxidant
toxicity and shortens lifespan. Methionine
oxidation is involved in regulating proteases
inhibitors, signaling proteins, and ion channels
(Hoshi and Heinemann, 2001).
28
Irreversible protein oxidation protein carbonyls
Mechanisms of carbonyl formation Oxidative
protein cleavage by a-amidation or by oxidation
of glutamyl side chains Direct oxidation of
lysine, arginine, proline or threonine
Reaction with lipid oxidation products
(aldehydes) Reaction of reducing sugars or
their oxidation products with e.g. lysine
(advanced glycation end products
AGEs) Detection of carbonyl formation
Reaction with 2,4-dinitrophenylhydrazine
(DNPH) Detection of chromophore by UV/Vis
spectroscopy Immunological detection using
a-DNP antibodies Berlett and Stadtman (1997)
J. Biol. Chem. 272 20313-20316.
29
Irreversible protein modification by lipid
oxidation products
H
H
a,b-unsaturated carbonyls (e.g. acrolein, HNE,
cyclopentenone prostaglandins)
R
O
H
O
O
O
Cysteine
Lysine
H
Histidine
N
H
H
N
N
S
N
HN
N
R
O
R
O
R
O
Strong electrophilic products react with various
amino acids by Michael addition. Can be detected
by specific antibodies,e.g. against HNE-modified
proteins (HNE-His) (Uchida, 1999)
30
Irreversible oxidative amino acid modifications
by ROS/RNS
Tyrosine
Tyrosine
Tryptophan
Phenylalanine
O
O
O
NHCHO
HO
2-OH-phenylalanine
N-formyl-kynurenine
3-NO2-tyrosine
o,o-dityrosine
31
Stable oxidation markers Tyrosine oxidation,
chlorination, and nitration

NH
3
OH
Ox
Tyr
OH
Tyrosyl radical
Tyrosine
o,o-dityrosine
NO2
HOCl, Cl2
Ox

NH
3
Cl
OH
NO2
3,4,-diOH-Phe (DOPA)
OH
3-Cl-tyrosine (RHS)
3-NO2-tyrosine (RNS)
32
Features of tyrosine nitration
Initially thought to represent a selective marker
for ONOO formation, now known as collective
marker for RNS formation (specifically
NO2). Tyrosine nitration occurs with some
selectivity, depending on protein and factors
such as surface exposure and electrostatic
factors. Protein nitration within mitochondria
may be dynamic (involving denitration and
renitration), suggesting some functional role
(Aulak et al., Am. J. Physiol. 2004 286,
H30-H38) Tyrosine nitration affects the pKa of
tyrosine and adds a bulky group, which
collectively could affect functional properties
of tyrosine in proteins.
33
Proposed consequences of tyrosine
nitration on protein function Enzyme Nitrotyros
ine analysis Tyrosine role Reference
Mn-SOD HPLC/UV, amino acid sequencing, MacMi
llan-Crow, 1998 MS/MS analysis of peptides
Catalytic activity? Yakamura,
1998 Fe-SOD HPLC/ESI-MS Catalytic
activity? Soulere, 2001 Tyrosine hydroxylase
Western blot, protein digestion, HPLC/UV
analysis Unknown Ara, 1998 GSH-S-Transferase
HPLC of tryptic fragments, MALDI/MS and
HPLC/ECD GSH binding and activation Wong,
2001 COX-1 Protein digestion, HPLC/UV and
amino acid sequencing Tyr radical in active site
Goodwin, 1998 Cytochrome P450 GC/MS and HPLC
of intact protein, sequencing of tryptic
fragments Unknown, electron transfer? Roberts,
1998 Glutamine synthetase HPLC and UV analysis
of tryptic peptide fragments Enzyme
(in)activation Berlett, 1998 Cytochrome
c Protein digestion, HPLC/UV and MS analysis
of peptides Catalytic activity? Cassina,
2000 Ribonucl. reductase HPLC/UV analysis,
protein digestion and amino acid sequencing
Catalytic activity Guittet, 2000 a1-proteinase
inhibitor Amino acid sequencing of peptide
fragments Catalytic domain Mierzwa,
1987 Surfactant protein A Protein digestion,
HPLC and MS analysis Receptor binding, lipid
aggregation Greis, 1996
34
Does irreversible protein modification affect
protein function in vivo?

• Biomarker Normal levels (range) Fold increase
  in disease
• Carboxymethyl-lysine pmol/mg 2-5
• DOPA pmol/mg 5-6
• o- and m-Tyr µmol/mol Phe 2-6
• dityrosine µmol/mol Tyr 100-500
• N-formyl-kynurenine pmol/mg 10
• aliphatic hydroxyls pmol/mg 1-10
• nitrotyrosine µmol/mol Tyr 10-100
• chlorotyrosine µmol/mol Tyr 10-100
• protein carbonyls nmol/mg 2-3
• Quantitatively, these modifications may be less
  significant than
• e.g. cysteine or methionine oxidation.

35
Removal of oxidized proteins proteosomal
degradation
                                                
                                                  
                      
Ubiquitin ligation and proteasomal degradation
require ATP, and will be impaired in energy
depleted cells (e.g. as a result of excessive
repair of oxidative DNA damage).
From Goldberg (2003) Nature 426 895-899
36
Irreversible protein modification and degradation
Protein modification/oxidation
Structural change Increased exposure of
hydrophobic regions
Proteasome
Protein aggregation Accumulation of unfolded or
mutated proteins Loss of cell function Cell
death/Apoptosis
Protein degradation Repair
37
Summary and major conclusions
Lipid oxidation may produce bioactive
intermediates that can act as second messengers
in oxidant signaling. Can form adducts with
specific targets in DNA or proteins.
Reversible protein oxidations (transition metal
ions, cysteine or methionine residues) are
important in cell signaling. Because of their
reversible nature, quantitation of these
modifications has been difficult.
Irreversible protein modifications are more
easily quantified and immunochemical and
analytical methods have been developed to detect
them. Useful as diagnostic markers of oxidant
production, but biological significance still
unclear, because of protein degradation and
turnover. Nevertheless, various irreversible
protein modifications accumulate with age.
Biological consequence of excessive oxidative
injury and repair in many cases due to metabolic
disturbance in the cell (ATP depletion and/or
Ca2 influx) leading to apoptotic or necrotic
cell death.
38
Metabolic disorder induced by oxidative stress
? NAD(P)H
? PARP (DNA injury)
? Ca2ATPase
? ATP-Syn
? NOS
? Mitochondrial electron transfer
? XO
? Ca2ATPase
? DYm
39
Recommended reading
Adams J (2003) The proteasome structure,
function, and role in the cell. Cancer Treat.
Rev. 29 (Suppl 1) 3-9. Berlett BS and Stadtman
ER (1997) Protein oxidation in ageing, disease,
and oxidative stress. J. Biol. Chem. 272
20313-20316. Bjelland S, Seeberg E (2003)
Mutagenicity, toxicity and repair of DNA base
damage induced by oxidation. Mutat. Res. 531
37-80. Poole LB, Karplus PA, and Claiborne A.
(2004) Protein sulfenic acids in redox signaling.
Ann. Rev. Pharmacol. Toxicol. 44
325-347. Giulivi C, Traaseth NJ, and Davies KJ
(2003) Tyrosine oxidation products analysis and
biological relevance. Amino Acids 25
227-232. Goldberg AL (2003) Protein degradation
and protection against misfolded or damaged
proteins. Nature 426 895-899. Grune T, Merker K,
Sandig G, and Davies KJ (2003) Selective
degradation of oxidatively modified protein
substrates by the proteasome. Biochem. Biophys.
Res. Commun. 305 709-718. Halliwell B and
Gutteridge JMC (1999) Free radicals in biology
and medicine. Oxford Science Publishers, New
York. Hamann M, Zhang T, Hendrich S, Thomas JA
(2002) Quantitation of protein sulfinic and
sulfonic acid, irreversibly oxidized protein
cysteine sites in cellular proteins. Meth.
Enzymol. 348 146-156. Heinecke JW (1999) Mass
spectrometric quantification of amino acid
oxidation products in proteins insights into
pathways that promote LDL oxidation in the human
artery wall. FASEB J. 13 1113-1120.
40
Recommended reading
Hoshi T, Heinemann SH (2001) Regulation of cell
function by methionine oxidation and reduction.
J. Physiol. 531 1-11. Ischiropoulos H (2003)
Biological selectivity and functional aspects of
protein tyrosine nitration. Biochem. Biophys.
Res. Commun. 305 776-783. Jaffrey SP, Snyder SH
(2001) The biotin switch method for the detection
of S-nitrosylated proteins. Sci. STKE 86 PL1
Lawson JA, Bokach J, and FitzGerald GA (1999)
Isoprostanes formation, analysis and use as
indices of lipid peroxidation in vivo. J. Biol.
Chem. 274 24441-24444. Levine RL (2002) Carbonyl
modified proteins in cellular regulation, ageing
and disease. Free Rad. Biol. Med. 32
790-796. Moskovitz J, Bar-Noy, Williams WM,
Requena J, Berlett BS, and Stadtman ER (2001)
Methionine sulfoxide reductase (MsrA) is a
regulator of antioxidant defense and lifespan in
mammals. Proc. Natl. Acad. Sci. USA 98
12920-12925. Roberts LJ 2nd, Morrow JD (2002)
Products of the isoprostane pathway unique
bioactive compounds and markers of lipid
oxidation. Cell Mol. Life Sci. 59
808-820. Stamler JS, Lamas S, Fang FC (2001)
Nitrosylation, the prototypic redox-based
signaling mechanism. Cell 106 675-683. Uchida K
(2000) Role of reactive aldehyde in
cardiovascular diseases. Free Rad. Biol. Med. 28
1685-1696. Wong PS, van der Vliet A (2002)
Quantitation and localization of tyrosine
nitration in proteins. Meth. Enzymol. 359
399-410.