NO Detection via Chemiluminescence

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
Rigorous Detection and Identification of Free
Radicals In Biology and Medicine, Workshop, 12th
Annual Meeting of the Society for Free Radical
Biology and Medicine, Hilton, Austin, Nov 16, 2005
2
Part I. NO Detection Using Gas Phase
Chemiluminescence
3
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
   
4
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. D
etection 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
5
Photolysis/Chemiluminescence Approach for
Detection of Nitroso Species
Reaction principle RSNO RS.
NO. R2NNO 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
light
light
6
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)
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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 Samouilov Zweier,
1998 56 mM I-/ 2 mM I2, 4mM CuCl, CH3COOH,
68C Marley et al., 2000 60 mM I-/10 mM I2,
CH3COOH, 60C Feelisch et al.,
2002 Cysteine/CuCl 1 mM
L-cysteine, 0.1 mM CuCl Fang et al.,
1998 Hydroqinone/Quinone 0.1/0.01
mM Samouilov Zweier, 1998 VCl3/H
0.1 M in 2M HCl 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
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Reaction Chambers Come in Many Different Designs
Menon et al., 1991
Cox Frank, 1982
Dunham et al., 1995
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The Most Frequently Used Type of
Chemiluminescence Set-up
Samouilov Zweier, 1998
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Application Example Direct Measurement of NO
Release from NO Donor Compounds
expected 100 pmol found 107 pmol
1 µM MAHMA/NONOate (direct injection of 100 µL
into PBS, 37C)
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Which NO-Related Species are Detected and How can
they be Discriminated from One Another?
Without Reduction Step NO (direct injection
into buffer or water) Upon Acidification NO2-
(disproportionation of HNO2) RONO (acid-catalyse
d decompos.) With Sample Reduction NO2-/NO3- (K
I/CH3COOH, RT for nitrite, VCl3/H, 90C for
nitrate) RSNO RNNO (I3-/CH3COOH,
60C) NO-Heme detection limit 1-50 nM,
depending on flow and inj. volume 250
fmoles NO (_at_ 50 µL injection vol.) Discrimination
between different species Selective NO2-
removal Sulfanilamide/H RSNOs from other
Nitroso-Species HgCl2/sulfanilamide Nitroso from
Nitrosyl Species Reducing vs. Oxidizing Reaction
Mix
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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,
(Iodine/Iodide Reaction Mix, neutral pH)
acidic pH)
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Application Example Detection of Nitrosyl-Heme
Species in Rat Tissues Using Ferricyanide
Brain
Ferricyanide Fe3
Fe2
Fe3
NO-Hb
MetHb NO
Ferrocyanide Fe2
RBC lysate
Plasma
Heart
Liver
Kidney
Lung Aorta
5 10 15 20 25
30 35 40
0.05M ferricyanide in PBS, pH 7.5 _at_ 37C
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Discrimination Between Nitrite and Different
Nitroso Species Using a Reducing Iodine/Iodide
Reaction Mix in Combination with Group-Specific
Reagents
biological sample
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Discrimination Between Nitrite and Nitroso
Species Using Group-Specific Sample Processing
1
2
3

HgCl2/
Sulf/H
30
Sulf/H
untreated
20
Nitrite
NO ppb

10
RSNO
RNNO (and possibly other species)
0
0
5
10
15
Time min
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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 Solution Alkylatio
n of SH groups with either NEM or
iodacetamide (5-10 mM in PBS, 15
min) Nitrite Removal using
Sulfanilamide/H (15 min RT azide, urea,
sulfamic acid dont work!) using Size
Exclusion Chromatography (Sephadex G-25),
followed by Sulfanilamide/H
SNO
SNO
NO2-
HS
HS
SH
SNO
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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)
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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)
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Part II. NO Detection Bioimaging Using
Fluorescence
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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
DAF-2 T
NOx
esterases
fluorescent, Ex 495 nm, Em 515 nm
non-fluorescent, cell-permeable
non-fluorescent
Advantages 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
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Yes, You can produce lots of pretty images,
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Bioimaging of Nitric Oxide Using DAF-2 in
Endothelial Cells
Control
Unloaded cells
Time course of NO formation in response to BK
(100 nM)
60s
5s
120s
180s
t0
HUVECs P4, labelling DAF-2 DA 10 µM for 60 min
incubationHBSS L-Arg (1 mM), 37C
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Propagation of NO Wave during Stimulation of
Endothelial Cells with the Calcium Ionophor,
A23187 (1 µM)
t0 0.5 min 1 min
1.5 min 2 min 5 min
10 min after stimulation of cells with BK
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Yes, You can produce lots of pretty images,
But
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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 compartment- alizatio
n in the tissue Complex metabolism and
susceptibility to oxidation renders quantitative
comparisons problematic DAF-2 may undergo
oxidative transformation to a radical
intermediate (Jourdheuil 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)
   
   
   
   
   
   
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Why does a probe that requires nitrosation work
at all in vascular tissue and other biological
environments?
How does it compete with endogenous antioxidants?
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)
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Compartmentalization of the Probe Around Elastic
Lamina Limits its Potential to Characterize the
Subcellular Site of NO Production in the
Vasculature
HE stain
DAF-2 NO Donor
Basal UV light
UV illumination leads to levels of nitrosating
species that interfere with NO detection by
enzymatic sources
(Rodriguez et al, 2005)
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Can Targets of NO Be Detected Through Photolysis?
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)
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Recent Developments
Development and commercial availability of red
fluorescent chromophores (diamino-rhodamine-based
DAR-4M) increases flexibility for combinations
with other green-fluorescent 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)