Transcript Slide 1

NO Detection via Chemiluminescence
and Fluorescence
Martin Feelisch, Ph.D.
Boston University School of Medicine
Department of Medicine/Section of Molecular Medicine
Whitaker Cardiovascular Institute
12th
“Rigorous Detection and Identification of Free Radicals In Biology and Medicine”, Workshop,
Annual Meeting of the Society for Free Radical Biology and Medicine, Hilton, Austin, Nov 16, 2005
Part I.
NO Detection Using
Gas Phase Chemiluminescence
Advantages and Disadvantages of Chemiluminescence
Compared to Other Methods of NO Detection
 Advantages
High Sensitivity and Linearity over a Broad Concentration Range
Good Reproducibility and High Specificity for NO
(few other gaseous substances (DMSO, ethylene) react with ozone)
Measurements Possible in Turbid or Colored Samples, Even at
Extreme pH
(in solution, in the headspace, in expired air)
Besides Mass Spectrometry the Only Other Method that Allows
Quantification of Absolute Amounts of Nitroso Species
Moderate Running Costs
 Disadvantages
With Some Biological Samples Difficult to Extract NO into Gas Phase
Provides Limited Structural Information
Limited Sample Throughput, High Purchase Price for Detector
Quantification of NO, Nitroso and Nitrosyl Species
Using Gas Phase Chemiluminescence
NO present or generated in an aqueous system has to be purged out of solution by an inert
gas (N2, Ar, He) to be available for analysis in the gas phase.
Biological samples containing nitroso and nitrosyl compounds are processed using either
Photolysis to cleave NO-adducts or Chemical Reaction (i.e., injection into a denitrosating
reaction mixture) to convert these species into NO. The NO-containing gas is then transfered
to the analyzer for quantification.
Detection Principle:
In the reaction chamber NO is mixed, at a defined flow rate and under
reduced pressure, with ozone.
NO + O3
NO2* + O2
NO2*
NO2 + h . 
The light that is emitted by the fraction of excited NO2 on returning to the ground
state (chemiluminescence; 640-3000 nm) is measured by photon counting.
With O3 being present in large excess, light intensity is directly proportional to [NO]
Photolysis/Chemiluminescence Approach for
Detection of Nitroso Species
Reaction principle:
RSNO
R2NNO
light
light
RS. + NO.
R2N. + NO.
Discrimination of RSNOs from other nitroso species and nitrite by measurement
before and after HgCl2 treatment, and with lamp ON and OFF
Stamler et al., 1992, Alpert et al., 1997
Advantages and Problems with Photolysis-Based Techniques
Advantages
Low interference by contamination with nitrite
Fewer interferences with components of the redox-active reaction mix
Problems
Extremely high temperatures are reached inside of the photolysis cell
Reported RSNO values using photolysis-based technques are orders of magnitude higher
than those using reductive methods (µM rather than nM)
Artefactual generation of nitroso species by the photolysis of nitrate (NO3-) and the trapping of
RNOS by thiols
(Dejam et al., FRBM, 2004)
Photolysis of nitroso species other than S-nitrosothiols also generates a signal
Controls with nitrite and mercuric chloride are pointless due to low photolysis yield of nitrite
and the ability of mercuric salts to complex sulfhydryl groups
( blockage of the targets of artefactual nitrosation naturally leads to lower levels of nitroso-related signals in the presence of
HgCl2, but this does not neccessarily indicate involvement of RSNOs)
Sample Processing Using Redox-Active Reaction Mixtures
Many Choices, Many Pitfalls
Most techniques use Chemical Reactions to convert nitroso and nitrosyl species into NO,
which is then detected by chemiluminescence
Reducing mixtures differ largely in reducing strengths and reduction capacity
Iodine/iodide (I3-)
60 mM I-/6-20 mM I2/ 1M HCl, RT
56 mM I-/ 2 mM I2, 4mM CuCl, CH3COOH, 68°C
60 mM I-/10 mM I2, CH3COOH, 60°C
Cysteine/CuCl
Hydroqinone/Quinone
VCl3/H+
1 mM L-cysteine, 0.1 mM CuCl
0.1/0.01 mM
0.1 M in 2M HCl
Samouilov & Zweier, 1998
Marley et al., 2000
Feelisch et al., 2002
Fang et al., 1998
Samouilov & Zweier, 1998
Ewing et al., 1998
Oxidizing mixture for determination of NO-hemes
Ferricyanide
0.2 M in PBS pH 7.5
Gladwin et al., 2002
Bryan et al, 2004
General Problem:
Neither method is absolutely specific and bears the potential to produce false positive
(nitrate, L-NitroArg, ...) or negative signals
Reaction Chambers Come in Many Different Designs
Menon et al., 1991
Cox & Frank, 1982
Dunham et al., 1995
The Most Frequently Used Type of Chemiluminescence Set-up
Samouilov & Zweier, 1998
Application Example
Direct Measurement of NO Release from NO Donor Compounds
8
NO [ppb]
6
expected:
found:
4
100 pmol
107 pmol
2
0
2
4
6
8
Time (min)
10
12
1 µM MAHMA/NONOate
(direct injection of 100 µL into PBS, 37°C)
Which NO-Related Species are Detected and
How can they be Discriminated from One Another?
Without Reduction Step:
NO
Upon Acidification:
NO2RONO
(direct injection into buffer or water)
30
(disproportionation of HNO2)
Nitrite
GSNO
Nitrite
With Sample Reduction:
NO2-/NO3RSNO
RNNO
NO-Heme
(KI/CH3COOH, RT for nitrite,
+
VCl3/H , 90°C for nitrate)
-
(I3 /CH3COOH, 60°C)
NO [pbb]
(acid-catalysed decompos.)
20
SNOAlb
10
NOHb
NOW
0
0
5
10
15
Time [min]
detection limit:
1-50 nM, depending on flow and inj. volume
250 fmoles NO (@ 50 µL injection vol.)
Discrimination between different species:
-
Selective NO2 removal
RSNOs from other Nitroso-Species
Nitroso from Nitrosyl Species
+
Sulfanilamide/H
HgCl2/sulfanilamide
Reducing vs. Oxidizing Reaction Mix
60
65
Now, how am I going to do this practically?
biological sample
Split into aliquots
Nitrosyl Species
Nitrite and Nitroso Species
(direct injection)
(split into further aliquots, depending on
requirements, differentiation between different
types of compounds by reaction with
group-specific reagents)
Oxidation
Reduction
(Ferricyanide Solution,
neutral pH)
(Iodine/Iodide Reaction Mix,
acidic pH)
Application Example
Detection of Nitrosyl-Heme Species in Rat Tissues Using Ferricyanide
4
Brain
Ferricyanide
2+
Fe
NO-Hb
NO [ppb]
3
Fe
3+
3+
Fe
MetHb + NO
Ferrocyanide
Fe
2+
2
RBC
lysate
1
Plasma
Heart
Liver
Kidney Lung
Aorta
0
5
10
15
20
25
30
35
40
Time (min)
0.05M ferricyanide in PBS, pH 7.5 @ 37°C
Discrimination Between Nitrite and Different Nitroso
Species Using a Reducing Iodine/Iodide Reaction Mix
in Combination with Group-Specific Reagents
biological sample
1
2
3
untreated sample
sulfanilamide/H+
HgCl2+sulfanilamide/H+
(15 min)
(45 min)
nitrite
nitrite
nitrite
RSNO
RSNO
RSNO
other
nitroso compounds
other
nitroso compounds
other
nitroso compounds
Discrimination Between Nitrite and Nitroso Species
Using Group-Specific Sample Processing
1
untreated
NO [ppb]
30
2
3
+Sulf/H +
20
+ HgCl2/Sulf/H+
Nitrite
10
RSNO
RNNO
0
(and possibly
other species)
0
5
Time [min]
10
15
Thiol Alkylation and Nitrite Removal are Required to Prevent
Artifactual Nitrosation of Cellular Constituents
Problem:
Reaction of protein thiols (or GSH) with nitrite to form RSNO
SNO
SNO
+ NO2-
HS
Solution:
SH
Alkylation of –SH groups
HS
SNO
with either NEM or iodacetamide
(5-10 mM in PBS, 15 min)
Nitrite Removal
using Sulfanilamide/H+
(15 min RT; azide, urea, sulfamic acid don’t work!)
using Size Exclusion Chromatography
(Sephadex G-25), followed by Sulfanilamide/H+
Most Important Factors Affecting Assay Sensitivity
Injection Volume
10-1000 µL, depending on sample availability, size of reaction vessel and gas flow
Rate of Reduction
Molarity and Temperature of the Reaction Mixture
(rapid reduction produces sharp peaks)
Flow Rate of Purging Gas and Dead Space of the System
50 mL/min-3000 mL/min (depending on whether developed for environmental monitoring or research)
100-200 µL/min represents a good compromise between short analysis time (high flow) and
high sensitivity (low flow)
Dead space should be as small as possible
Detector Sensitivity, Integration Time and Baseline Noise
Largely determined by instrument noise (dependent on photomultiplier type and temperature as well
as on reaction chamber design; Dasibi, Sievers and EcoPhysics machines differ by a facor of 2-10)
Longer integration time increases sensitivity
Baseline noise increases with fluctuations in pressure
Nitrite Background
Nitrite contamination (water, glass- and plasticware incl. pipette tips, ultrafiltration membranes)
Recent Challenges to the Validity of This Analytical Method
Mercury-stable nitroso signal in the iodine/iodide assay may be nitrated lipids rather
than N-nitrosamines
(Schopfer et al., J Biol Chem 2005)
However,
spiking with a final concentration of 75 µM (!) of a nitrated lipid standard was required to
produce a response similar to the Hg-stable signal in human plasma, while the endogenous
concentration of these species was estimated (by the same authors) not to exceed 1-2 µM.
The „harsh chemistry“ required to completely remove nitrite from biological samples
when working with reductive chemiluminescence-based assay (i.e. the pretreatment
with sulfanilamide/H+) renders S-nitrosothiols unstable
(Stamler et al., repeated editorial claims without data)
(Rogers et al., J Biol Chem, 2005)
However,
all tested S-nitrosothiols are rock-stable under these conditions
(Feelisch et al. & Gladwin et al., unpublished)
Heme autocapture may be responsible for the previous lack of detection of nitroso
species in human red blood cells
(Rogers et al., J Biol Chem, 2005)
However,
no problem other than peak broadening was observed in the presence of very high conc of Hb
(Feelisch et al. & Gladwin et al., unpublished)
Part II.
NO Detection & Bioimaging Using
Fluorescence
Bioimaging of Nitric Oxide Using DAF-2
Detection principle:
Reaction of aromatic vicinal diamines with NO in the presence of oxygen to produce the
corresponding triazenes
cell membrane
DAF-2 DA
DAF-2
NH 2
DAF-2 T
N NH
N
NH 2
H 2N
H 2N
esterases
O
NOx
-
COO
-
COO
O
AcO
O
OAc
non-fluorescent,
cell-permeable
Advantages:
-
O
O
non-fluorescent
O
-
O
O
O
fluorescent,
Ex 495 nm, Em 515 nm
Sensitivity for NO (5 nM in vitro) with high temporal and spatial resolution
No cross-reactivity to NO2-/NO3- and ONOO-
Assay limitations: Possible interference by reducing agents and divalent cations, pH sensitive,
subject to photobleaching, requiring standardized illumination conditions
Yes,
You can produce lots of pretty images, …
Bioimaging of Nitric Oxide Using DAF-2 in Endothelial Cells
Control
Unloaded cells
Time course of NO formation in response to BK (100 nM)
t=0
5s
60s
120s
180s
HUVECs P4, labelling: DAF-2 DA 10 µM for 60 min; incubation:HBSS + L-Arg (1 mM), 37°C
Propagation of NO Wave during Stimulation of Endothelial Cells
with the Calcium Ionophor, A23187 (1 µM)
t=0
0.5 min
1 min
1.5 min
2 min
5 min
10 min after stimulation of cells with BK
Yes,
You can produce lots of pretty images, …
But …
Problems and Pitfalls with DAF-2 as an NO Probe
 Unclear what species exactly is detected in biological systems
More likely an indicator of nitrosative events (i.e. of RNOS) than of NO per se
 The true sensitivity for “NO” in tissues is compromised by the presence of thiols and
other antioxidants and autofluorescence of the probe
(Rodriguez et al, 2005)
 Specificity for “subcellular formation of NO” depends on the degree of compartmentalization in the tissue
 Complex metabolism and susceptibility to oxidation renders quantitative comparisons
problematic
DAF-2 may undergo oxidative transformation to a radical intermediate (Jourd’heuil; 2002)
DAF-2T may undergo rapid reduction or quenching (producing transient signals)
 DAF-2 forms adducts with ascorbate and dehydroascorbate
(Zhang et al, 2002)
 There is a light-sensitive component in cells/tissues the nature of which is unclear
Nitrate/thiol interaction?
 Formation of adducts with mercuric salts and glutathione results in spectral changes
that may be misinterpreted as NO signals
(Rodriguez et al, 2005)
Why does a probe that requires nitrosation work at all
in vascular tissue and other biological environments?
100
DAF-2 10µM
DAF-2 50µM
60000
80
Fluorescence counts
Fluorescence (% of control)
How does it compete with endogenous antioxidants?
60
40
20
50000
40000
30000
20000
10000
0
0
0
0.01 0.03 0.1
0.3
1
3
10
Ascorbate concentration (mM)
0
0.1
0.5
2.5
GSH concentration (mM)
How does it compete with other cellular targets (e.g. reactive protein moieties)?
Using incubation conditions frequently used in the literature (10 µM) intracellular
DAF-2 concentrations approach the millimolar concentration range
(Rodriguez et al, 2005)
Compartmentalization of the Probe Around Elastic Lamina
Limits its Potential to Characterize the Subcellular Site of
NO Production in the Vasculature
DAF-2 +
NO Donor
Basal
H&E stain
UV light
UV illumination leads to levels of nitrosating species that interfere with NO
detection by enzymatic sources
(Rodriguez et al, 2005)
400
350
DAF-2 Fluorescence
cGMP Level
300
250
200
150
100
50
0
30
0
32
0
34
0
36
0
38
0
40
0
42
0
44
0
46
0
48
0
50
0
52
0
Changes from control (%)
Can Targets of NO Be Detected Through Photolysis?
Irradiation wavelength (nm)
NO is generated via photolysis from a UV-absorbing species with an absorption peak
below 310 nm, consistent with the characteristics of nitrate (NO3-)
(Rodriguez et al, 2005)
Recent Developments
Development and commercial availability of red fluorescent chromophores (diaminorhodamine-based; DAR-4M) increases flexibility for combinations with other greenfluorescent probes and shows reduced interference with tissue autofluorescence,
but is otherwise very similar to DAF-2
Difluoroboradiaza-s-indacene based fluorophore (similar chemistry)
Detection of nitroso peptides and proteins on diaminofluoresceine gels
(standard SDS-PAGE followed by UV photolysis in the presence of DAF-2 or DAF-FM for detection of C-,
O-, N- ans S-nitrosated compounds)
(Mannick et al, 2005)
Near-Infrared fluorescent probes for „NO“ detection in isolated organs
(tricarbocyanine as NIR fluorochrome coupled to o-phenylenediamine as NO sensor; NIR is potentially very
interesting for in vivo imaging approaches as it allows deeper penetration of light into tissues and shows no
interference with tissue autofluorescence; promising novel approach
(Nagano et al, 2005)
Amplifier-coupled fluorescent NO indicator with nanomolar sensitivity in living cells
(genetically encoded fluorescent indicator based on the binding of NO to soluble guanyly cyclyase and
detection of formed cGMP by FRET; interesting, but potentially problematic cross-talk with cGMP generated
by particulate GC and modulation of sensitivity by PDE activity)
(Sato et al, 2005)