Sunrise Free Radical School
Society for Free Radical Biology and Medicine
Washington, DC November 2007
How to Measure ROS and RNS in Biology
Some generality and a singlet molecular oxygen case
Paolo Di Mascio
Departamento de Bioquímica
Instituto de Química
Universidade de São Paulo, Brasil
Reactive species have , in generally, a very short half-life, are produced in
low amounts. Multiple methods of measurement are available today, each
with their own benefits and limits.
Reactive Species measurement methods must be:
-Very, very sensitive
-Highly selective
-Fast
-In situ
How to Measure Reactive Species?
Direct
Electron paramagnetic resonance, EPR (free
radical detection)
+Trapping (Spin-traps, “chemical”)
Indirect (but specific)
Mass spectrometry, MS/MS (ESI, MALDI)
+Scavengers
Probes (spectroscopic investigation)
Luminescence (direct and indirect)
Combination of Different Techniques !
Free Radicals in Biology and Medicine
B. Halliwell ans J.M.C. Gutteridge, 2007.
Electron Paramagnetic Resonance (EPR)
We can detect & measure free radicals and paramagnetic species
• “High” sensitivity (nanomolar concentrations)
Direct detection
e.g.: semiquinones, nitroxides, thiyl, ROO•…
Indirect detection
Spin-trapping
Species: superoxide, hydroxyl, alkyl, NO
Spin-traps: DMPO, PBN, DEPMPO, Fe-DTCs
We can use EPR to measure free radicals from biological systems
(in vivo or ex vivo)
Intact tissues, organs … can be measured.
Other Trapping methods
Hydroxyl radical OH▪ [Trapping methods (without EPR)]
-Reaction with aromatic compounds (Phenylalanine ‘Tyrosine’, Salicylate, …
-Attack of OH▪ on 2-deoxyribose produces a range of products…malondialdehyde
Superoxide O2 ▪- [Fluorescent probes] Dihydroethidium (DHE) conversion to 2-hydroxyethidium and
lucigenin =
- Ability to reduce cytochrome c or nitroblue tetrazolium
- Superoxide electrodes
- Histochemical detection: Conversion of diaminobenzidine (DAB) to an insoluble product
Peroxynitrite and other nitrogen species ….Nitric oxide NO ▪
-NO ▪ :
-Light emission in the presence of O3 (excited NO2▪)
-NO electrodes (porphyrinic sensors)
-Haemoglobin trapping (A is measured)
-Spin trapping (Haemoglobin, other haem proteins, …)
-4,5-Diaminofluorescein diacetate (DAF-2-diacetate), fluorescent product
Diaminoanthroquinone, red fluorescent product
-“indirect methods”: NO2- measurement, use NOS inhibitors, …
Nitration assays
ONOO- : ONOO- nitrates many aromatic compounds: tyrosine (3nitrotyrosine), tryptophan, phenylalanine
Reactive halogen species
HOCl / HOBr
- [taurine assay] Conversion of taurine to a chloramine
-bromo- and chlorotyrosine ?
Hydrogen peroxide H2O2
- [fluorescent products] Amplex red and DCF-DA
- [non fluorescent products] Fluorescent compound scopoletin
-Inactivation of catalase by aminotriazole….in cells…
Singlet oxygen 1O2……..O2 (1g) in biological system
-Monomol and dimol Light emission
-Use of scavengers (azide), traps (histidine, anthracene derivatives)
and D2O effect
Studies using low-level / ultraweak / dark chemiluminescence ….
Many methods are available to identify Reactive Species in cultured cells
1- Cell culture process itself causes oxidative stress
2-Trypsinization increases ROS
3- Artifacts are produced
-Dichlorofluorescein diacetate (DCF-DA) is deacetylated by esterases to
dichlorofluorescein (DCFH) which can be visualized by fluorescence at 525 nm
More specific for H2O2?
-Dihydrorhodamine 123 (DHR) is used to detect several reactive species
Conversion to rhodamine 123, highly fluorescent 536 nm
-Luminol and lucigenin
“Biomarkers”
Of oxidative DNA damage
Of lipid peroxidation
Of protein damage
by reactive species
•
•
•
“Biomarkers”
Single and double strand breaks (Comet assay)
Oxidative modification of DNA-bases (e.g. 8-oxo-dGuo… LC/MS/MS)Of oxidative DNA damage
Formation of DNA adducts with oxidized lipids or proteins (e.g. dGuo-MDA,
dT-Tyr….LC/MS/MS).
O
Guan(os)ine
H2N
O
N•
HN
OH
H
N
H2N
Oxidation
Ring opening
N
DNA base oxidation:
oxidation of guanine by
OH•
N
HN
N
OH•
G8OH•
N
O
R
N
H2N
Reduction
R
H2N
N
OH
H
R
O
O
H
N
HN
N•
HN
O
N
HN
OH
N
FAPy-Guanine
NH
R
H2N
N
8-oxo-Guanine
N
R
Arachidonic acid oxidation products:
“Biomarkers”
Of lipid peroxidation
Isoprostanes: Relatively stable endproducts that are currently
viewed as most reliable marker of lipid oxidation in vivo
Isoprostanes comprise four regioisomers with eight stereoisomers.
in vivo (Lawson et al., J. Biol. Chem. 1999: 274, 2444-24444).
“Biomarkers”
Of lipid peroxidation
Metabolic Fate of 15-F2t-IsoP (8-Iso-PGF2) in Humans
OH
COOH
OH
OH
15-F2t-IsoP
-oxidation 5-reduction
OH
COOH
Major urinary metabolite
OH
A GC/MS assay for F2-IsoP-M
has been developed
OH
2,3-Dinor-5,6-Dihydro-15-F2t-IsoP
(F2-IsoP-M)
(Roberts LJ, II, et. al. J. Biol. Chem :271:20617, 1996)
“Biomarkers”
Stable oxidation markers:
Tyrosine oxidation, chlorination, and nitration
Of protein damage
NH3+
NH3+
COO–
NH3+
OH
COO–
–
COO
Ox
Tyr•
OH
Tyrosine
O
OH
Ox
Tyrosyl
radical
•
NO2•
HOCl, Cl2
NH3+
OH
COO–
NH3+
NH3+
o,o’-dityrosine
NH3+
COO–
COO–
COO–
OH
Cl
NO2
3,4,-diOH-Phe
(DOPA)
OH
3-Cl-tyrosine
(RHS)
OH
3-NO2-tyrosine
(RNS)
H
DNA / Protein Adducts
O
R
,-unsaturated carbonyls
(e.g. acrolein, HNE, cyclopentenone
prostaglandins)
O
H
N
O
deoxyGuanosine
H2N
HO
N
H
OH
N
N
N
N
dR
Acr-dGuo 1&2
N
H
N
N
N
dR
Acr-dG3
Cro-dG
HNE-dG
R
O
N
R’
H
N
O
R
dR
Acrolein
Crotonaldehyde
Hydroxynonenal (HNE)
O
O
Cysteine
O
H
N
Lysine
S
N
N
N
Histidine
H
N
N
HN
H
-R’
-H
-CH3
-CH(OH)-CH2-CH2-CH2-CH2-CH3
HN
O
R
O
Half-life of some reactive species
Reactive species
Half-life
(s)
Hydroxyl radical (OH)
Alcoxyl radical (RO)
Singlet oxygen (1O2)
Peroxynitrite anion (ONOO-)
Peroxyl radical (ROO)
Nitric oxide (NO)
Semiquinone radical
Hydrogen peroxide (H2O2)
10-9
10-6
10-5
0.05 – 1.0
7
1 - 10
minutes/hours
spontan. hours/days
Superoxide anion (O2-)
(accelerated by enzymes)
Hypochlorous acid (HOCl)
spontan. hours/days
(by SOD accel. to 10-6)
dep. on substrate
Physiol
conc. (mol/l)
10-9
10-9 - 10-7
10-12 - 10-11
Free Radical Reaction Rate
ROS generated by physical or chemical processes can damage biomolecules.
-difficulties and how to measure ROS with the singlet oxygen case
Photosensitization
Enzyme
Myeloperoxidase
1O
-
2
-e
Ionizing Radiation
Oxidative Metabolism
UV Laser Pulses
Xenobiotics
•OH
H2O2
O2•-
ROOH
DNA
Diseases Lethality
Proteins
Lipids
Mutagenesis Carcinogenesis Aging
Singlet Molecular Oxygen in biological systems as an example
What’s the Problem?
?
X = HOCl, ONOOH, ….
ROOH
?
O2•¯
•
HO
H2O2
.
ROO
1O
2
?
Singlet Molecular Oxygen in biological systems as an example
Direct
Luminescence (Light emission) +Quenchers (N3-), Solvent
effect (Deuterated solvent, D2O)
Indirect but specific
Isotopically labeled oxygen
Chemical trapping and HPLC-mass spectrometry
(18O-labeled compounds)
EPR
Synthesis / Characterization (LAOOH, PCOOH)
18O-Labeled Compounds
Chemical source of 18[1O2]
-Development of a pure source of isotopically labeled singlet oxygen,
18[1O ]
2
Tool for mechanistic studies !
Photosensitized
generation of singlet O2
3O
SO 2
2
sens
S* + sens
3O
Energy transfer
S - + sens +
Eletron
transfer
Hydrogen transfer
S + sens
[S +
3sens * ]
Type I
S
3sens
*
3O
2
ISC
1sens *
sens + hv
2
sens + 1O 2
Type II
S
SO 2
Porphyria Patient
Porphyria is a diseases in which pigments called porphyrins accumulate
in the skin, bones and teeth.
R1
R8
R2
R3
N
NH
X
HN
N
R7
R6
R4
R5
Porphyrin Nucleus
Angew-Chem.Int. Ed., 21, 343 ,1982
Activated leukocytes
Bacterial killing: Respiratory burst
Fagocytosis
Steinbeck et al. (1992) J. Biol. Chem., 267, 13425
Wentworth et al. (2002) Science, 298, 2195.
Babior et al. (2003) Proc. Natl. Acad. Sci. USA, 100, 3031.
Lipid peroxidation
Russell Mechanism?
Is singlet oxygen generated from lipid hydroperoxides?
X
O2
LH

OO L
XH
LH
ONOO-,
OOH
X  = OH, RO , ROO , CO3 
L
LOO
LOOH
1O
2
?
Generation of Singlet Oxygen by the “Russell Mechanism”
1957
Russell proposed that
termination reaction of 2
peroxyl radicals involves a
tetraoxide intermediary state.
1968
Howard & Ingold showed the formation
of singlet oxygen in the reaction of
sec-butylhydroperoxide with Ce4+.
Russel J. Am. Chem. Soc., 79, 3871, 1957
Howard & Ingold J. Am. Chem. Soc., 90, 1057, 1968
O O•
R1
2
C
R2
C O + CH OH + 1O2
C
H
(22 kcal/mol)
O O
O
H
O
!
C
H
(100 kcal/mol)
LOO• + LOO•
LOOOOL
or
3
C O* + CH OH + O2
(75-80 kcal/mol)
L=O
L-OH
O2
Experimental Strategy
I- Synthesis of LA18O18OH
Incubation: LA18O18OH + Metal ion (Cerium, Iron, peroxynitrite)
LA18O18O
→
18O 18O 
-
18O
+
LA18O18O
IV-
18O
LA18O18O
[ LA18O18O 18O18OLA ]
18 1
[ O2]
Chemiluminescence
III
II
Chemical Trapping of [ xOxO(1g)] with DPA
EPR
R
R
+ xO2(1g)
R
Ox
Ox
R
R: –C6H5
DPA
X :: 18O or 16O atoms
DPA xOxO
Part I
Synthesis of 18O-Labelled Linoleic Acid
Hydroperoxide (LA18O18OH)
Structures of the 4 LAOOH isomers generated
from linoleic acid photooxidation under 18O2 atmosphere
13
12 10
O
9
18O
18
18
OH
2
Methylene Blue
Irradiation
OH
O
Linoleic Acid (LA)
O
O
OH
OH
18
9-LA18O18OH
18
18
O
18
OH
10-LA18O18OH
OH
O
O
O
OH
OH
13-LA18O18OH
18
O
18
OH
12-LA18O18OH
Synthesis of Phosphatidylcholine Hydroperoxides by
Photooxidation using Methylene Blue as a Photosensitizer
1O
2
OOH
Phosphatidylcholine
(PC)
Phosphatidylcholine Hydroperoxides
(PCOOH)
Electrospray ionization mass spectra of LAOOH and
LA18O18OH obtained in the negative ion mode.
100
A
[M-H2O-H]– = 293
[M-H]– = 311
311
[M-H]–
OH
O
O
LA16O16OH
(M = 312)
Relative abundance (%)
OH
[M-H2O-H]–
293
+4
0
100
B
[M-H218O-H]– = 295
18
315
[M-H]– = 315
18
OH
[M-H]–
O
O
LA18O18OH
(M = 316)
OH
[M-H218O-H]–
295
0
60
80
100 120 140 160 180 200 220 240 260 280 300 320 340 360
m/z
Generation of [18(1O2)] from LA18O18O
LA18O18OH / Metal ion (Cerium, Iron) or (peroxynitrite) → LA18O18O
R1
R2
C
18
H 18
O
O
18
2
H
18
O
O
18
O
(1g)
18
C

LA18O18O
R2
O
R1
LA18O18O 18O18OLA
1 18
[ O18O]
?
Part II
Detection and quantification of 1O2 by
chemical trapping with
9,10-diphenylanthrancene (DPA)
Mass spectrometry
HPLC/MS-MS
(18O-labeled compounds)
A
+
1
1O
A O2
2
O2
O
“ene” type reaction
O
H
H
Cycloaddition of Singlet Oxygen
1
O2
Cycloaddition [2+2]
O
O
1
O2
O
O
Cycloaddition [4+2]
Singlet Molecular Oxygen Quenching
Chemical Quenching
Q +
O2(1g)
kr
QO2
Physical Quenching
Q +
O2(1g)
Lycopene
kq
Q + O2(3S g) + heat
Chemical Trapping of [ xOxO(1g)] with DPA
-
18O
+
18O
[ O2]
.
.
LA18O18O
18 1
LA18O18O
[ LA18O18O 18O18OLA ]
Chemical Trapping of [ xOxO(1g)] with DPA
R
R
+ xO2(1g)
R
Ox
Ox
R
–C6H5
R: –
DPA
X :: 18O or 16O atoms
DPA xOxO
ROOH
ROS / RNS “Conversion” !
?
Ce4+, Fe2+
+ ONOOHOCl
Russell
1O
PC-OOH
LAOOH
2
Chemical Trapping of [ xOxO(1g)] by EAS and
Detection of EASxOxO by HPLC-ESI-MS
Probe
OSO3-
OSO3xOXO (1
+
EAS
g)
x= 16 or 18
OSO3-
OSO3-
EAS16O16O
%
0
222
224
226
228
230
m/z
EAS16O18O
%
EAS16O16O
0
232
234
236
238
230
100
LA18O18OH
+ HOCl
xO
EASxO2
228
100
LA16O16OH
+ HOCl
xO
222
224
226
EAS18O18O
228
229
228
230
232
m/z
234
236
238
Part III
Luminescence
Light Emission (spectroscopic investigation)
Dimol emission
O2(1g) + O2(1g)  2O2(3Sg) + hn (l= 634 and 703 nm)
Monomol emission
O2(1g)  O2(3S g) + hn (l= 1274 nm)
Detection and Characterization of 1O2
Improvement / “new” systems
Singlet Oxygen Monomol light Emission
O2(1g)  O2(3S g) + hn (l= 1270 nm)
O 2(1 g)
Sample Chamber
Monochromator
Ge-Diode
1O
2
hn
Photomultiplier
O2(3S g )
Emission spectrum of the
hydrogen peroxide-hypochlorite
reaction
H2O2 + OCl-  HOCl + HO2HOCl + HO2-  HOOCl + HOHOOCl + HO-  H2O + ClOOClOO-  1O2 + Cl------------------------------------------H2O2 + OCl-  1O2 + Cl- + H2O
Dimol emission
O2(1g) + O2(1g)  2 O2(3Sg) + hn
(l= 634 and 703 nm)
Khan and Kasha, 1963
Monomol emission
Near-IR
O2(1g)  O2(3S g) + hn (l= 1274 nm)
9000.00
8000.00
7000.00
6000.00
5000.00
4000.00
3000.00
Time (ms)
Wavelength (nm)
1200.00
1227.00
1241.00
1255.00
1282.00
1295.60
1309.00
0 5
10 15
20 25
30 35
40 45
50 55
60 65
1322.90
0.00
1268.30
1000.00
1213.70
2000.00
1337.00
Light Emission
(cps)
Characterization of singlet oxygen generated in the reaction
of LAOOH and Ceric ion by chemiluminescence
Dimol light emission at l > 570 nm
O2(1g) + O2(1g)  2O2(3Sg) + hn (l= 634 and 703 nm)
injection
Monomol light emission at l=1270 nm
H2O2 + OCl- (B)
12
O2(1g)  O2(3S g) + hn (l= 1270 nm)
10
8
NaN3
injection
injection
Ce4+
stop
injection
18
6
LAOOH + Ce4+ (A)
4
2
0
0
10
20
30
40
Time (seconds)
50
65
16
Ge-Diode Signal (104 counts)
Light Emission (10-2 mV)
l>570 nm
14
14
LAOOH + Ce4+ (A)
12
10
8
LAOOH + Ce4+ + NaN3 (B)
6
4
2
0
0
20
40
60
80
Time (seconds)
100
120
The presence of a hydrogen- at the carbon to which the
hydroperoxide is attached is essential for the generation
of O2 (1g).
ROOH / HOCl
Emissão de luz em 1270 nm (102 counts)
O2 (1g)
O2 (3Sg-) + hn (l=1270 nm)
160
A
140
6
H2O2
120
C
5
100
t-BuOOH
H3 C
H3 C C O OH
H3 C
4
80
100 %
60
40
3
2
HOCl
20
HOCl
1
0
0
6
30
60
90
B
5
HO
O
4
120
0
150
0
6
LAOOH
3
60
D
5
O
OH
30
90
14 %
HOCl
4
H3 C
2
1
150
CuOOH
3
2
120
O OH
C H3
C
HOCl
1
0
0
30
60
90
120
150
0
0
Tempo (s)
30
60
90
120
150
EPR spectrum of linoleate peroxyl radicals, LAOO.
Reaction of LAOOH with HOCl
g = 2.014
Part IV
A
Direct detection
B
3400
3410
3420
3430
Magnetic Field (G)
3440
Take-Home Message
Conclusion: Combination of Different Techniques !
Source
For example, lipids?
V- Clean chemical source of 18O-labeled reacive species, [1O2]
Clean source
OH
OH
H
N
I- Synthesis of
18O-labeled
II- Chemiluminescence
18
O
O
DHPN18O2
O18
source
Near-IR
H
N
Light Emission ( 103 cps)
2.4
OH
O
HO
18
+
18
O
(0,0) 1 g  3Sg-
and Visible
1270
2.1
1.8
1.5
1.2
0.9
0.6
O
(1
1200 1250 1300 1350
l (nm)
g)
trapping
HPLC-MS/MS
[M+H]+ = 367
C 6H 5
2
[ LA18O18O
C 6H 5
367
320
340
360
380
400
420
440
m/z
IV- EPR
3430
[M+H]+
%
III- Mass Spectrometry
B
3420
18[1O ]
2
300
A
Magnetic Field (G)
]
O
O
0
g = 2.014
3410
18O18OLA
18
330
100
EPR
3400
18
[M - 18O18O]+
LA18O18O
3440
Chemical source
R
18
O
of [18O] isotopically labeled singlet oxygen
18[1O ]
2
R
18
O
18
1
[O ]
2
+
R
R
? ..to elucidate mechanism of 1O2 reaction
towards biological target
Excellent tool (+MS) for mechanistic
studies of the reaction of singlet molecular
oxygen in biological media.
Positive APCI-mass spectra in the MS/MS mode of the endoperoxides
of 9,10-diphenylanthracene (DPAxOxO, x: 16O or 18O)
Probe
[M 100
DPA16O16O
A
[M+H]+ = 363
16O16O]+
C6 H5
330
16
O
[M+H]+
16
O
363
C6 H5
Relative abundance (%)
0
[M+H]+ = 365
[M - 16O18O]+
100
330
DPA18O16O
B
C6 H5
16
O
[M+H]+
18
O
C6 H5
365
0
100
[M -
DPA18O18O
C
[M+H]+ = 367
18O18O]+
C6 H5
330
18
[M+H]+
O
18
O
367
C6 H5
0
160
180
200
220
240
260
280
300
320
340
360
m/z
25 mM LA18O18OH + 25 mM Ce4+ + 60 mM DPA/37C, 1 hour
380
400
420
440
Energy transfer between singlet (1g) and triplet (3Sg-)
molecular oxygen in aqueous solution.
“propagation” !