AHA Scientific Statement
Measurement of Reactive Oxygen Species, Reactive
Nitrogen Species, and Redox-Dependent Signaling
in the Cardiovascular System
A Scientific Statement From the American Heart Association
Kathy K. Griendling, PhD, FAHA, Co-Chair*; Rhian M. Touyz, MD, PhD, FAHA, Co-Chair*;
Jay L. Zweier, MD, FAHA; Sergey Dikalov, PhD; William Chilian, PhD, FAHA;
Yeong-Renn Chen, PhD; David G. Harrison, MD, FAHA; Aruni Bhatnagar, PhD, FAHA;
on behalf of the American Heart Association Council on Basic Cardiovascular Sciences
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Abstract—Reactive oxygen species and reactive nitrogen species are biological molecules that play important roles in
cardiovascular physiology and contribute to disease initiation, progression, and severity. Because of their ephemeral nature
and rapid reactivity, these species are difficult to measure directly with high accuracy and precision. In this statement, we
review current methods for measuring these species and the secondary products they generate and suggest approaches
for measuring redox status, oxidative stress, and the production of individual reactive oxygen and nitrogen species. We
discuss the strengths and limitations of different methods and the relative specificity and suitability of these methods for
measuring the concentrations of reactive oxygen and reactive nitrogen species in cells, tissues, and biological fluids. We
provide specific guidelines, through expert opinion, for choosing reliable and reproducible assays for different experimental
and clinical situations. These guidelines are intended to help investigators and clinical researchers avoid experimental
error and ensure high-quality measurements of these important biological species. (Circ Res. 2016;119:e39-e75.
DOI: 10.1161/RES.0000000000000110.)
Key Words: AHA Scientific Statements ◼ free radicals ◼ oxidants ◼ peroxides ◼ reactive nitrogen species
◼ reactive oxygen species
R
eactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced in virtually all eukaryotic cells.
These molecules regulate several physiological processes including proliferation, migration, hypertrophy, differentiation,
cytoskeletal dynamics, and metabolism, but when available
in excess, they react with lipids, proteins, and nucleic acids,
thereby altering structural and functional properties of target
molecules and leading to extensive tissue dysfunction and injury. ROS and RNS have been implicated in the development
of many cardiovascular diseases, including hypertension, heart
failure, atherosclerosis, and cardiovascular and renal complications of diabetes mellitus. Cardiovascular injury caused by
ischemia-reperfusion or exposure to environmental pollutants,
tobacco smoke, and ionizing radiation has also been linked to
excessive ROS or RNS accumulation.
Despite extensive data implicating oxidative stress in animal models of cardiovascular disease and dysfunction, there
is still no conclusive evidence that ROS/RNS are fundamentally involved in the pathogenesis of cardiovascular disease
in humans, and there is a paucity of convincing evidence that
*Drs Griendling and Touyz contributed equally to this work.
The American Heart Association makes every effort to avoid any actual or potential conflicts of interest that may arise as a result of an outside relationship
or a personal, professional, or business interest of a member of the writing panel. Specifically, all members of the writing group are required to complete
and submit a Disclosure Questionnaire showing all such relationships that might be perceived as real or potential conflicts of interest.
This statement was approved by the American Heart Association Science Advisory and Coordinating Committee on February 15, 2016, and the American
Heart Association Executive Committee on April 25, 2016. A copy of the document is available at http://professional.heart.org/statements by using either
“Search for Guidelines & Statements” or the “Browse by Topic” area. To purchase additional reprints, call 843-216-2533 or e-mail kelle.ramsay@
wolterskluwer.com.
The American Heart Association requests that this document be cited as follows: Griendling KK, Touyz RM, Zweier JL, Dikalov S, Chilian W, Chen
Y-R, Harrison DG, Bhatnagar A; on behalf of the American Heart Association Council on Basic Cardiovascular Sciences. Measurement of reactive oxygen
species, reactive nitrogen species, and redox-dependent signaling in the cardiovascular system: a scientific statement from the American Heart Association.
Circ Res. 2016;119:e39–e75. doi: 10.1161/RES.0000000000000110.
Expert peer review of AHA Scientific Statements is conducted by the AHA Office of Science Operations. For more on AHA statements and
guidelines development, visit http://professional.heart.org/statements. Select the “Guidelines & Statements” drop-down menu, then click “Publication
Development.”
Permissions: Multiple copies, modification, alteration, enhancement, and/or distribution of this document are not permitted without the express
permission of the American Heart Association. Instructions for obtaining permission are located at http://www.heart.org/HEARTORG/General/CopyrightPermission-Guidelines_UCM_300404_Article.jsp. A link to the “Copyright Permissions Request Form” appears on the right side of the page.
© 2016 American Heart Association, Inc.
Circulation Research is available at http://circ.ahajournals.org
DOI: 10.1161/RES.0000000000000110
e39
e40 Circulation Research August 19, 2016
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antioxidants are effective therapies in cardiovascular medicine. Yet the belief remains that oxidative stress contributes
to many cardiovascular pathologies. Putative reasons for this
paradox relate to the relatively weak nature of the antioxidants
used in clinical trials, an incomplete understanding of the
complex molecular mechanisms whereby ROS cause pathological changes, the difficulty in extrapolating findings from
experimental models to clinical scenarios, and the methodological challenges relating to accurate measurement of ROS
in the cardiovascular system.
Although myriad techniques, protocols, assays, and commercial kits have been developed to measure ROS, and numerous biomarkers have been used to study human oxidative
stress, many of these assays are used inappropriately or without due experimental diligence, resulting in potential inaccuracies and artifacts, as well as much confusion in the literature.
Moreover, the cardiovascular researcher who is a novice in the
field of free radical biology may be overwhelmed in choosing
from the many tools and methods currently available to measure ROS, RNS, and oxidative stress.
Objectives
The purpose of this scientific statement is to review current
methods for measuring ROS, RNS, and their secondary products; to discuss the strengths and limitations of specific methodological approaches; and to provide guidelines, through expert
opinion, for reliable and reproducible techniques for quantifying these species and for elucidating their role in redox-dependent signaling and cell injury. Because of the diversity of
experimental systems in which the roles of these species are
interrogated, we offer here only general methodological guidelines and assistance with decisions as to which assay to choose.
This review does not focus on specific protocols, and the reader
is referred to appropriate methodology-based articles for specific protocols. Measurements of ROS and RNS in clinical
studies are discussed separately to recommend a distinct set of
measurements that should be made to assess systemic oxidative
stress and to document oxidative injury in clinical scenarios.
Background
Chemically reactive molecules containing ROS or RNS are
generated endogenously in most cells during the course of
normal metabolism, during disease development, and in response to tissue injury. These species are also produced on
exposure to exogenous insults such as environmental pollutants, tobacco smoke, and ionizing radiation. Most ROS and
RNS are relatively unstable oxygen- or nitrogen-centered free
radicals that contain unpaired electrons (biological half-life is
microseconds to milliseconds); however, these species also include more stable species such as peroxides and nitrosothiols
(biological half-life is seconds). Major ROS and RNS generated in biological systems are listed in Table 1.
Although nitrogen itself is a stable diatomic gas, molecular oxygen in the ground state is a free radical with 2 unpaired
electrons in its outmost shell. Because both these electrons
have parallel spin, diatomic oxygen is relatively unreactive.
However, during its utilization in the mitochondria, oxygen
accepts 1 electron at a time, which leads to the generation of
.−
partially reduced species such as superoxide (O2 ). Most of
the oxygen is reduced to water, but incomplete reduction of
.−
O2 leads to the generation of O2 , hydrogen peroxide (H2O2),
and hydroxyl radical (•OH). Hence, normal utilization of oxygen during mitochondrial respiration results in continuous
ROS formation. Superoxide is unstable, with a lifetime of milliseconds at neutral pH, and in aqueous solution it spontaneously reacts or dismutates to yield H2O2 and O2.
Hydrogen peroxide by itself is fairly unreactive, but it
oxidizes Fe(II) to Fe(III) to generate hydroxyl radicals in a
.−
process known as the Fenton reaction.1,2 In vitro, O2 can
reduce the oxidized metal generated by the Fenton reaction
to regenerate Fe(II) (Haber-Weiss reaction); however, the in
vivo identity of the reductant is less clear, and Fe(III) can be
reduced with variable efficiency by a variety of cell reductants. In addition to iron, other metals such as Cu, Cr, Co, V,
and to a lesser extent Ni can catalyze Fenton chemistry,3 but
in most biological systems the role of iron is critical. In most
vertebrates, iron is bound to ferritin, which oxidizes Fe(II) at
the ferroxidase center, thereby decreasing Fe(II) available for
the Fenton reaction.4 Notably, dysregulation of iron homeostasis because of injury or hereditary ferritinopathies results
in increased oxidative damage. In the mitochondria, destruction of the [4Fe-4S] cluster (eg, aconitase) can also result in
the release of Fenton-active Fe(II), leading to increased ROS
production.5 This could be a mechanism whereby non-Fenton
metals (such as Ag and Hg) exert toxicity by increasing ROS
production.6 The hydroxyl radical generated by the Fenton
reaction is extremely reactive and short-lived. It reacts indiscriminately with most cell constituents and leads to the
generation of secondary free radical species that deplete antioxidants and oxidize thiols and unsaturated lipids. In contrast
to the indiscriminate reactivity of the hydroxyl radicals, ROS
generated by enzymatic reactions such as those catalyzed by
nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, cytochrome P450 enzymes, and glucose oxidase are
generally considered to serve signaling functions, although
localized generation of hydroxyl-like species (eg, in the endoplasmic reticulum) could also affect the expression of specific
genes or their coactivators.7
NO is a diatomic molecule containing 1 atom of oxygen
(O, 8 electrons) and 1 atom of nitrogen (N, 7 electrons) and
therefore contains 1 unpaired electron, which makes it a free
radical.8 NO is a π-radical where the unpaired electron is delocalized between nitrogen and oxygen atoms, rendering it
relatively stable. NO can be isolated as a gas and is stable in
the absence of oxygen. In mammalian cells, NO is enzymatically formed by nitric oxide synthases (NOS), which oxidize
l-arginine. NO acts as a second messenger to modulate numerous biological processes, including endothelial function,
vascular smooth muscle cell contraction/dilation, inflamma.−
tion, neuroplasticity, and cytotoxicity. In the presence of O2 ,
NO is converted into the strong oxidant peroxynitrite. NO can
also react with oxygen to form other RNS and with thiols to
generate nitrosothiols (RSNO).
Both ROS and RNS are highly reactive, and as a result, they have short half-lives in biological environments.
Therefore, these species are difficult to measure directly, and
even indirect estimates of their abundance and reactivity are
challenging. Although many approaches have been adopted
Griendling et al Measurement of Reactive Oxygen Species e41
Table 1. Major Biological Reactive Oxygen and Reactive Nitrogen Species
Formula
Name
Comment
Reactive oxygen species
O2
1
Singlet oxygen
H2O2
Hydrogen peroxide
HOO∙
Hydroperoxyl radical
HO∙
Hydroxy radical
O2
Superoxide anion
.-
O3
Ozone
Electronically excited state of molecular oxygen
Simplest peroxide with oxygen-oxygen single bond
Free radical; protonated form of superoxide
Free radical; neutral form of hydroxide ion (HO−)
Free radical; dioxide (1-); product of 1 e− reduction of
dioxygen
Trioxygen; an allotrope of oxygen
OCl−
Hypochloride anion
Salt of hypochlorous acid
ROOH
Hydroperoxide
Organic peroxide, protonated peroxyl radical
ROO∙
Peroxyl radical
Free radical; lipid peroxyl radical when R is a lipid carbon
RO∙
Alkoxyl radical
Free radical; lipid alkoxyl radical when R is a lipid carbon
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Reactive nitrogen species
∙NO
Nitric oxide
N2O
Nitrous oxide
“Laughing gas,” reacts with oxygen to generate nitric oxide
ONOO−
.
NO2
Peroxynitrite
Product of the reaction between superoxide and nitric oxide
Nitrogen dioxide
N2O3
Dinitrogen trioxide
ONOOH
Peroxynitrous acid
Nitrogen-centered free radical
Free radical, derived from peroxynitrite
Product of reaction between nitric oxide and nitrogen dioxide
Protonated form of peroxynitrite
NO−
Nitroxyl anion
Conjugate base of nitroxyl
NO+
Nitrosyl cation
Also, nitrosonium cation
HNO2
Nitrous acid
NO2Cl
Nitrosyl chloride
NO2−
Nitrite
NO2+
Nitronium ion
Also nitryl ion, generated from the removal of e- from
nitrogen dioxide or protonation of nitric acid
RSNOs
Nitrosothiols
Formed by covalent addition of nitric oxide to cysteine and
protein or nonprotein sulfhydryl residues
to circumvent these limitations, and a wide range of methods
have been devised to quantify ROS, RNS, and their secondary products, there are many considerations in the choice of
assay and its application to a particular system. Following
are specific methodological guidelines to assist researchers in
choosing appropriate assays and designing specific controls to
obtain reliable measures.
Measurement of Superoxide, Hydrogen
Peroxide, and Redox Status
Introduction
Many methods have been devised to measure oxygen free
radicals and their derived oxidants with various levels of sen.−
sitivity and accuracy. These include chemical assays for O2 ,
H2O2, or •OH generation, direct chemiluminescent assays or
measurement of signal in the presence of chemiluminigenic
probes, fluorescence detection in the presence of redox-sensitive probes, and either direct or spin trapping–based electron paramagnetic resonance (EPR) spectroscopy. Only EPR
Weak monobasic acid
Derived from nitrite and hypochlorous acid
Anion, generated from nitric oxide
enables the direct detection of free radicals, but other assays
can be informative if used with proper controls.
.
Measurement of Superoxide Anion Radical (O2− )
EPR Assay: EPR Spin-Trapping
EPR spectroscopy is the “gold standard” for unambiguous assessment of oxygen-centered free radical generation in biological systems. EPR, also known as electron spin resonance,
is a method to detect molecules with unpaired electrons. When
combined with an appropriate spin trap, it is a powerful and
.−
reliable technique to unequivocally measure O2 , •OH, and NO
in biological samples (for NO, see Measurement of RNS).
A variety of spin traps have been used to detect and
quantify free radicals in cardiovascular tissues. Spin trapping with DMPO (5, 5-dimethyl-1-pyrroline-N-oxide) is the
.−
strategy most widely used to detect O2 or •OH generated in
endothelial cells9,10 or myocytes (see Table 2 for comparison
of spin traps).11–14 DMPO has also been used to assay for
.−
mitochondria-derived O2 generation by EPR.13,15 Because
of the short half-life of DMPO/•OOH (t1/2 ≈45 seconds) and
e42 Circulation Research August 19, 2016
Table 2. Summary of EPR Spin Probes Used to Measure Oxygen Free Radical(s) and Redox Status In Vitro, Ex Vivo, and In Vivo
Spin Probes
Nature of Probes
System Applied
DMPO
Nitrone spin trap
In vitro and ex vivo
O2 , OH, and carbon-centered radicals in vitro and ex vivo
DEPMPO
Nitrone spin trap
In vitro
O2 and •OH generation mediated by the enzymes of cellular
systems of endothelium and myocytes
PBN or POBN
Nitrone spin trap
In vitro, ex vivo, and in vivo
In vivo spin trapping of O2 -derived hydroxyl and alkyl radicals
TAM radical (OXO63
and CT02-H)
Trityl free radical
In vitro and ex vivo
Simultaneous determination of O2 and oxygen consumption
in vitro and ex vivo
TEMPOL
Stable nitroxide
In vitro and ex vivo
Redox status in vitro and ex vivo with X-band EPR
PCA
Stable nitroxide
In vitro and in vivo
In vivo redoximetry with L-band EPR
TEMPONE
Stable nitroxide
In vivo
Biradical spin probe
In vitro and ex vivo
RSSR
.−
Measurements
•
.−
.−
.−
In vivo redoximetry with PEDRI
Detection of reduced thiols with X-band EPR
.−
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CM-H
Hydroxylamine spin probe
In vitro, ex vivo, and In vivo
Detection of total cellular and tissue O2 by X-band EPR
CP-H
Hydroxylamine spin probe
In vitro, ex vivo, and in vivo
Measurements of O2 in membrane fractions, cells, tissue
PP-H
Hydroxylamine spin probe
In vitro, ex vivo, and in vivo
Measurements of extracellular O2 using X-band EPR
CAT1-H
Hydroxylamine spin probe
In vitro and ex vivo
Measurements of extracellular O2
TM-H
Hydroxylamine spin probe
In vitro and ex vivo
Detection of cellular and mitochondrial O2
mitoTEMPO-H
Hydroxylamine spin probe
In vitro, ex vivo, and in vivo
.−
.−
.−
.−
.−
Mitochondria-targeted detection of O2
EPR indicates electron paramagnetic resonance spectroscopy; and PEDRI, proton-electron double-resonance imaging.
.−
the possibility that the O2 adduct can decompose to give an
•
OH adduct, a superoxide dismutase (SOD)–sensitive 4-line
spectrum of DMPO/•OH is often detected. Therefore, a suitable SOD, polyethylene glycol–conjugated SOD (PEG-SOD),
or SOD mimetic must be used to confirm that the detected
.−
DMPO/•OH is dependent on the formation of O2 .16–18 To dis.−
tinguish O2 from •OH in biological systems or for in vivo
•
OH detection, DMSO (dimethyl sulfoxide), a specific hydroxyl radical scavenger, is used.19 An alternative to DMPO is
DEPMPO [5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-Noxide], a diethoxylphosphoryl derivative of DMPO, which
.−
has the advantage that it forms a more stable O2 adduct (t1/2
≈15 minutes).20 PBN (α-phenyl-N-t-butylnitrone) or POBN
[α-(4-pyridyl-1-oxide)-N-tert-butylnitrone] have been used
effectively for in vivo spin trapping of ROS production.21,22
.−
Although these traps are useful for trapping O2 -derived hy.−
droxyl and alkyl radicals, stable O2 adducts are not typically
formed with PBN or POBN.
In vivo or in situ direct EPR or EPR spin trapping with
DMPO has been demonstrated in the Langendorff-perfused
.−
heart, which provides direct evidence of O2 generation during myocardial ischemia and reperfusion injury.11,14,23,24 These
measurements also detect the carbon-centered radical formed
by ischemia and reperfusion injury–mediated lipid oxidation or lipid peroxidation (DMPO/•R).14,24 Ex vivo EPR spin
trapping with DMPO has been used recently to quantify mi.−
tochondria-derived O2 in the rat heart.15 DEPMPO has been
used extensively to investigate molecular mechanisms of ROS
generation mediated by enzymes such as endothelial NOS
(eNOS),25 NADPH oxidases, xanthine oxidase,26 and the mitochondrial electron transport chain in vitro.27–30
In summary, EPR spin trapping with either DMPO or
DEPMPO is a useful approach for measuring oxygen-, carbon-, nitrogen-, and sulfur-centered radicals. These spin traps
.−
.−
can measure O2 generation in cells, as well as O2 or •OH
generation mediated by cardiovascular enzymes and mitochondria in vitro and in the isolated heart ex vivo. However,
when used with tissues or cells, they have the limitation that
spin adducts can be converted to EPR silent products, so that
failure to detect a signal does not rule out low-level radical
generation.31 In vivo spin trapping of ROS with nitrones can
be performed but might not provide the adduct stability and
resultant sensitivity to detect low levels of radical generation.
Recent advances in the development of stabilized/protected nitrone spin traps bound to cyclodextran or calixpyrrole groups
offer promise in addressing this limitation.32,33 Alternative approaches for the detection of spin trap adducts or their metabolites include high-performance liquid chromatography
(HPLC) and mass spectroscopy,34,35 high-field nuclear magnetic resonance,36 and antibody-immune based detection.37,38
EPR Assay: Spin Probes With Cyclic Hydroxylamine or
Other Hydroxylamines
Several cyclic hydroxylamines, including CM-H (1-hydroxy3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine), CP-H,
PP-H, CAT1-H, and mitochondria-targeted mitoTEMPO-H,
.−
have been used widely as spin probes to measure O2 production in the cardiovascular system, an approach that requires
confirmation with SOD, PEG-SOD, or SOD mimetics.31,39,40
.−
Cyclic hydroxylamines react rapidly with O2 , producing
.−
stable nitroxides, and allow site-specific O2 detection in the
intracellular, extracellular, and mitochondrial compartments.
However, 1-electron oxidation of cyclic hydroxylamine to
nitroxide is also mediated by other cellular oxidants, including H2O2, peroxynitrite, hypochlorite, and compound I- and
compound II-like species. This makes it necessary to use
specific scavengers to identify the oxidant generating the
signal. Because of their favorable cell permeability, cyclic
Griendling et al Measurement of Reactive Oxygen Species e43
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hydroxylamines can be used as spin probes to determine the
redox status of the cardiovascular system in situ by EPR redoximetry as described in Measurement of Redox Status.
.−
CM-H has been used to detect intracellular O2 in cultured
cells and tissue samples. To minimize hydroxylamine autoxidation, the spin probe solution must be prepared in argonbubbled ice-cold NaCl in the presence of the chelating agent
diethylenetriamine-penta-acetic acid or a combination of deferoxamine and diethyldithiocarbamate.31 In complex in vivo
.−
systems, O2 -independent oxidation of the hydroxylamine to
the nitroxide is possible, and the nitroxide product could be
reduced enzymatically. Hence, these possibilities should be
considered and appropriate controls included, such as SOD,
PEG-SOD, or SOD mimetics.31,39,40
EPR with the spin probe CM-H has been used to detect
and quantify angiotensin II (Ang II)–induced and Nox1.−
dependent O2 production in the aorta,39 as well as NADPH.−
and xanthine oxidase–dependent O2 production in models of
atrial fibrillation.40 EPR with the cyclic hydroxylamine spin
.−
probe is an effective approach for studying intracellular O2 in
vessels and other tissues.31
EPR Assay: Triarylmethyl (Trityl) Free Radical Probes
Triarylmethyl (trityl)-based radicals (also called TAM radicals or probes) can be used for simultaneous measurements
.−
of O2 and oxygen.41 These probes provide >10-fold higher
EPR sensitivity compared with all other types of spin probes
and are well suited for in vivo EPR spectroscopy and imaging.
CT02-H is an analogue of the fully substituted TAM radical
.−
CT-03, with a higher rate of reaction with O2 and an EPR
doublet signal because of its aromatic hydrogen.42 On reac.−
tion with O2 , CT02-H loses its EPR signal, accompanied by
a change in color from green to purple, which provides an
.−
additional indication of O2 flux. In the cardiovascular system,
stimulated decay of the EPR signal of TAMs (CT-03, CT-02,
.−
or OXO63) can be initiated by O2 , •OH, and alkyl peroxyl
radical (ROO•).41 In vitro studies suggest that the reactivity of
.−
O2 with these TAM probes is much higher than that of •OH
and ROO•. However, these probes are stable in the presence
of biological reductants or oxidants, including ascorbate, glutathione, NADPH, H2O2, and NO. In combination with suitable SOD, PEG-SOD, or SOD mimetic controls, the use of
TAM or CT02-H is advantageous over cyclic hydroxylamines
.−
for the detection of O2 because the probe does not react with
H2O2, and very low concentrations of the probe are needed
for the measurement. TAMs can also be used for EPR oximetry to determine the oxygen concentration and consumption
rate in cardiovascular systems of cultured cells or tissue homogenates.41 Unlike cyclic hydroxylamine and nitrone spin
traps, conventional TAM radical probes (OXO63, CT03, and
CT02-H) have 3 or 2 carboxylate groups, and their negative
.−
charge limits their use to extracellular O2 detection. This
limitation can be overcome by esterified TAM probes, such as
AMT-02, that are taken up in cells and can provide intracellular
.−
O2 detection.
EPR with TAM has been applied to monitor NADPH
.−
oxidase–mediated O2 release from stimulated neutrophils, endothelial cells, and other cellular systems.41 TAM spin
.−
probes enable simultaneous measurements of O2 and oxygen
concentration and consumption in biological systems associated with respiratory burst and anoxia-reoxygenation.42 The
probe CT02-H is more sensitive than nitrone spin traps in mea.−
suring low O2 flux. It is also uniquely well suited for in vivo
EPR imaging applications (see Measurement of Redox Status).
Cytochrome c Reduction Assay
Cytochrome c (or acetylated cytochrome c) reduction is an.−
other assay used to measure O2 production in vascular and
myocardial cells and tissue.40,43,44 Ferricytochrome c accepts
.−
an electron from O2 to form ferrocytochrome c, which results in an increase in absorbance at 550 nm. As a control,
the assay must also be performed in the presence of SOD or
PEG-SOD. Acetylation of cytochrome c reduces the charge
effect of the lysine residues of cytochrome c; thus, use of
acetylated cytochrome c improves the electron transfer
.−
specificity for O2 .45 Potential drawbacks of the use of cytochrome c relate to its reactivity in biological systems. Both
cytochrome c and acetylated cytochrome c show peroxidase
activity in the presence of ROS because of oxidation of the
sixth heme ligand methionine.46 Furthermore, cytochrome c
can be reduced by a variety of biological reducing agents, including the NOS and P450 reductases. In the mitochondrial
system, higher electron transfer efficiency mediated by com.−
plex III than from O2 to cytochrome c limits its usefulness.
A suitable mitochondrial inhibitor and SOD should be used
to distinguish whether the reduction of ferrocytochrome
c is caused by electron leakage or electron transfer. Thus,
depending on the system, specificity could be a concern.
Controls with SOD, PEG-SOD, or an SOD mimetic or other
appropriate scavengers are essential.
The cytochrome c reduction assay has been used to mea.−
sure O2 production in biological systems in vitro and oc.−
casionally ex vivo.44 It is best suited for measuring O2 in
controlled systems that are free of enzymes that can directly
.−
reduce it or for measuring extracellular O2 in cellular systems. A major limitation of the cytochrome c assay is its rela.−
tive insensitivity except for high levels of O2 production. For
this reason, it has been used predominantly for measurement
.−
of O2 production by phagocytic cells and is not often used to
.−
detect the low levels of O2 produced by nonphagocytic cells.
Nitroblue Tetrazolium Assay
The nitroblue tetrazolium assay is based on the conversion
of the water-soluble yellow-colored dye to its blue formazan
.−
product by O2 . This assay is well suited for use in in situ
.−
microscopic assays detecting O2 release from individual
cells. The method can be modified for quantitative purposes
by dissolving nitroblue tetrazolium in DMSO under alkaline conditions and measuring its absorbance at 620 nm
47
with
. − a microplate reader, and it has been used to measure
O2 generation by neutrophils or phagocytes during the
inflammatory process.47,48 Unfortunately, this assay is not
.−
specific for O2 because of the enzyme-mediated (NADPH
oxidase and NOS reductase) formation of blue formazan.
Because nonspecific oxidation of nitroblue tetrazolium can
occur, appropriate controls with SOD, PEG-SOD, or SOD
mimetics are essential. Therefore, it is recommended that
this assay be used mostly for high-throughput, in vitro ROS
measurements.
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Fluorescence Analysis of Dihydroethidium or
Hydroethidium
.−
Dihydroethidium (DHE) is widely used to detect O2 generation in situ, in vitro, and ex vivo in cardiovascular systems
because the DHE probe is user-friendly and has high sensi.−
tivity. Oxidation of DHE by O2 yields 2-hydoxyethidium
+ 49
(2-OH-E ). However, other oxidants such as ONOO−, •OH,
H2O2, compound I, and compound II can produce a 2-electron oxidation product, E+, with similar fluorescence charac.−
teristics. Detection of O2 by these fluorescent probes is not
specific because of overlapping fluorescence of the superoxide-specific 2OH-E+ and the nonspecific oxidation ethidium
(E+) product.49 DHE can also be oxidized spontaneously because of atmospheric oxidation or light-induced oxidation,
resulting in the formation of ethidium. Therefore, to avoid
artificial signals, it is essential to pretreat DHE with Dowex
cation exchange beads immediately before use to absorb any
ethidium contamination. New protocols in which fluorescent
.−
settings are optimized to measure the O2 -specific product and
to minimize interference by nonspecific DHE oxidation products have been developed by changing the excitation from 480
to 405 nm, which significantly reduces the emission intensity
of E+ compared with 2OH-E+.50 The most unequivocal and
.−
quantitative detection of intracellular O2 by 2-OH-E+ in tissue specimens can be obtained with an HPLC equipped with a
fluorescence or electrochemical detector, or by liquid chromatography (LC)/mass spectrometry (MS).31,51
In combination with the technique of EPR redoximetry,
.−
DHE or hydroethidium has been used to monitor O2 production during myocardial ischemia and reperfusion in vivo52
.−
and ex vivo.53 In situ DHE imaging of O2 generation in the
myocardium has been applied in experimental models of cardiac hypertrophy induced by pressure overload.54 DHE has
also been used extensively in the vasculature.55–57 Combined
with HPLC instrumentation, 2-OH-E+ formation from frozen
myocardial tissue extracts preincubated with DHE has been
.−
reported as the indicator of O2 production associated with
pathological cardiac hypertrophy.58
In all of the above applications, it is critical that appropriate controls with SOD, PEG-SOD, or SOD mimetics are
used.59 When combined with HPLC and LC/MS instrumentation, the DHE probe and the fingerprint of 2-OH-E+ are a
.−
useful approach to measure and quantify intracellular O2 production in cells and tissues. The approach is highly sensitive
and valuable in a range of applications including translational
.−
research in obtaining O2 measurements from patient-derived
cells and tissues. When used with microscopy, it is imperative
to understand that DHE is detected as blue fluorescence within
the cytoplasm and only becomes a bright fluorescent red when
it intercalates within the DNA. Hence, in the absence of DNA,
for example, in platelets, DHE should not be used for ROS
measurement.
Fluorescence Analysis of Hydrocyans
Hydrocyanines or deuterocyanines are reduced dyes capable
of detecting 1 or more types of ROS.60,61 These dyes exhibit little or negligible fluorescence in the reduced state; however, on
reaction with ROS, they are oxidized, which results in a large
increase in fluorescence intensity. The stability of reduced
dyes is enhanced by replacing hydrogen with deuterium.
Deuterocyanines are thus more protected from auto-oxidation
and exhibit lower levels of background fluorescence in cell
culture. The cyanine dyes have emission wavelengths from
500 to 1100 nm, and the corresponding reduced dyes have
longer half-lives than DHE and thus have potential for in vivo
application. In many cases, hydrocyanines or deuterocyanines
are membrane permeable and can accumulate in cells or tissues. Once inside cells, hydrocyanines or deuterocyanines are
reoxidized by ROS, become membrane impermeable, and are
trapped within the cells. For example, the reduced dye hydroIR-676 exhibits negligible fluorescence; however, on reaction
.−
with O2 , IR-676 exhibits a 100-fold increase in fluorescence
(λex=675 nm and λem=693 nm). In vitro studies indicate that
hydro-Cy7 and hydro-Cy3 exhibit much higher selectivity for
.−
O2 and •OH than other ROS. Hydro-Cy3 and deuteron-Cy3
have been used to detect Ang II–mediated intracellular ROS
production from aortic muscle cells and ROS production in
en face preparations of live and explanted aortas.60 In the mouse
model of lipopolysaccharide-induced acute inflammation,
hydro-Cy7 has been used to image in vivo ROS generation by
activated macrophages and neutrophils.60 As with other fluorescent dyes, SOD controls are important to ensure specificity.
Mitochondria-Specific Fluorescent Probes
Mitochondria-targeted hydroethidium and Mito-SOX
(3,8-phenanthridinediamine, 5-[6′-triphenylphosphoniumhexyl]5,6 dihydro-6-phenyl), which show a chemistry similar to DHE
or hydroethidium,49 are widely used to measure mitochondrial
.−
O2 in situ. Their use and limitations are similar to those
described above for DHE.49 Measurement of mitochondrial
.−
O2 by Mito-SOX tracks well with electron spin resonance
measurements and the signal produced by the more sensitive MitoTracker Red CM-H2XRos.62 Both Mito-SOX and
MitoTracker Red CM-H2XRos are nonfluorescent in their
reduced form and fluoresce red when oxidized. The advantage of these probes is that they provide information about
.−
mitochondria-localized O2 production; the disadvantage is
that like other fluorescent probes, they are not fully quantitative and must be used with appropriate controls, such as mitochondria-targeted antioxidants. Mito-SOX has been used with
flow cytometry to better quantify ROS, as described in H9c2
myocytes and coronary artery endothelial cells.63
Chemiluminescence Assays
Several chemiluminescent probes have been used to measure
.−
O2 , including lucigenin, coelenterazine, and the luminol analogue L-012. Lucigenin (N,N′-dimethyl-9,9′-biacridinium dinitrate) undergoes 1 electron reduction to emit an amplified
chemiluminescence signal that correlates with the amount of
.−
O2 present in the sample. This assay has been widely used
.−
for O2 detection in the cardiovascular system.40 In general,
it is more applicable to cell-free assays than to intracellular
.−
O2 because of its limited permeability. Coelenterazine
(2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo [1,2-a]pyrazin-3-1) is the luciferin chromophore
in aquaporin. It has been used in membrane preparations to
.−
detect O2 and purportedly produces 15 times more light than
.−
lucigenin.64 Coelenterazine has been used to detect O2 in vas65,66
L-012
cular smooth muscle cells and adventitial fibroblasts.
Griendling et al Measurement of Reactive Oxygen Species e45
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(8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4(2H,3H)dione sodium salt) produces luminescence at long
wavelengths (530 nm) and is more sensitive than lucigenin.67
.−
It has been used to detect extracellular O2 in whole blood and
in membrane fractions from cells and tissue.67 However, L-012
has recently been shown to react with H2O2 and peroxidase to
.−
produce O2 , which makes the signal artificially inhibitable by
.−
SOD and thus unsuitable for O2 detection.68
The validity of the lucigenin assay has been questioned
.−
because O2 is generated by lucigenin itself.69 Lucigenin can
.−
uncouple flavoenzymes, thus triggering O2 in a concentration-dependent manner. The concentration of lucigenin used
is therefore a critical parameter affecting the validity of this
assay.70 It has been shown that the concentration of lucigenin
should be ≤5 μmol/L, and extensive controls are required to
rule out redox cycling.71,72 It is also important to document
that the concentration used does not significantly increase
oxygen consumption. Higher concentrations of lucigenin
can attenuate endothelium-dependent relaxation by acetylcholine and have been shown to uncouple eNOS.70 An additional consideration is the concentration of NADPH used in
cell-free assays. NADPH at a concentration of 100 μmol/L
is most commonly used, because higher concentrations can
lead to aberrant results. Thus, lucigenin is only useful if used
at low concentrations (≤5 μmol/L) in fractionated cells or tissues with the appropriate concentration of NADPH, as well as
SOD controls.
.
Recommendations for Measurement of O2−
Figure 1 provides a comparison of methods for measuring
.−
O2 and H2O2. The cytochrome c reduction assay is suitable
.−
for measurements of O2 in controlled systems free of reducing equivalents or enzymes that couple to it. Therefore, cytochrome c is not recommended for use in cellular and tissue
applications. In contrast, the nitroblue tetrazolium assay is ef.−
.−
fective in measuring O2 generation from cells with high O2
production and can be used as a high- throughput ROS assay system in vitro. Suitable SOD controls are important to
establish specificity. Lucigenin is not recommended for use in
.−
measuring intracellular O2 because of its ability to uncouple
.−
flavoenzymes, resulting in O2 generation, but it has been used
extensively to measure ROS generation in membrane fractions using NADPH as a substrate. For such an application,
the concentration of lucigenin (≤5 μmol/L) is critical, because
high concentrations of lucigenin can produce anomalous results.70 If lucigenin is used, it should be complemented with a
second, independent assay. Similar considerations should also
be applied to coelenterazine.
Combined with appropriate SOD controls, fluorescence
analysis with DHE is a useful approach to measure and image
.−
intracellular O2 production from cells and tissues because of
its high sensitivity. Ideally, it should be combined with HPLC
to ensure specificity. DHE should only be used in cells containing DNA. Mitochondria-specific fluorescent probes are useful
for qualitative assessment, for example, ROS localization, but
not for quantitative studies. Localization of ROS production
can be confirmed by supplementation with cell-impermeable
Cu,Zn-SOD, cell-permeable PEG-SOD, or mitochondria-targeted antioxidants such as mitoTEMPO.57 Hydrocyanines and
deuterocyanines are useful as fluorescence probes with high
persistence and high sensitivity, but further work is needed to
characterize their specificity. These fluorescence-based methods are useful in a wide range of applications including translational research from patient-derived cells and tissues.
The EPR spin-trapping technique provides the most direct
and specific identification of oxygen free radicals, including
.−
O2 and •OH, in cellular and ex vivo systems; however, EPR
with nitrone spin traps is not feasible with intact tissue samples. Failure to detect a signal does not rule out free radical
or other ROS generation. Hydoxylamine probes with appro.−
priate SOD controls are useful for the measurement of O2 .
Hydroxylamine probes and nitroxides also enable assessment
of redox state and stress and are useful in ex vivo and in vivo
systems. Newly developed trityl probes or trityl-nitroxide bi.−
radicals can provide more sensitive O2 measurements and are
well suited for in vivo EPR and EPR imaging.
Figure 1. Choice of assays to measure
.−
O2 and H2O2.. When choosing an assay
to measure O2− or H2O2, one must
first consider whether reactive oxygen
species (ROS) production is intracellular
or extracellular. Assays listed as primary
(1°) are the most well accepted, secondary
(2°) are more widely used, and tertiary
(3°) are either less sensitive (cytochrome
c) or should be verified with a second
method (chemiluminescence). Cyt c
indicates cytochrome c; DCFH-DA,
dichlorodihydrofluorescein diacetate; DHE,
dihydroethidium; EC, electrochemical;
EPR, electron paramagnetic resonance
spectroscopy; HPLC, high-performance
liquid chromatography; and NBT, nitroblue
tetrazolium.
e46 Circulation Research August 19, 2016
Measurement of H2O2
Electrochemical Detection With H2O2 Electrode
Electrochemical detection of H2O2 using an H2O2-specific
electrode is a direct and reliable method for measuring H2O2
in vitro and has been used to assess H2O2 produced in endothelial cells and neutrophils.73 In combination with spin-trapping
agents, electrochemical detection is effective for distinguish.−
ing the production of H2O2 and O2 that results from NOS
uncoupling.74 Electrochemical detection is useful in assessing
H2O2 generated by isolated enzymes and for measuring H2O2
metabolism in the isolated mitochondria and cellular systems.
However, this method is not widely used for in vivo measurements because of its poor sensitivity and difficulties with in
vivo use of polarographic electrodes.
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Amplex Red
In the presence of H2O2, Amplex Red is oxidized efficiently
by horseradish peroxidase to a fluorescent product, resorufin,
which can be measured using a fluorescent plate reader. The
assay is a feasible and reliable method to measure extracellular H2O2 in vessels75 and the heart.76,77 The assay is also highly
effective for assessing extramitochondrial H2O2 generated
from isolated myocardial mitochondria76,77 and has been used
to measure H2O2 released from mouse aorta.31,75 It has also
been used to quantify external H2O2 released from mitochondria of the ischemic heart.76 The Amplex Red assay is highly
recommended for measuring H2O2 released from mitochondria, cells, vessels, and cell-free systems. However, in tissues,
the product (resorufin) induces nonspecific oxidation via other
oxidant and radical-mediated reactions. Therefore, controls
to correct for the fluorescent background must be performed
with addition of SOD and catalase.
Dichlorodihydrofluorescein Diacetate
Dichlorodihydrofluorescein diacetate (DCFH-DA) is a cellpermeable and widely used probe to detect intracellular production of H2O2. It is commonly used for measuring H2O2 or
oxidant stress in cells or tissues in combination with confocal microscopy or flow cytometry. DCFH has been used to
image H2O2 in vessels and to evaluate mitochondrial H2O2
as the source of flow-induced dilation in coronary resistance
arteries,78 as well as H2O2 levels of carotid artery and aortic
smooth muscle cells during the progression of atherosclerotic
lesion development.79 DCFH-DA has also been used to explore iron-mediated redox-signaling mechanisms in the cardiovascular system80 and to investigate doxorubicin-induced
cardiotoxicity.81
Because DCF does not directly react with H2O2 to form
a fluorescent product, DCF fluorescence must be used with
caution as an indicator of H2O2. Compounds I and II are reactive 1-electron oxidizing intermediates that are formed from
peroxidase or heme proteins on reaction with H2O2.82,83 They
can oxidize DCFH to DCF, leading to artificial fluorescence
in cells and tissue. Fe2+, a redox-active metal, can promote
DCFH oxidation in the presence of O2 or H2O2, which can
pose a serious problem when the assay is applied to myocardial reperfusion or vascular inflammation. The 1-electron oxidation intermediate, DCF− (DCF semiquinone anion radical),
.−
is a source of O2 ,83,84 which triggers redox cycling and leads
to artificial amplification of DCF fluorescence. In view of
these limitations, assays using DCFH or DCFH-DA must be
conducted along with SOD and catalase controls to establish
specificity; however, because of the potential for artifacts, this
probe is not recommended for measuring intracellular ROS.59
HyPer Probe
A redox-active biosensor, HyPer, is another approach for
monitoring intracellular H2O2 levels. The design of HyPer is
based on OxyR, a natural bacterial H2O2 sensor. HyPer is the
first genetically encoded fluorescent sensor to detect intracellular H2O2. HyPer is highly sensitive to H2O2 (~ submicromo.−
lar) but is insensitive to other oxidants, such as O2 , GSSG
(oxidized glutathione), NO, and peroxynitrite. It does not
cause artificial ROS generation and is capable of detecting fast
changes in H2O2 concentration. HyPer has been used for in
vivo imaging of H2O2 in cardiac myocytes from eNOS−/− mice
and mice deficient in neuronal NOS (nNOS−/−)85 and for detecting in situ H2O2 synthesis stimulated by Ang II in cardiac
myocytes. HyPer can be expressed in cardiac myocytes of
adult mice through tail vein injection of HyPer lentivirus.85,86
HyPer is useful in monitoring intracellular H2O2 in situ.
A major advantage is that it is genetically encoded and so can
be targeted to specific compartments, such as the endoplasmic
reticulum and mitochondria.87 Care must be taken, however, to
avoid quenching of H2O2 in these compartments by the presence of the probe. Moreover, despite its high specificity for
H2O2, the fluorescence signal of cytosolic HyPer can respond
to pH changes, decreasing with acidosis and increasing with
alkalosis. Thus, the HyPer assay should be conducted with
side-by-side pH monitoring.88 Furthermore, the HyPer fluorescent response can adapt to repeated oxidation and can be
insensitive to reducing stimuli. HyPer responses thus must
be interpreted carefully.88 Recent modifications (eg, HyPer-3)
have overcome some of these difficulties and allow expanded
dynamic range and ratiometric imaging.89
Mitochondrial Probes
MitoB ([3-hydroxybenzyl]triphenylphosphonium bromide)
accumulates in the mitochondria by virtue of a triphenylphosphonium cation component. The arylboronic moiety of MitoB
reacts with H2O2 to form a phenol product, MitoP, in the mitochondrial matrix. H2O2 concentration is determined by assessing the ratio of MitoP to MitoB as measured by MS with
d15-MitoP and d15-MitoB used as standards.90,91 As such, this
assay can be used in cultured cells and tissue. A limitation of
this method is the need for sophisticated equipment not readily available in many laboratories.
Dickinson and Chang92 recently described a mitochondriatargeted boronate-based peroxy yellow 1 probe containing a
triphenylphosphonium group that allows selective detection
of H2O2 within the mitochondria (MitoPY1). This new fluorescent probe permits imaging H2O2 within the mitochondria
of living cells using confocal microscopy and flow cytometry.
Recommendations for Measurement of H2O2
Figure 1 provides decision support for choosing a method to
measure H2O2. Electrochemical detection is useful in assessing H2O2 generation by isolated enzymatic, mitochondrial and
cellular systems; however, it is not suited for in vivo systems
Griendling et al Measurement of Reactive Oxygen Species e47
because of its limited sensitivity and difficulties associated
with in vivo use of polarigraphic electrodes. The Amplex Red
assay is suitable for measuring H2O2 released from mitochondria, cells, vessels, and cell-free systems; however, in tissues,
there is the potential for nonspecific oxidation so that controls
to correct for the fluorescent background must be performed
with addition of SOD and catalase. HyPer can be used to monitor intracellular H2O2 in situ. Because it is genetically encoded,
the protein can be targeted to specific cellular compartments.
The fluorescence ratio of HyPer changes with pH; therefore,
it should be used with parallel pH monitoring. Mitochondriaspecific production of H2O2 can be measured with MitoB.
Measurement of Redox Status
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Redox status refers to the tendency or ability of the tissue
or cell to accept (more oxidized) or donate (more reduced)
electrons. The biological redox status is often reflected by
the balance of GSH (reduced glutathione)/GSSG, NAD+/
NADH, and NADP+/NADPH in a cell or organ, as well as
by the balance of several sets of metabolites (eg, lactate and
pyruvate, β-hydroxybutyrate, and acetoacetate) whose interconversion is dependent on these ratios, free radical reactions, and reversible/irreversible modifications of redox
proteins. Although abnormal redox status has been linked to
several conditions such as hypoxia, ischemia and reperfusion, hypertension, atherosclerosis, shock, and sepsis, accurate assessment of redox status is difficult. EPR-based
techniques and measurement of redox couples such as GSH/
GSSG and cysteine/cystine (Cys/CySS) represent the most
common approaches. The NADPH/NADP+ ratio is also an
important determinant of the cellular redox status, but it is
difficult to measure because no methods are available to
measure the concentration of free NADPH or NADP+ in the
cell. The NADH:NAD+ ratio, however, can be estimated indirectly by measuring the pyruvate to lactate ratio, which
is regulated by the reaction catalyzed by lactate dehydrogenase, although in such measurements it is important to
ensure that the reaction is at or near equilibrium.93 Levels
of free NAD+/NADH can also be measured with newly developed biosensors or nuclear magnetic resonance using
the magnetic enhancement technique,94,95 but these methods
will not be discussed further here.
EPR Redoximetry With Spin Probe Hydroxylamines and
Nitroxides
In oxidized conditions such as those in the postischemic
myocardium, cyclic hydroxylamines can serve as a redox
substrate to determine the redox activity of tissue specimens
using EPR. The assay can be combined with a nitroxide
probe such as TEMPOL to assess the level of reductive stress
in the tissue specimen. This approach is applicable to in vivo
measurements of whole animals and translational research
using human samples; however, it cannot determine the redox status of thiols that are involved in oxidative posttranslational modifications (Oxidative Modification of Proteins). To
obtain a complete picture of redox status, additional assays
should be conducted, including analysis of GSH and GSSG
(by an enzymatic recycling assay), measurement of free thiols by the biradical spin probe RSSR [bis-(2,2,5,5-tetramethyl3-imidazoline-1-oxyl-4-yl) disulfide], redox immunoblotting
to assess protein oxidation, and HPLC analysis of oxidatively
modified amino acids.
In Vivo Redoximetry With Low-Frequency EPR
Spectroscopy and Imaging
In vivo redoximetry provides noninvasive in situ measurement of redox status. With the use of nitroxide probes, this
approach has proved useful in monitoring in situ redox alterations of the myocardium during ischemia and reperfusion.96–98 Nitroxides are paramagnetic and are reduced or
reoxidized depending on the redox environment. By measuring the shift of redox status from more reduced to more
oxidized, one can determine how redox alterations correlate
with disease progression.52,96 Trityl-nitroxide is a biradical
that can also be used for in vivo redoximetry. With this biradical, the sharp trityl signal increases with nitroxide reduction as opposed to the loss of the nitroxide EPR signal. It
can be used to simultaneously monitor oxygenation and redox alterations.99 With the addition of magnetic field gradients, EPR images of the free radical probe and its reductive
clearance can be performed.100–102 This oxy-redoximetric assay is highly promising, and ex vivo cardiac applications
have been performed with nitroxide probes.103
For in vivo redoximetry using nitroxides, it is important
to confirm that the signal decay is caused by reduction of the
nitroxides to the diamagnetic hydroxylamine. The hydroxylamine can be reoxidized to paramagnetic nitroxide by ferricyanide. Ferricyanide is thus used to confirm reduction to the
hydroxylamine. In vivo EPR redoximetry is considered to be
the “gold standard” for measuring redox physiology in the in
vivo setting under physiological and pathological conditions.
In Vivo Redox Imaging Using Proton-Electron DoubleResonance Imaging
This approach utilizes the Overhauser effect to enable in
vivo imaging of free radicals in the heart. The redox state of
the tissue is measured by combining a nitroxide spin probe
or biradical with proton-electron double-resonance imaging (PEDRI) instrumentation, enabling nuclear magnetic
resonance/magnetic resonance imaging–based detection and
imaging of the redox state based on saturation of the EPR
resonance. As described above, nitroxide spin probes enable measurement of the tissue redox state based on their
reduction to diamagnetic hydroxylamines.93,98,101 PEDRI
instrumentation enables imaging of the in vivo distribution
and metabolism of nitroxide radicals in living mice, which
enables the redox status of the vascular system to be visualized.98 PEDRI has been applied to image nitroxide radicals
in the isolated beating rat heart, with visualization of radical
uptake, distribution, and clearance.101
When PEDRI is applied to acquire in vivo redox images of
the beating heart using nitroxides as the redox-sensitive probe,
a nitroxide radical with longer relaxation time and sharp EPR
line width can offer better sensitivity. In vivo, PEDRI provides
an enhanced form of in vivo redoximetry along with spatial
mapping. It is a powerful tool for measuring and visualizing
the redox distribution and clearance in the isolated heart and
in animal models of disease, but it requires very specialized
equipment and expertise that are not commonly available in
most laboratories.
e48 Circulation Research August 19, 2016
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GSH/GSSG and Cys/CySS
A simpler method for estimating redox potential is assessment
of the ratio of the concentration of reduced to oxidized glutathione or reduced to oxidized Cys. As the most abundant
redox couple in the cell, and because it is maintained by the
combined activity of several redox couples, GSH/GSSG is a
useful indicator of redox status and correlates with many cell
functions, such as proliferation, differentiation, and apoptosis.104 Virtually all cardiovascular cell types express transporters for GSH, GSSG, Cys, and CySS, so that redox potentials
measured in plasma reflect tissue redox state, but only indirectly. GSH can be measured using enzymatic recycling of
GSSG reductase, HPLC with UV detection, and electrochemical detection, but these methods are not sensitive enough to
detect low levels of GSSG. A better assay uses iodoacetate to
alkylate free thiols, derivatization with dansyl chloride to fluorescently tag amino groups, and HPLC and fluorescence to
separate, detect, and quantify GSH, GSSG, Cys, and CySS.105
Subsequent calculation of the nonequilibrium redox potential
(EhGSH or EhCys) is a reflection of the redox state of the tissue of interest. During the assay, care must be taken to prevent oxidation and degradation. These measurements can be
performed in plasma, tissue biopsy specimens, and cultured
cells.105 Ashfaq et al106 showed that EhGSH/GSSG in plasma
correlates with changes in intima-media thickness, a subclinical indicator of atherosclerosis.
RoGFP
Another fluorescence-based method to measure redox status
involves the use of reduction-oxidation sensitive green fluorescent protein (RoGFP) probes.107 These probes consist of
a green fluorescent protein in which 2 cysteines were introduced, the oxidation state of which serves as a readout of redox status in the particular subcellular compartment in which
the RoGFP is expressed. When a disulfide bond is formed
within the molecule, its fluorescent properties are altered such
that there is a reciprocal change in emission (525 nm) after
excitation at 2 different wavelengths (400 and 484 nm).108 This
ratiometric property makes the signal independent of expression levels of the probe. Another advantage is the ability to add
a targeting sequence so that the probe is expressed in specific
subcellular compartments.109,110 Early versions of this probe
(RoGFP2) are sufficient for reducing environments such as
the cytosol and mitochondria. Subsequent modifications, in
which the thermodynamic stability of the disulfide is lowered
by inserting a single amino acid adjacent to one of the cysteines (eg, roGFP1-iL),111 have rendered the redox potential
closer to that of the endoplasmic reticulum. Thus, one must
know the redox potential of the compartment to be studied and
choose the version of RoGFP that most closely aligns with it.
RoGFP has been used successfully to measure redox status in
the cytosol, mitochondria, and mitochondrial inner membrane
space in vascular smooth muscle cells exposed to hypoxia as
well as glutathione-induced reductive stress in the heart.110,112
Recommendations for Measurement
of Redox Status
If EPR is not available, measurement of GSH/GSSG or Cys/
CySS couples can be used instead. Care should be taken during
sample preparation to avoid oxidation and degradation, as well
as contributions from inadvertent lysis of cells. RoGFP probes
are a useful addition to our armamentarium and allow measurement of redox status in specific subcellular compartments.
Measurement of RNS
Introduction
The short half-life of NO in biological systems presents unique
challenges for its detection and quantification.113 NO is lipophilic and diffuses rapidly through cells. The main sources of
NO include NOS and reductive metabolism of nitrite (NO2−)
and RSNO.114 In biological systems, NO is rapidly oxidized
by several nonenzymatic and enzymatic pathways. In the vasculature, NO is oxidized by oxyhemoglobin to nitrate (NO3−).
NO bioavailability can be reduced by its reaction with free
.−
.−
radicals such as O2 . One such reaction between NO and O2
occurs at a diffusion-limited rate and yields OONO−, a strong
oxidant implicated in numerous pathological processes.115
In addition, O2 reacts with NO to produce N2O3. Other RNS
include N-nitrosation products and nitrated unsaturated fatty
acids (Oxidative Modification of Proteins).116
Detection of NO
Detection of NO in biological systems is complicated because
of its short half-life, contaminants in laboratory reagents, and
the multiple reactions that it undergoes, leading to a variety
of end products. Because of their short half-lives, most RNS
cannot be detected in frozen or fixed tissue samples, but they
can be detected in live tissue or intact cells.
Colorimetric (Griess Reaction) and Related Methods
In the Griess reaction, nitrite-containing media is first treated
with sulfanilamide in acidic media to form a transient diazonium salt, which then reacts with a coupling reagent, Nnaphthyl-ethylenediamine, to form a stable azocompound.
These products can be measured by spectrophotometry, fluorescence,117 or HPLC.118 A major problem with the unmodified Griess reaction is its relative insensitivity (≈500 nmol/L),
which makes measurements of endothelium-derived nitrite
difficult. With this method, it is feasible to measure the production of nitrite generated by the inducible or neuronal NOS
isoforms, which have higher Vmax values. The sensitivity of the
method can be enhanced 10-fold by fluorometric detection,
which makes it suitable for clinical applications.117 Another
drawback of the Griess reaction is that a substantial portion of
NO production by NOS enzymes is oxidized to nitrate, which
is not detected by this technique unless NO is reduced to nitrite by a nitrate reductase reaction.
Nitrite and nitrate contaminants in laboratory reagents
often yield strong signals that can overwhelm true biological
responses. In pathological states in which NO is oxidized rapidly, its bioavailability is reduced, but nitrate and nitrite levels
are not affected. Diet is a major source of nitrite and nitrate
and must be controlled for, particularly when measurements
are made in the plasma. The main advantage of measuring
nitrite and nitrate is that these products are relatively stable.
Nitrate and nitrite are incapable of eliciting vasodilatation or
other signals themselves; however, they can be biologically
relevant because they can generate NO via nitrate reductases
or on reaction with deoxyhemoglobin.119
Griendling et al Measurement of Reactive Oxygen Species e49
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Fluorescent Techniques
There are several fluorescent probes for detecting NO based on
diaminofluorescein (DAF), including DAF-2 and DAF-FM.
DAF-FM is significantly more photostable than DAF-2 and
is thus more sensitive for NO detection.120 The NO detection
limit for DAF-FM is ≈3 nmol/L120 versus ≈5 nmol/L for DAF2.121 In this assay, cells are loaded with DAF-FM, washed, and
analyzed for DAF-FM fluorescence by either a fluorescent
plate reader, fluorescence microscopy, HPLC, or occasionally
by flow cytometry.122 NO donors are generally used as a positive control. Some samples should also be preincubated with
NOS inhibitors such as L-NAME (Nω-nitro-l-arginine methyl
ester) or L-NIO [N5-(1-iminoethyl)-l-ornithine hydrochloride] to ensure specificity of the signal.
A potential problem with DAF-2 and DAF-FM is that
ascorbic acid and dihydroascorbic acid react with these probes
and reduce their ability to detect NO.123,124 This reaction is
concentration dependent, and as long as the concentrations of
ascorbic acid and dihydroascorbic acid are stable, this issue
does not affect the semiquantitative value of these probes.
Chemiluminescence
Ozone-based chemiluminescence provides sensitive NO detection.125 Liquid samples from either cells or vessels are injected
into a reflux chamber that contains inert gas (nitrogen or argon),
which carries NO to the NO analyzer where it reacts with ozone
to produce the excited state NO2*, which emits light on decay.126
Typically, the reaction chamber that contains either sodium iodide (NaI) in acetic acid or vanadium in hydrochloric acid is
maintained at 100°C. When NaI is used, nitrite is converted to
NO, whereas vanadium can reduce both nitrite and nitrate. Other
1-electron oxidation state nitroso species, such as nitrosothiols,
can also be detected with this approach. To differentiate between
RNS without having to change reaction solutions, samples can
be pretreated with RNS-specific reagents before analysis.127
Recently, an improved method for detecting NO with chemiluminescence has been reported that uses a reaction with
soluble guanylyl cyclase-luciferin-luciferase.128 This approach
is based on NO-mediated activation of soluble guanylyl cyclase, which catalyzes the conversion of GTP to cGMP and
inorganic pyrophosphate. The assay measures the pyrophosphate, which is converted into ATP with the help of ATP sulfurylase. The resulting ATP activates the luciferin-luciferase
reaction, which yields light. This approach allows detection of
NO in the low nanomolar range.
Electrochemical Approaches
NO can be detected with microelectrodes, most often coated to
be selectively permeable to NO and to eliminate other oxidizable substances.126 For example, polymerization of o-phenylenediamine on the electrode can increase NO specificity.126 In
electrochemical detection, the voltage of an electrode is controlled to catalyze NO oxidation on the electrode surface. The
current generated is a linear function of NO concentration at
the electrode surface.126 This approach requires shielding from
extraneous electrical signals, usually by a Faraday cage. In addition, it is essential that the electrode be placed at a constant
distance from the source of the NO, for example, a set distance
from the cell surface, using a micromanipulator. An advantage
of electrochemical detection is that the sample size can be quite
small; however, the commercially available devices purported
to measure NO are insensitive to the low levels released by endothelial cells. Measurements of NO by this approach are less
sensitive than those obtained with lipophilic probes, such as
Fe[DETC]2 (DETC=N, N-diethyldithiocarbamate) (see Electron
Paramagnetic Resonance below). This is likely because of the
lipophilic nature of NO and the integrative effect of the trap.
NO can also be measured with hemin-functionalized graphene transistors with subnanomolar sensitivity.129 The graphene is noncovalently functionalized with hemin chloride to
form an NO sensor, because hemin has a high binding constant and high selectivity toward NO.
Electrochemical detection with NO electrodes has been
used in studies of cultured cells and tissue.126,130 Common
challenges in such measurements include the specificity of the
electrode to NO, the reproducibility of results with different
electrodes, and the dependence of data on the distance of the
electrode from the cell surface.
Electron Paramagnetic Resonance
EPR is an extremely specific and sensitive approach to detect
NO in biological systems. Because of its short half-life and
very short relaxation time, NO cannot be directly detected by
EPR; however, compounds such as the nitronyl nitroxides PTIO
(2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide) and
CPTIO [2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline1-oxyl-3-oxide]131,132 and the iron-dithiocarbamate complexes
Fe[DETC]2 and Fe[MGD]2 (MGD=N-methyl-D-glucaminedithiocarbamate) that form stable adducts with the NO radical can be used to detect NO.133 Detection of NO by nitronyl
nitroxides is limited by bioreduction of nitroxides32 and potential cross-reaction with HNO and NO2.134 Fe[DETC]2 and
Fe[MGD]2, in contrast, specifically react with NO radical to
produce relatively stable complexes with specific EPR spectra,
which provides unambiguous NO detection.
A natural spin trap for NO is hemoglobin. On reaction
with NO, ferrous hemoglobin forms a stable hemoglobin-NO
complex that yields specific high-resolution spectra in rat and
mouse blood.133,135 Typically, blood is snap-frozen in a tuberculin syringe and placed in a liquid nitrogen quartz Dewar flask
designed for low-temperature EPR.135 The average lifetime of
hemoglobin-NO is 4 hours, and it can accumulate in the blood
in substantial concentrations. Blood hemoglobin-NO levels
have been used to estimate vascular NO production; however,
other sources of NO, such as inducible NOS or neuronal NOS,
also likely contribute to the signal.133 Indeed, endotoxin-induced inflammation has been shown to increase the electron
spin resonance hemoglobin-NO signal.136 The contribution of
inducible NOS and eNOS in the circulating hemoglobin-NO
can be determined by treating experimental animals with specific NOS inhibitors such as 1400W and L-NIO or by using
mice deficient in specific NOS enzymes.133 It should also be
kept in mind that dietary nitrite can be reduced to NO by deoxygenated hemoglobin and yield an identical hemoglobin-NO
signal; however, this can be minimized by use of a standard
or low-nitrate/nitrite diet.133 The hemoglobin-NO signal in humans is very difficult to detect, likely because of lower levels
of hemoglobin-NO and a shift to the low-resolution EPR spectra of 6-coordinate hemoglobin-NO.
e50 Circulation Research August 19, 2016
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Fe[DETC]2 is used in a second method to detect NO with
EPR and can be produced by reaction of ferrous iron with DETC
under anaerobic conditions. The resulting colloid is lipophilic
and appears to detect NO that accumulates in cell membranes.
The maximum signals from vessels or cultured endothelial
cells are obtained when they are stimulated with the calcium
ionophore A23187. Fe[DETC]2 can be used for in vitro, ex
vivo, and in vivo NO measurements.126,133,137 The stability of
the NO-Fe[DETC]2 complex allows measurement of the accumulation of bioactive NO released from the tissue or cells over
time. Limitations of this technique include the special handling
required for Fe[DETC]2 colloid to prevent oxidation and the
requirement for an EPR spectrometer and expertise with such
measurements.138
The third EPR method to detect NO uses Fe[MGD]2, which
is water soluble and much more hydrophilic than Fe[DETC]2
and does not penetrate cell membranes. It remains in the extracellular compartment, which allows detection of NO released
from cells.138,139 A limitation of Fe[MGD]2 and also to some
extent Fe[DETC]2 is that the ferrous state can reduce nitrite to
NO.140 This problem is largely prevented when these traps are
present in the ferric state, but the trapping efficiency drops by a
factor of 2.67
Detection of Peroxynitrite
.−
Peroxynitrite, formed by the rapid reaction of NO with O2 ,
reacts with thiols, lipids, proteins, and DNA, which makes it
very short-lived.141 Excessive production of OONO− contributes to cellular and end-organ dysfunction in inflammation and
cardiovascular disease.141 Peroxynitrite rapidly oxidizes tetrahydrobiopterin and thus induces eNOS uncoupling and vascular
dysfunction.142 Its reaction with lipids leads to formation of isoprostanes and highly reactive ketoaldehydes, which can be used
as indirect measures of OONO−. Peroxynitrite is also frequently
measured by accumulation of stable nitration products such as
8-nitroguanine143 and nitrotyrosine.144 Other methods include
EPR detection with spin probes and spin traps such as CM-H
and EMPO [5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide].
The recently developed boronate probes have proved useful in
direct detection of peroxynitrite and related species.66 Uric acid
has been used as a relatively specific scavenger of peroxynitrite.
Thus, inhibition of boronate, CM-H or luminol signals with BH4
or uric acid can provide confirmation of peroxynitrite detection.
Dihydrorhodamine and luminol chemiluminescence have
often been used to detect peroxynitrite; however, these probes
undergo nonspecific oxidation in the presence of other oxidants and are therefore not specific for peroxynitrite. Their
use is not recommended unless accompanied by controls to
validate specificity.59
Citrulline Assay
NOS convert l-arginine to l-citrulline; therefore, accumulation of l-citrulline was used as a marker of NOS activity and
NO production in the past.145,146 The main source of citrulline,
however, is the urea cycle, and alteration of urea production
affects citrulline levels.147 Furthermore, citrulline is a marker
of a wide range of pathologies such as bowel diseases148 and
rheumatoid arthritis.149 Hence, l-citrulline is not a specific biomarker of NO bioavailability and as such should not be used
as an index of NO status.
Recommendations for RNS Measurement
The selection of an assay depends on the experimental conditions and which RNS the investigator wishes to detect. Given
the caveats presented above, several assays are available to
measure either NO, oxidation products of NO, or other RNS
(Figure 2). Either EPR with the NO spin trap Fe[DETC]2 or
the fluorescent dye DAF-2 can be used to detect NO with suitable accuracy. Ozone-based chemiluminescence is also quite
accurate and can be used to detect not only NO but also nitrite,
nitrate, and nitrosothiols. Proper controls, such as inhibition of
the NOS enzymes, control for contaminants in the media used,
and in some cases, dietary controls for nitrate-rich foods, are essential to yield valid experimental results. For detection of peroxynitrite, no probe or detection method is entirely specific, but
when combined with known scavengers of peroxynitrite such
as uric acid or BH4, oxidation of CM-H or boronate probes can
be used. Although peroxynitrite can react with protein tyrosines
to form nitrotyrosines, immunostaining for nitrotyrosine is not
recommended as a specific signal for peroxynitrite, because nitrotyrosine can also be formed via other pathways. Variations of
the Griess assay can be used to detect nitrite, but one needs to be
Figure 2. Decision tree for measuring
reactive nitrogen species (RNS). When
choosing an assay, one must first consider
the identity of the RNS to be measured,
the type of sample (cells, tissue, or in vivo),
and the product to be analyzed. Assays
are listed along with the appropriate
probe. Ab indicates antibody; DAF-FM,
diaminofluorescein-FM; EPR, electron
paramagnetic resonance spectroscopy;
Fe[DETC]2, Fe-diethyldithiocarbamate;
Hgb, hemoglobin; and Mass Spec, mass
spectrometry.
Griendling et al Measurement of Reactive Oxygen Species e51
aware of nitrite and nitrate in laboratory reagents, plasticware,
and the diet as potential contaminants.
Oxidative Modification of Proteins
Introduction
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Amino acid residues of proteins can act as electron donors,
leading to covalent modifications of the amino acid chain.
Oxidation of proteins can be induced by many stimuli, including ROS/RNS, metal cations, γ-irradiation, ultraviolet
light and ozone, leakage of the electron transport chain in
mitochondria, oxidoreductase enzymes, and products of lipid
peroxidation.150,151 Increased bioavailability of ROS/RNS can
lead to protein oxidation in cardiovascular cells. Oxidative
modification of proteins has functional consequences such as
gain or loss of protein function or enzymatic activity, altered
protein-protein interactions, conformational changes, changes
in protein localization, susceptibility to proteolysis, increased
immunogenicity, and modified gene transcription. Such biochemical modifications alter cell signaling and impair or promote cell functions.150,152–154
Common Protein Oxidative Modifications in
Cardiovascular Cells
Proteins are highly sensitive to posttranslational oxidative
modifications, which can be reversible (by biological antioxidant systems) or irreversible.155 Whereas irreversible
modification usually leads to loss of protein function and
protein degradation, reversible oxidative modification influences cell signaling and regulatory and homeostatic functions. Most amino acids can be irreversibly modified either
directly via oxidation by ROS/RNS or indirectly by secondary by-products of oxidative stress. Some amino acids, such
as Cys and methionine (Met), are more susceptible to oxidation than other amino acids because of the nucleophilic
nature of sulfur (S) atoms present on their side chains.156,157
N-nitrosation of lysine, arginine, and histidine also contributes to protein modification. The most common type of irreversible modification is the formation of carbonyl groups
(C=O). Amino acids prone to carbonylation include proline,
arginine, threonine, lysine, histidine, and Cys. Assessment
of the extent of such generalized oxidation serves as a marker of oxidative stress.158
Sulfur-containing Cys and Met, the amino acids most susceptible to oxidation, undergo reversible modifications by cellular enzymes and accordingly have a regulatory role in redox
signaling.153 Oxidation of Cys leads to production of sulfenic
acid (SOH), which could further react with another Cys residue to form an intermolecular or intramolecular disulfide bond
or which could be further oxidized in a stepwise manner to
sulfinic acid and sulfonic acid (Figure 3).161 Sulfenic acid can
be redox recycled back to thiol by the glutaredoxin or thioredoxin systems.161,162 Cys sulfinic acids, previously thought to
be irreversibly oxidized, can be reduced to the catalytically
active thiol by sulfiredoxin.162 Because Cys is both an e- donor
and acceptor, it is considered a redox-active amino acid that
plays an important role in the regulation of protein function by
ROS in cardiovascular cells.
Met is readily oxidized to form methionine sulfoxide
(MetO; Figure 4), which can be further oxidized to produce
methionine sulfone or radicals. These latter molecules contribute to oxidative damage.164 Protein methionine oxidation can
be reversed enzymatically by methionine sulfoxide reductases.164 Methionine sulfoxide reductases are repair enzymes that
reduce methionine sulfoxide residues in oxidatively injured
proteins to methionine residues. Accordingly, these enzymes
protect cells from oxidative stress and have been considered
as antioxidant and protein repair enzymes. Proteins with oxidation-sensitive methionine residues that are important in the
cardiovascular system include calcium/calmodulin-dependent
protein kinase II, α2-macroglobulin, 15-lipoxygenases, apolipoprotein A-1, thrombomodulin, and von Willebrand factor.
Approaches for Detection of Oxidative
Modifications
Protein oxidation can be assessed in cultured cells, circulating cells, and tissue by direct and indirect approaches.
Direct approaches detect changes by exploiting the properties of oxidized forms of proteins or structural changes induced by oxidation. Such approaches detect both reversible
and irreversible modifications with specific antibodies165,166
or chemoselective probes167 and can be applied at a global
or targeted level. Cell-permeable probes have the advantage
of labeling within the system, thus preventing any artifactual labeling caused by oxidation on cell lysis.168 Indirect approaches are most commonly used in cardiovascular research
because they exploit conserved biochemical properties of the
reversible oxidative modifications, as well as the catalytic
activity of certain proteins. Most of these assays are based on
the ability of unmodified, active sites (eg, free thiols, S−) to
be irreversibly alkylated and the resistance of oxidized sites
(eg, SOH, SNO[S-nitrosothiol]) to alkylation. Indirect approaches monitor reversible modifications by using labeled
substrates169 or probes170 and can be classified as negative
and positive methods. In negative methods, the unmodified
sites (eg, S−) are labeled with the probe, and oxidation is
measured as loss of signal. On the other hand, in positive
methods, the unmodified sites are alkylated initially, and the
reversibly oxidized sites are then reduced and labeled with
the probe; therefore, an increase in signal intensity indicates
elevated levels of oxidation. Indirect approaches are advantageous because they can provide a relative quantification
of the active versus the oxidized state. The major limitation
of indirect approaches is that the fraction of reversibly oxidized proteins is overestimated if a significant proportion of
oxidized proteins within the sample are in the irreversible
state.171 The sensitivity of these methods can be improved by
preparing subcellular enrichments before the assay.
Methods to Assess Protein Oxidation
Irreversible Oxidative Modification: Measurement of
Protein Carbonylation (C=O)
The most commonly used direct method to assess carbonylation levels is based on the derivatization of the stable carbonyl groups by 2,4-dinitrophenylhydrazine, which results in
the formation of a stable product, dinitrophenyl hydrazone.172
Dinitrophenyl hydrazone is generally detected either by spectrophotometric assay (absorbance at 370 nm)173 or by immunoassays. Development of dinitrophenyl hydrazone–specific
e52 Circulation Research August 19, 2016
Figure 3. Oxidative posttranslational
modifications on protein thiols (cysteine).159,160
The side chain of cysteine residues possess
a terminal thiol (-SH) functional group. The
sulfur at the core of the thiol is electron rich,
allowing multiple oxidation states as shown
in the Figure. Protein thiols can form various
oxidative modifications, including reversible
and irreversible forms. SNO indicates
S-nitrosylation; and SOH, S-sulfenylation.
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antibodies has permitted quantification of carbonyl content
by ELISA,174 immunohistochemistry,175 or Western blotting.176
Immunodetection by Western blotting offers the advantage of
increased specificity compared with all other assays, because
it allows for the assessment of carbonylation of individual proteins, which can further be extracted as gel bands and identified by tandem MS (MS/MS). Alternatively, carbonyl groups
can be quantified after reduction by sodium borotritide to the
corresponding 3H-radiolabeled hydroxy compound, which is
detected by standard methods.177 An advantage of the methods
used for assessment of protein carbonylation is that only standard laboratory equipment is needed.
Reversible Oxidation: Measurement of Cys Thiol Oxidation
Most approaches used to monitor reversible oxidative modifications of Cys thiols rely generally on the efficient trapping of
the native redox state of the thiol proteome. For this purpose,
all free thiols are selectively alkylated (or trapped) by alkylating agents, such as iodoacetamide and N-ethylmaleimide.178
Modified Cys residues can be labeled by direct or indirect approaches. The labeled probes used in these strategies can be
fluorescently tagged, biotinylated, or isotopic derivatives of
thiol alkylating agents. Labeled proteins are typically either
separated by 2-dimensional gel electrophoresis and subjected
to MS or LC/MS identification in proteomics approaches;
pulled down and detected by immunoblotting; or analyzed by
Western blotting with specific antibodies, as in the case of a
modified cysteinyl-labeling assay.179
The most commonly used approaches involve alkylation
of free thiols with subsequent reduction of the reversibly oxidized thiols and labeling with a probe. Increase in the signal
is indicative of elevated oxidation.170 This approach can assess either global or specific types of reversible thiol modifications, depending on the selectivity of the reductant being
used. Dithiothreitol and Tris(2-carboxyethyl)phosphine are
considered general reductants and are therefore used for untargeted screening of protein thiol modifications. Detection
of sulfenylation, which occurs when free thiols react with
H2O2 to form sulfenic acid (R-SOH), is typically based on
the specific reduction of sulfenic acid with arsenite.162 Snitrosylation (RSNO) of free thiols on reaction with NOderived species such as N2O3 (NO+) or by interaction with
Figure 4. Oxidative posttranslational
modifications on methionine residues in
proteins. Addition of O2 to the sulfur atom
of methionine (Met) leads to production of
methionine sulfoxide (MetO).163,164 MetO
is reduced back to Met by methioinine
sulfoxide reductases. Methionine sulfoxide
residues can be further targeted by H2O2 to
methionine sulfone, which is an irreversible
form of methionine oxidation.
Griendling et al Measurement of Reactive Oxygen Species e53
metalloprotein-linked enzymes is another Cys thiol modification that is discussed in more detail below in Modification of
Proteins by NO. In addition, formation of mixed disulfides,
referred to as S-glutathionylation as a result of other thiol redox reactions can be detected via reduction by glutaredoxin.162
Although this methodology is widely used, especially during
proteomic screenings of the thiol proteome, it does not allow
for identification of the specific site of the thiol modification.
A more direct approach in assessing Cys thiol oxidation
under nonreducing conditions is the antibody-based strategy.
Specific antibodies have been raised to detect S-glutathiolated
proteins180; however, their use has limitations because of their
low sensitivity. Antibodies have also been generated to react with the hyperoxidized (SO3H) form of proteins, such as
peroxiredoxin.181
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Measurement of Methionine Oxidation
Methionine oxidation is a promising marker of oxidative
stress. Conventional methods for assessing MetO generation
and reduction depend on HPLC and MS. More recently, novel
antibodies and Met-rich proteins have been used to measure
methionine/selenomethionine oxidation and methionine sulfoxide reductase.182
Measurement of Sulfenylation
Sulfenylation is typically measured with a specific alkylating
agent, 5,5-dimethyl-1,3-cyclohexanedione (dimedone). This
approach is based on the principle that sulfenylated proteins
react irreversibly with dimedone and can then be isolated by
affinity-tagged purification and identified by MS/MS, Western
blotting, or fluorescence measurements.
Several dimedone-based probes and antibodies have
been developed to provide direct methods for assessment
of protein sulfenylation. The probes typically consist of the
dimedone reactive group, a linker, and a reporter/affinity
tag or an azide group.183,184 The tag is usually a fluorophore
or biotin, which enables detection of the oxidized proteins
with fluorescence. The introduction of an azide group has
improved the reactivity of probes (eg, DAz1, DCP-N3), because it allows connection of the bulky reporter/affinity tag
to the probe by “click chemistry” after the probe is already
bound to the Cys-SOH.184 Moreover, the recent development
of cell-permeable probes (eg, DCP-Rho1, DYn-2) has permitted capture of sulfenylation in living cells, which can be
analyzed by confocal or fluorescence imaging.168,183 A limitation of using dimedone-based probes is their reduced reactivity with certain protein families, such as protein tyrosine
phosphatases, important in signaling in vascular and cardiac
cells185; however, this issue has been addressed by generation of dimedone-based probes with a protein tyrosine phosphatase–binding module, which binds irreversibly to the
oxidized catalytic site of protein tyrosine phosphatases (eg,
BiPhaz-1, NAPhaz-1).186 In the case of dimedone-specific
antibodies, cells need to be treated with dimedone before
they are lysed. A limitation of this method is that dimedone
reacts irreversibly with SOH groups; therefore, treatment
with dimedone for long periods of time will alter the cellular
redox state and could generate false-positive results.187
Another more targeted approach monitors sulfenylation
of specific proteins of interest using conformation-specific
antibodies, which recognize the oxidized structure of the
protein.188 The limitation of this method is that the crystallographic structure of the unmodified and modified form of the
protein has to be known in order for the specific conformation-sensing antibody to be developed.
Most studies examining oxidative modifications of proteins
have detected protein S-glutathionylation S-sulfenylation, and
S-nitrosylation (see Modification of Proteins by NO). These
modifications are increasingly recognized as important signaling events in cardiovascular physiology and disease. The significance of protein S-glutathionylation and S-sulfenylation in
cardiovascular disease has been suggested in various experimental models. In rat aortic rings, S-glutathiolated eNOS is
associated with impaired vasodilatation.189 In atherosclerosis,
S-glutathionylation induced activation of sarco(endo)plasmic
reticulum Ca2+ ATPase (SERCA) is substituted by irreversible
sulfonylation.190 The cardiac sarcolemmal Na+-K+ pump is inhibited by S-glutathionylation on the β1-subunit in ventricular
myocytes.191 In models of ischemia-reperfusion, there is a loss
of function of GAPDH,159 aldose reductase,192 and actin because of S-glutathionylation193
Studies that used selective reduction of sulfenic acid and
specific probes to assess S-sulfenylation levels have shown
that multiple structural and mitochondrial proteins of cardiac
myocytes, including cytoskeletal components, undergo sulfenylation after exposure to H2O2.194,195 However, it is still unclear whether sulfenic acids can act as signaling molecules or
whether they are intermediates in the process of irreversible
oxidation that results in the formation of sulfinic and sulfonic
acids.
Modification of Proteins by NO
Nitrosothiols are important mediators of NO signaling pathways196 and are likely formed by multiple mechanisms, including nitrosation by N2O3, radical recombination of thiyl radicals
with NO, and transition metal-catalyzed reactions.197 Cellular
levels of nitrosothiols can be reduced by denitrosylating enzymes such as S-nitrosoglutathione reductase.198 The major
cellular S-nitrosothiol is S-nitrosoglutathione reductase.199 An
important reaction is transnitrosylation, in which NO is transferred from one thiol to another.200 Several methods have been
developed for detection of protein and low-molecular-weight
nitrosothiols; however, these techniques are challenging and
prone to artifacts.201 These measurements can be affected by
light, reducing agents, and pH because of an effect on nitrosothiol stability. Chung et al202 offer a comprehensive review
on the utility of Cys tandem mass tag (cysTMT) to detect and
determine the reactivity of an individual Cys’s reactivity for
S-nitrosylation. This approach has facilitated the identification
and quantification of Cys oxidative modifications in cardiovascular cells and tissue.202 Other major methods of nitrosothiol detection include chemiluminescence-based methods and
switch-based methods,201 and recently, nitrosothiol-specific
antibodies have been developed.203,204
The biotin-switch method of detecting nitrosothiols is similar to that described in Reversible Oxidation: Measurement of
Cys Thiol Oxidation. For measurement of nitrosothiols, this
method consists of 4 major steps: (1) thiol blocking, (2) selective reduction of RSNO, (3) labeling of resultant free thiols,
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and (4) detection of biotin-labeled thiols.201 Significant concerns have arisen regarding the potential artifacts associated
with high levels of ascorbate that can potentially reduce some
protein disulfides.205 Phosphine-mediated selective reduction
appears to be preferable because it is specific for nitrosothiols
and does not reduce protein disulfides.206
Both polyclonal and monoclonal antibodies have been
developed that can detect RSNO moieties.203,204,207 An example is the anti-S-nitrosocysteine antibody, which detects
S-nitrosocysteines but not reduced cysteines.203 It is generally
recommended that one use positive and negative controls with
chemical nitrosation and cleaving of S-NO bonds in situ.208 If
cleaving the S-NO bonds does not reduce the signal, then immunostaining is likely nonspecific.
A third method of detecting NO-mediated protein modification is anaerobic reductive chemiluminescence, which
uses excess Cys to drive the transnitrosation equilibrium in
favor of CySS-NO and reduce Cu2+ to Cu+. Reductive RSNO
assays are performed under helium or argon in a purge vessel that contains Cys and CuCl.201,208 The free NO that is
released is purged in the inert gas stream to a chemiluminescence-based NO analyzer. Photolysis-chemiluminescence is
based on photosensitive homolytic cleavage of RSNO after
exposure to light; however, ultraviolet photolysis has been
criticized for overestimating RSNO values because of the
presence of endogenous nitrite and nitrate that can also be
converted to NO by ultraviolet irradiation.209 The Cu-Cys
assay is not useful for solutions with high protein content
(plasma, hemoglobin) and has been replaced by triodiode
(KI3)-based reductive chemiluminescence, which provides a
more specific assay of RSNO levels.210,211 Triodiode-based
assays have become the most accepted and widely used
method for RSNO measurement, providing sensitive and
specific measurements in complex systems.
A cardioprotective role of S-nitrosylation has been demonstrated for cyclooxygenase 2212 and mitochondria complex I,213
translated as attenuated infarct size during ischemia-reperfusion. In rat hypoxic myocardium, decreased S-nitrosylation of
Na+-K+ ATPase was followed by increased S-glutathionylation
and loss of activity.214 S-nitrosylation of p47phox by NO has
been shown to suppress Nox-induced ROS production in
human endothelial cells.215 On the other hand, impaired relaxation in mouse aorta has been related to protein kinase C
S-nitrosylation,216 as well as to augmented global S-nitrosylation
in Ang II–induced hypertension.217 Studies in bovine aortic
endothelial cells demonstrated increased levels of S-nitrosylated
endothelial NOS, which was reduced on stimulation with vascular endothelial growth factor.218
Measurement of Protein-Derived Radicals
Immuno-spin trapping with anti-DMPO antibodies is a useful
approach for detecting protein-derived radical intermediates
both in vitro and in vivo. Antibodies produced in rabbits immunized against the hapten 5,5-dimethyl-2-octanoic acid-1-pyrroline N-oxide (OA-DMPO, a structural analog of DMPO
with an additional carbon chain terminating in a carboxylgroup for amide linking) bind specifically to the protein adduct of DMPO.219–221 This approach expands the utility of spin
trapping without EPR and can be highly sensitive in detecting
protein radicals formed under physiological or pathological
conditions. It can be used in conjunction with immunoblotting analysis, immunofluorescent staining, flow cytometry,
and even magnetic resonance imaging. In combination with
MS, immuno-spin trapping has been used to define the role
of protein thiol radicals in mediating S-glutathionylation of
mitochondrial complex I at the level of isolated enzymes,
myocytes, and hearts of eNOS−/− mice.13,30,222 In endothelial
cells, the same approach has been used to examine a similar mechanism in regulating protein S-glutathionylation coupling, and function of eNOS.223 Anti-DMPO antibodies can
also be covalently bound to an albumin-gadolinium-diethylenetriaminepentaacetic acid-biotin magnetic resonance imaging contrast agent and used for molecular magnetic resonance
imaging.224,225 This approach has been used to detect in vivo
levels of membrane-bound protein/lipid radicals in kidney,
liver, and lung of diabetic mice.225 Immuno-spin trapping is
a unique and powerful approach to detect protein radical intermediates in time and space; however, the approach is limited to the specific protein radical adducts of DMPO, mainly
Cys- and tyrosine-based protein radicals. Additional antibodies against different type of spin traps (eg, nitroso spin traps)
could provide broader utility in determining other types of
protein radicals.
Tyrosine or Tryptophan Modifications
Tyrosine and tryptophan nitration are common posttranslational modifications that affect protein structure and function.
3-Nitrotyrosine and nitrotryptophan are formed on reaction
of OONO− with proteins containing tyrosine or tryptophan
residues. Alternatively, these modifications can occur by peroxidase-mediated reactions between protein tyrosines or tryptophans and nitrite. Such changes have been detected by MS
and immunostaining in various disease processes, including
aging.226,227 They have also been used to serve as a footprint of
peroxynitrite formation in vivo228; however, peroxidase-based
reactions of nitrite with tyrosines can also yield nitrotyrosine
and in fact might be its major source in vivo. HPLC, biotin
tagging, MS/MS, and immunological methods have been used
for nitrotyrosine and nitrotryptophan detection.144,229 MS/MSbased peptide sequencing provides unambiguous confirmation of nitrotyrosine formation. Antibodies purported to detect
nitrotyrosine also detect nitrotryptophan,230 and so must be
used with caution.
Formation of dityrosine residues as a result of oxidative
damage has also been used as a marker of oxidant stress. In
this method, dityrosines are first released by acid hydrolysis
in antioxidant-containing buffers and then derivatized with
heptafluorobutyric anhydride/ethyl acetate before analysis by
MS. This method can be used on whole tissue, but usually
dityrosine formation on individual proteins is below the detection limit of the assay.
Recommendations for Measurement
Considering the importance of oxidative stress and redox signaling in cardiovascular pathophysiology, unraveling exactly
how protein function is changed by oxidation is critical to elucidate molecular mechanisms of disease. Accordingly assessment of oxidative modifications of proteins, a posttranslational
Griendling et al Measurement of Reactive Oxygen Species e55
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phenomenon, should be a priority in the tool kit of redox methodologies in cardiovascular research.
Although MS- and electrophoresis-based proteomics are
commonly used, these approaches separate proteins as individual polypeptides rather than as functional complexes.
Moreover, data generated are rarely quantitative and do
not indicate modification levels per functional unit, and
full resolution of basic or hydrophobic proteins, including
membrane-associated proteins, cannot be attained with current technology. Hence, these techniques provide information on global oxidative changes. Another system that is
gaining popularity in cardiovascular research is the OxyBlot
(Merck Millipore) protein oxidation assay, which is ideal
for rapid detection of proteins modified by ROS. This assay,
which can be performed in crude or purified protein preparation, measures carbonyl groups introduced into protein side
chains and also gives an assessment of global rather than
specific protein oxidation.
In-depth characterization and quantification of oxidative modification of individual proteins requires complex
oxidative proteomics. Such studies should be performed
in collaboration with specialized proteomics laboratories.
However, it is becoming relatively simple to detect individual oxidized proteins within a mixture of proteins using immune-based techniques, such as Western blotting,
ELISA, and immunohistochemistry/immunocytochemistry.
Numerous commercial antibodies to assess nitrosylation,
glutathionylation, carbonylation, and sulfenation of proteins
are now available, which makes assessment of protein oxidation in cardiovascular cells and tissues relatively simple.
Immunostaining can also be used to measure 3-nitrotyrosine, a marker of RNS. Other immune-based methods rely
on 5-iodoacetamido fluorescein to label oxidized sulfhydryl
groups, which can then be pulled down with fluorescein antibody.154 Alternatively, proteins such as thioredoxin can be
derivatized at the active-site Cys thiols using a large maleimide derivative, and then nonreducing gel electrophoresis can be used to separate reduced (labeled) and oxidized
(unlabeled) proteins based on their size.231 Choice of method depends on the availability of species-specific antibodies, the type of modification to be studied, and the particular
protein of interest. As with other redox methods, appropriate controls must be included to demonstrate specificity of
the protein modification under study.
Measurement of Lipid Peroxidation
Introduction
Because of their highly reactive state, ROS are unable to diffuse away from their site of formation, which often leads to
only site-specific alterations. Thus, it has been suggested that
lipid peroxidation products amplify and propagate the cellular effects of ROS.232,233 Measurements of lipid peroxidation products are therefore not only sensitive assessments of
ROS generation but also provide mechanistic insights into the
pathogenesis of ROS-induced injury.
Common Lipid Oxidative Modifications
Oxidation of lipids (peroxidation when biological material
is involved) is a complex process, with several free radicals,
metastable intermediates, and end products.234,235 Several
products of lipid peroxidation can be measured to document
ROS-mediated lipid peroxidation in biological tissue. Because
of their extremely short half-life, C-centered radicals or peroxyl radicals are rarely measured, except in detailed mechanistic studies.
Measurement of Lipid Peroxidation
Lipid Hydroperoxides
Lipid hydroperoxides (LOOH) are usually measured by HPLC,
because they are largely degraded during MS. Older methods
for LOOH separation/detection used either chemiluminescence236 or mercury cathode electrochemical detection.237
These methods have been improved considerably by the inclusion of appropriate stable isotope internal standards238,239 and
are suitable for the measurement of LOOH in plasma, lowdensity lipoprotein, and tissue samples. Phospholipid LOOH
in tissues and cells can also be identified by HPLC followed
by fluorescence detection.240 The method has high sensitivity
(1–2 pmol of LOOH), although its specificity remains unclear. The formation of specific hydroperoxides can be used
to study the reactivity of specific ROS; for instance, singlet
oxygen, ozone, or ionizing radiation in biological tissues can
be specifically documented by measuring specific cholesterol
hydroperoxides.233
Unsaturated Aldehydes
Aldehydes such as 4-hydroxy-trans-2-nonenal (HNE),
malondialdehyde (MDA), isoprostanes, and acrolein are
major end products of lipid peroxidation.232 Among the free
aldehydes generated from lipid peroxidation, the C9 unsaturated aldehyde HNE is the most abundant product of
arachidonic acid241 and is one of the most studied products
of lipid peroxidation. Tissue and plasma concentrations of
free lipid peroxidation-derived aldehydes such as HNE can
be measured by HPLC or gas chromatography (GC)/MS.
HPLC methods usually use aldehyde-reactive probes such
as 2,4-dinitrophenylhydrazine and 1,3-cyclohexandione or
other similar chemical probes.242 Derivatization by reagents
such as pentafluorobenzyl oxime followed by silylation is
also required for detection with negative chemical ionization
by GC/MS.243,244 With the use of an appropriate deuterated internal standard, GC/MS can be a sensitive and reliable method for the measurement of free HNE245,246; however, current
approaches that use LC/MS247,248 are preferable because they
do not require derivatization to enhance optical activity or
to generate a volatile product. In addition, measurement of
HNE-modified proteins has been widely used as a measure
of oxidative stress and ROS generation in a variety of disease
conditions, and coupled with MS identification of the modified proteins,249–251 it can provide mechanistic understanding
of oxidative stress–induced injury and dysfunction.
Proteins modified by lipid peroxidation–derived aldehydes such as HNE, MDA, and acrolein are commonly detected by Western analysis or immunohistochemistry with
antibodies against aldehyde-modified proteins. Several anti-protein MDA and anti-protein HNE antibodies are commercially available and suitable for immunohistochemistry,
ELISA, and Western blotting. Polyclonal or monoclonal
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antibodies raised against HNE-treated keyhole limpet haemocyanin252,253 or bovine serum albumin254 have been used
to identify HNE-histidine adducts, with little cross-reactivity
with HNE-lys or HNE-cys.
Increased formation of HNE and related aldehydes such
as acrolein in animal models and humans can also be measured by quantification of their urinary metabolites. The
major urinary metabolites are mercapturic acid derivatives.
In mouse and rat urine, the most abundant HNE metabolite
is 1,4-dihydroxynonane-mercapturic acid (DHN-MA),255,256
which is derived from the reduction of the glutathione conjugate of HNE (GS-HNE) to GS-DHN by the enzyme aldose
reductase.257 Similarly, the reduced form of the mercapturate
derivative (3-hydroxypropanal mercapturic acid; 3-HPMA)
is also the most abundant urinary metabolite of acrolein in
rats.258 Both DHN-MA255,259 and 3-HPMA260,261 have also
been detected in human urine and have been linked to oxidative stress; however, because in addition to lipid peroxidation
acrolein can also be derived from exposure to combustion
products and cigarette smoke, DHN-MA might be a more
selective marker of lipid peroxidation than 3-HPMA in
humans.
Oxidized Phospholipids
In addition to free aldehydes, peroxidation of phospholipids
generates carbonyls that remain esterified to the phospholipid
backbone. The most commonly studied oxidized phospholipids are 1-palmitoyl-2-oxo-valeroyl-sn-glycero-phosphocholine
(POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-phosphocholine
(PGPC), and 1-palmitoyl-2-(5,6-epoxyisopro-stane E2)-snglycero-phosphocholine (PEIPC).262,263 These phospholipids
are derived from the oxidation of 1-palmitoyl-2-arachidonylglycerol-3-phosphocholine (PAPC), one of the most abundant phospholipids in low-density lipoprotein. The levels of
these oxidized phospholipids can be measured by electrospray ionization MS in tissues after chloroform-methanol
extraction and solid-phase extraction chromatography using
dimyristoylphosphatidylcholine as an internal standard.262
Such measurements have shown that aortas obtained from
rabbits fed an atherogenic diet contain higher levels of
POVPC, PGPC, and PEIPC than those from rabbits fed a
control diet.262 Moreover, oxidation products of cardiolipin,
which is abundant in the mitochondria, can be measured as
specific biomarkers of mitochondrial oxidative stress with
LC/MS.264
Because oxidation creates new epitopes, oxidized phospholipids are recognized by antibodies of innate immunity. Several natural monoclonal IgM antibodies have been
cloned, including E06,265–267 which was cloned from apolipoprotein E–null mice.268 These IgM autoantibodies have
been shown to identify oxidized phospholipids in arterial
plaques268 and in lipoproteins, although the specific epitopes
recognized by these antibodies have not been well-defined
because they also recognize MDA-modified low-density lipoprotein or high-density lipoprotein, which suggests that
they recognize the adduct between oxidized lipids and apoproteins.268 Of these antibodies, E06 appears to specifically
recognize the perturbed phosphorylcholine headgroup of
oxidized phospholipids and can be used to recognize these
phospholipids in cells, tissues, and lipoproteins. Therefore,
positive reactivity to the antibody can be a useful indicator
of the oxidation of phosphocholine phospholipids in cells
and tissues. However, in most cells, oxidized phospholipids are readily removed, either by cytosolic enzymes with
phospholipase A2 activity, which removes the oxidized sn2 side chain,269 or by the reduction of sn-2 aldehydes by
aldose reductase.270 Hence, the absence of oxidized phospholipids does not necessarily indicate a lack of lipid peroxidation, and the measured levels are reflective of a steady
state achieved between the generation and the metabolism
of these phospholipids.
Oxidized phospholipids can also be measured in the plasma, and the levels of oxidized phospholipids bound to lipoproteins correlate with the extent of angiographically documented
atherosclerotic disease,266 which suggests that oxidized phospholipid might be involved in the formation of atherosclerotic
lesions.271
Isoprostanes
Nonenzymatic peroxidation of arachidonic acid generates
prostaglandin-like compounds that have been measured as reliable biomarkers of oxidative stress in general and lipid peroxidation in particular. Several chemically distinct species of
isoprostanes are generated from the oxidation of arachidonic
acid.272,273 From 4 F2-isoprostane (IsoP) regioisomers, each
composed of 8 racemic diastereomers, 64 different species are
generated. Although oxidation products of arachidonic acid
have been classically measured as markers of lipid peroxidation, other isoprostanes derived from eicosapentaenoic acid,
docosahexaenoic acid, adrenic acid, and α-linolenic acid provide a more comprehensive picture of fatty acid–specific lipid
peroxidation.274 In principle, any or all of these isoprostanes
could be measured; however, practically, only a few of these
species are quantified, of which the 5-series F2-IsoPs and the
15-series F2-IsoPs derived from arachidonic acid are most frequently measured.
Because of the high structural diversity of isoprostanes,
GC/MS or LC/MS is the method of choice for measuring
these species, although ELISA-based immunoassays are
also used frequently. The immunoassays, although simpler,
cheaper, and faster, have high variability. There is poor correlation between different immunoassays, and the values
obtained from ELISA often do not correspond to those
measured by GC or LC/MS.275 Moreover, the antibodies
used in these assays exhibit cross-reactivity with molecules
of related structure, such as the products of cyclooxygenase.276 Immunoassays are also highly sensitive to interference by biological impurities present in complex biological
systems, such as cell extracts, urine, or blood. Hence, the
results obtained from ELISA and other immunoassays
should be interpreted with caution. The most sensitive and
accurate measurements of isoprostanes involve quantification by GC/MS with appropriate deuterium-labeled internal
standards.277,278 These methods can measure isoprostanes to
a lower limit of quantification of 2 to 100 pg/ml and can be
optimized to measure multiple species of isoprostanes and
isofurans.277 However, these techniques require elaborate
sample preparation steps, such as thin-layer chromatography,
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solid-phase extraction, liquid-liquid extraction, or affinity chromatography. Moreover, the samples must undergo
chemical derivatization to pentafluorobenzyl ester, trimethylsilyl ether analogs before GC/MS. As a result, only a few
specialized laboratories have the necessary experience and
infrastructure to reliably measure isoprostanes by GC/MS.
In contrast with the GC/MS methods, LC/MS measurements of isoprostanes require shorter procedures for sample
preparation, with no derivatization step. Although earlier
methods suffered from poor resolution, long run times, and
low sensitivity, newer methods using ultraperformance LC
with tandem MS detection using multiple reaction monitoring rival or exceed the sensitivity and specificity of GC/MS
techniques.279,280 These methods generate high resolution of
analytes to minimize matrix effects281 and can measure isoprostanes in the low picomolar range. With such high-sensitivity methods, changes in the levels of 15-F2-IsoP can be
measured in as few as 10 000 cells.280 These methods can also
be adapted to simultaneously measure both enzymatically and
nonenzymatically generated eicosanoids.282 Although these
techniques require sophisticated mass spectrometers and ultraperformance LC, they enable rapid, accurate, and sensitive
detection of a wider range of isoprostanes and related species
than has been possible thus far.
Isoprostanes are more stable than other products of lipid
peroxidation and are therefore measured as reliable indices
of oxidative stress. Levels of F2-IsoP in blood and urine have
been measured to assess systemic oxidative stress in both
animals283 and humans284 under conditions of validated oxidative stress and in animal models of atherosclerosis, transplant arteriosclerosis, flow-induced vascular remodeling,285
and age-accelerated atherosclerosis.286 Additionally, urinary
levels of isoprostanes have been used to document an increase in oxidative stress in humans with high cardiovascular disease risk,287 diabetes mellitus,288 or pulmonary
hypertension289 and to document increased free radical generation after reperfusion in patients undergoing acute coronary angioplasty.290,291
In the plasma, F2-IsoPs circulate in their free unesterified
form, although they can also be bound to the phospholipid
backbone, from which they can be released by platelet-activating factor acetylhydrolase. Because the levels of free and
bound isoprostanes are highly correlated, usually only levels
of free isoprostanes are measured. The metabolism of F2-IsoPs
in the liver generates multiple metabolites, including those
generated by their conjugation with taurine or glucuronide. In
human studies, these metabolites are measured along with the
unmetabolized parent compound.273
Recommendations for Measurement
In the design of experiments, the choice of the lipid peroxidation product to be measured will depend on the overall
objective of the study (Figure 5); however, in general, measurement of lipid peroxidation products can provide a useful
and informative index of oxidative stress. Direct measurements of lipid peroxidation products such as HNE, acrolein,
or lipid hydroperoxides, however, are difficult because they
are rapidly metabolized or react readily with other cell constituents. Therefore, the formation of these products is usually assessed indirectly by measuring their covalent protein
adducts. Measurements of protein-HNE or protein-MDA adducts provide unambiguous evidence for lipid peroxidation,
Figure 5. Analytical methods for
measuring products of lipid peroxidation
in experimental and clinical studies. The
choice of the assay depends on the
experimental system. Assays suitable
for different experimental systems are
shown in horizontal bars. Measurements
of conjugated dienes are suitable only for
isolated membranes and cells, whereas
thiobarbituric acid reactive substances
(TBARS), free 4-hydroxy-trans-2-nonenal
(HNE) and malonaldehyde (MDA) levels,
hydroperoxides, isoprostanes, oxidized
lipids, and protein-aldehyde adducts can be
measured in most experimental systems.
Autoantibodies against protein conjugates
of aldehydes can be measured in the
animal and human plasma, whereas urinary
isoprostanes and metabolites of aldehydes
(mercapurates) can be measured in the
urine. Assays listed as primary (1°) are the
most well accepted, secondary (2°) are more
widely used, and tertiary (3°) are either less
sensitive or less specific (TBARS) or should
be verified with a second method.
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and an increase in abundance of these adducts is likely to
be reflective of increased oxidative stress. However, these
assays, although qualitative, are not very quantitative. When
generated, HNE and MDA react with multiple proteins that
show variable levels of modification, making quantification
of the total extent of lipid peroxidation somewhat challenging. Moreover, some proteins are adducted to HNE or MDA
at baseline, and therefore their presence interferes with
quantification of the total adduct formation. In addition, proteins covalently modified by HNE are readily degraded by
proteolysis,292 and thus, measurements of the levels of these
adducts at a single time point could underestimate their formation. Hence, it is preferable to measure more stable products of lipid peroxidation, such as isoprostanes, in urine,
blood, or tissue if the intent is to document total local or
systemic oxidative stress. F2-IsoPs have been well validated
as markers of oxidative stress both in animal and clinical
studies.273 Nevertheless, measurements of specific products
of lipid peroxidation such as lipid hydroperoxides, proteinHNE adducts, or oxidized phospholipids are useful in addressing specific mechanistic questions.
Oxidative Modification of DNA
Introduction
One of the more severe consequences of excessive ROS production is oxidative modification of DNA.293 Characteristic
DNA modification has been detected under several pathological conditions associated with increased oxidative
stress; however, whether these modifications contribute
to disease progression or are a consequence of pathologically increased ROS levels remains to be determined.294
Techniques to measure DNA oxidation can be applied to
both nuclear and mitochondrial DNA, and many are applicable to RNA, which also undergoes oxidative modification.295 Oxidation of guanine has been considered to be
a surrogate measure of oxidative DNA modification, but
there are several other chemical modifications of DNA that
are induced by ROS and RNS, including (1) purine and pyrimidine base lesions generated by oxidation, deamination,
depurination, and depyrimidination; (2) modification of the
sugar moiety; (3) formation of protein-DNA cross-links;
(4) formation of DNA-DNA cross links; and (5) single- and
double-strand breaks.296
Typically, strand breaks occur when a base is oxidized,
for instance, conversion of guanosine to 8-hydroxy-deoxyguanosine, on which endogenous enzymes excise the modified base, creating an abasic site. Sometimes, the newly
added base is a mismatch, which can lead to errors in replication. Modification of the sugar moiety by ROS such the
alkoxyl radical generates reactive aldehydes such as base
propenals and MDA.297 These aldehydes, generated from
radical oxidation of 2-deoxyribose, like those generated
from lipid peroxidation, react with nucleophilic centers in
the DNA and modify DNA bases. In aggregate, between
50 and 75 base modifications have been reported, although
to date, some of these have been found only under in vitro
conditions in which oxidative modification was specifically
induced.293,294,298
Under normal conditions, there appears to be a background level of ROS-induced damage in both nuclear and
mitochondrial DNA that is normally repaired by enzymes
such as the DNA glycosylases. These repair mechanisms can
be broadly summarized as base excision repair, nucleotide
excision repair, and mismatch repair. Although estimates of
DNA damage range from 100 000 to 1 000 000 lesions per
genome per cell division,299 the overall rate in humans appears to be about 1 mutation per 30 million base pairs per
cell generation.300
Mechanisms of Oxidative Damage to Nucleotides
Although several ROS can modify proteins, their reactivity
with DNA is more limited. Some species such as the •OH
radicals and crypto-OH or ferryl-OH react avidly with DNA,
.−
whereas other species such as O2 or H2O2 are not known
to directly modify DNA.298,301 Additionally, RNS such as
OONO− can react directly with DNA.302 In vitro, the •OH
radical reacts with DNA by addition to double bonds of
DNA bases and by abstraction of an H atom (also known
as hydrogen atom transfer) from the methyl group of thymine and the C-H bonds of 2′-deoxyribose. The •OH radical
also reacts with carbons at the C5-C6 double bond of pyrimidines, which results in the production of C5-OH and C6OH adduct radicals. Hydrogen atom transfer from thymine
results in the allyl radical. Adduct radicals differ in terms of
their redox properties, with some being reducing (C5-OH)
whereas others are oxidizing (C6-OH).298 These radicals can
form many different products (see references 294, 296, and
298 for detailed lists of the many products) depending on
their redox properties and environment (eg, presence or absence of oxygen or reaction partners), but for the purposes
of this statement, we will focus on the measurement of oxidation modification in broad categories: chemical modification, fragmentation, and abasic sites.
Measurement of DNA Damage
Oxidative DNA base damage is measured by either direct or
indirect approaches.303 Direct approaches use sophisticated
analytical procedures, for example, MS, to evaluate chemical modification of extracted and purified DNA. Indirect approaches involve the use of a specific DNA repair enzyme and
measurement of DNA strand breaks. For indirect analysis, a
DNA N-glycosylase repair enzyme is used to excise the modified bases, which induces a strand break that can be measured
by reliable methods such as the comet assay and the alkaline
elution technique. More recent techniques use polymerase
chain reaction (PCR) to evaluate strand breaks in mitochondrial DNA or antibodies directed against oxidized bases to
quantify base modification.
Among DNA bases, guanine has the lowest ionization potential, which makes it more nucleophilic and therefore more
susceptible to oxidation than other bases.296 For this reason,
some assays have focused specifically on the oxidation of guanine, or 8-oxoguanine; however, a caveat for these measurements is that sometimes the nature of the procedure affects
the measurements. For instance, LC or LC/MS techniques can
potentially overestimate 8-oxoguanine levels in the sample
because of spontaneous oxidation of the guanine during sample preparation.304 Hence, it is imperative that all methods of
Griendling et al Measurement of Reactive Oxygen Species e59
procedure are performed with extreme care to avoid spurious
oxidation during sample preparation.
Comet Assay
The comet assay is a simple method based on gel electrophoresis that enables measurement of DNA strand breaks in cells.305
Isolated cells are mixed with an agarose solution, which is
then allowed to gel. The imbedded cells are then lysed and
electrophoresed at an alkaline pH, which produces comet-like
structures when the DNA is observed by fluorescence microscopy. The “intensity” of the fluorescent signal of the comet
tail relative to the head reflects the number of DNA breaks.306
The comet assay has been used to evaluate DNA strand breaks
in cardiac myocytes subjected to oxidative injury.307 Although
this assay is simple and easy to use, it provides only general
information about strand breaks and does not provide insight
into the nature of the oxidative modification.
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Mass Spectrometry
Typically, MS analysis of ROS-induced DNA damage involves GC-MS, HPLC-MS, or electrospray ionization MS
analysis.308,309 MS can detect a range of specific oxidations
of DNA bases, including 8-oxoguanine, thymidine hydroperoxides, and oxidizing or reducing radicals. Although MS is
highly sensitive (femtomolar range), the approach is not without limitations. For example, conditions of sample preparation
(derivatization of the nucleotides) for GC-MS are conducive to
oxidation and can lead to overestimates of the amount of oxidized DNA. Therefore, for GC-MS analysis of ROS-induced
DNA modification, it is important to incorporate procedures
to prevent excessive sample oxidation, such as the addition
of antioxidants. Other MS methods, HPLC-MS and electrospray ionization MS, are less prone to induce sample oxidation, and these approaches also allow precise identification of
the nature of the chemical modifications. MS has been used to
study oxidative and DNA damage in experimental models of
cardiovascular disease. For example, in Ang II–induced hy.−
pertension in mice, O2 formation, double-strand DNA breaks,
and mutagenic DNA base modification 7,8-dihydro-8-oxoguanine were increased in the heart and kidney, effects that
were attenuated by TEMPOL treatment.310 Similarly, Daehn
et al311 described 8-oxoguanosine accumulation and evidence
for DNA fragmentation linked to endothelial dysfunction in
glomerulosclerosis.
High-Pressure Liquid Chromatography
HPLC coupled to electrochemical detection is another useful
technique for measuring oxidatively modified DNA.309 It is a
fairly sensitive technique and is also relatively inexpensive to
use; however, it is not as sensitive as the LC/MS methods and
does not enable identification of the molecular modification
of the base.
Immunoassays
The use of antibodies that recognize 8-oxoguanine is another useful technique for analyzing oxidatively modified
guanine.312,313 This approach has been used to “visualize” the
base lesions with immunostaining or to provide a more comprehensive view of the modifications via Western blotting.
For example, Martinet et al314 showed elevated 8-oxo-guanine
in rabbit atherosclerotic plaques, and Mayr et al315 used anti8-oxoguanine antibodies to detect DNA damage in vascular
smooth muscle cells exposed to mechanical stress. An ELISA
can also be performed to quantify these modifications with
a high-throughput plate reader approach. The caveats to this
approach are similar to any measurements that use antibodybased analytical approaches; namely, that it is imperative
to establish the specificity of the antibody because methods
reported in the literature often use different concentrations
of primary and secondary antibodies and different blocking
agents. Also, the use of a secondary antibody in the absence
of the primary antibody serves as an important control in immunostaining approaches to determine background or nonspecific staining. The principal advantage of this technique
is that the use of antibody-based analyses is routine in many
laboratories, and hence, these techniques can be readily extended to oxidatively modified DNA. The limitations are
that only 1 base modification is analyzed, and as mentioned
above, sometimes the sample preparation induces oxidative
modification of guanine; thus, these measurements could
overestimate the amount of injury that is occurring in a cell
or tissue. Another limitation is that some antibodies raised
against 8-oxoguanine cross-react with guanine, which would
overestimate the number of oxidized bases.316,317 Hence, this
approach is better suited for qualitative rather than quantitative analysis of DNA damage.
Southern Blotting
Oxidative damage to DNA can also be assessed by Southern
blotting,318–320 and this can be applied to nuclear or mitochondrial DNA. For this procedure, known amounts of isolated DNA are incubated in sodium hydroxide to produce
single-strand breaks at abasic sites present in the DNA because of repair of oxidatively modified bases. This mixture
is electrophoresed and transferred to a nylon membrane,
where it is hybridized with a 32P-labeled probe for DNA
sequencing. The break frequency can be derived from the
band intensity of the oxidatively modified DNA as a function of the intensity of the control DNA bands. An advantage of this technique is that there is no requirement for
expensive, high-end equipment; only routine electrophoresis equipment, commonly available in most laboratories, is
needed. The disadvantages are that the exact nature of the
base lesion or lesions is not revealed and that the site where
the lesion occurs cannot be identified.
Polymerase Chain Reaction
Several approaches have been adopted to evaluate oxidative DNA modification with PCR.321–323 One such approach
involves the treatment of DNA with formamidopyrimidine
glycosylase to produce strand cleavage at sites of oxidized
purines, thus creating single-strand breaks that block PCR
amplification. The difference in PCR amplification between formamidopyrimidine glycosylase–treated and untreated DNA is then used as an estimate of oxidative base
damage. Data can be expressed as the change in lesion
frequency normalized to a 1-kb sequence, calculated from
the quotient of band intensities in the formamidopyrimidine glycosylase–treated and untreated samples. Another
PCR strategy involves determining the concentration of
e60 Circulation Research August 19, 2016
different sized PCR products (0.2, 2.9, and 16 kb) amplified from mitochondrial DNA.324 If there are no lesions,
the products should all have the same concentration, but
if there are lesions, there will be less concentration of the
larger products than the smaller ones. Using this approach,
DNA lesion frequencies (break points in DNA) are calculated as the amplification of damaged (treated) samples
relative to the amplification of nondamaged controls (from
genomic DNA), which results in a relative amplification ratio (treated/control). The principal advantage of the PCR
approach is the relative ease of the assay; a disadvantage is
that the specific nature of the base lesion is not known, nor
is the identity of the nucleotide revealed by this approach.
Wosniak et al325 used this method to assess mitochondrial
DNA damage in rabbit vascular smooth muscle cells exposed to Ang II.
Recommendations for Measurement
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A variety of techniques can be used to assess oxidative
DNA base damage, but the choice of the method depends
on the nature of the research question (Figure 6). If the
question pertains to the formation of a particular sugar
or base modification, then an approach using MS is more
informative. If instead the question pertains to general
base modifications, then a less precise technique might
Figure 6. Analytical methods of determining oxidative
modification of DNA. If the desired measurement is chemical
modification of bases or sugars, the best method is mass
spectroscopic analysis. If this technique is not available,
then high-performance liquid chromatography (HPLC) can
be used, but with less capability of determining the specific
modification. Antibody-based methods are only applicable for
deoxyguanosine. If the goal is to determine reactive oxygen
species–induced fragmentation or base lesions that can
disrupt transcription, quantitative polymerase chain reaction
(qPCR) offers a reasonably simple and quantitative approach.
Southern analysis can also provide quantitative information
but requires the use of radioactivity. The comet assay can
show fragmentation but is difficult to quantify. Assays listed
as primary (1°) are the most well accepted, secondary (2°) are
more widely used, and tertiary (3°) are either less sensitive or
less specific or should be verified with a second method.
be applicable. The most important aspect of application
of these techniques is careful preparation of the sample,
as oxidation of bases during the analysis procedures overestimates oxidative modification of DNA and RNA. We
refer the reader to Collins et al (Table I),326 in which oxidation of guanine was evaluated by a number of methods,
with a range of nearly 4 orders of magnitude. This variation is in part attributable to the method used and to the
sample preparation. Thus, no matter which technique is
used to determine the oxidative modification of DNA or
RNA by ROS, the measurements are only as good as the
care and preparation of the samples. This is likely the most
critical and overlooked aspect of these measurements. As
a general measure of DNA damage, the comet assay or immunoassays for 8-oxoguanine can be used; alternatively,
HPLC-based approaches represent a sensitive and economical alternative. However, for more detailed analysis
and for identification and quantification of DNA oxidation, MS-based approaches (GC-MS, HPLC-MS, or electrospray ionization MS) are recommended.
Measuring ROS and RNS and Oxidative
Stress in Clinical/Translational Studies
On the basis of preclinical studies showing that ROS/RNSinduced oxidative stress plays a key role in cardiovascular
physiology and pathology, measurements of these species
and oxidative stress should be useful in understanding
cardiovascular function and disease in humans. However,
because free radicals and other ROS/RNS are very reactive, they are difficult to measure. As a result, accurate
.−
measurements of ROS/RNS such as O2 , H2O2, or NO in
human tissues and biological fluids is challenging. Hence,
indirect methods that involve the measurement of stable
end products or biomarkers of oxidative stress have been
used to assess oxidative status in clinical and translational
studies. Although several circulating biomarkers are currently used to assess oxidative stress in humans, an ideal
biomarker, as defined by Giustarini et al327 (Table 3), has not
yet been identified. Therefore, in human studies, measurements of multiple independent indices of oxidative stress
are recommended. Several such biomarkers are currently
being measured, primarily in research laboratories, but are
not yet routine assays in clinical laboratories because their
diagnostic value remains uncertain.
Lipid Peroxidation
Because of their relatively high stability, products of lipid peroxidation in blood and urine are frequently measured to assess
oxidative stress in clinical and translational studies, including
lipid hydroperoxides, MDA, F2-IsoPs, and HNE.
Lipid Hydroperoxides
Several clinical studies have used LOOH as an index of
peroxidative injury of membrane lipids and oxidative
stress.328,329 Such studies have shown that plasma levels of
lipid hydroperoxides are higher in patients with ischemic
heart disease, peripheral artery disease,330 heart failure,331
and type 1 diabetes mellitus.332 Increased levels of lipid hydroperoxides have also been detected in the venous effluent
Griendling et al Measurement of Reactive Oxygen Species e61
Table 3. Criteria for an Ideal Clinical Biomarker of ROS/RNS
1. Chemically stable molecule
2. Unsusceptible to artificial generation or loss during storage or
processing
3. Directly implicated in the onset or progression of disease
4. Specific for the ROS/RNS in question
5. Detectable in the target tissue
6. Present in sufficient and quantifiable concentrations
7. Low intravariability and intervariability in humans
8. Determined by an assay that is accurate, precise, specific, sensitive,
robust, and interference-free
9. Measurable within a detection range of a reliable and robust analytical
method
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10.Free of confounding from dietary input or environmental factors
11.Consensus and establishment of reference values and intervals
12.Consensus and establishment of animal models
RNS indicates reactive nitrogen species; and ROS, reactive oxygen species.
of reperfused myocardium333 and in the plasma after thrombolytic therapy,334 which indicates that measurements of lipid hydroperoxide can be a reliable index of acute oxidative
stress. Changes in plasma levels of lipid peroxides have also
been measured to document changes in oxidative stress after
drug intervention.335 In PREVENT (Prospective Randomized
Evaluation of the Vascular Effects of Norvasc Trial), baseline serum LOOH levels, measured by the ferrous oxidation
of xylenol orange (FOX) assay, were found to correlate with
thiobarbituric reactive substances (TBARS) and with higher
risk of vascular events.336 Overall, measurements of plasma
LOOH levels are a useful index of oxidative stress, although
it is unclear whether this is reflective of systemic oxidative
stress or local oxidation of circulating lipoproteins.
Malondialdehyde
MDA is a stable end product of lipid peroxidation and can
be measured as an index of oxidative stress; however, few
studies measure MDA directly but instead rely on measurement of TBARS in the plasma. It has been reported that
plasma TBARS levels are increased in patients with coronary artery disease, hypertension, atherosclerosis, diabetes mellitus, heart failure, stroke, and aging.337–341 Cigarette
smoking is also associated with elevated levels of TBARS,
which suggests that proatherogenic and vascular injury
effects of smoking might be related to oxidative damage
induced by lipid peroxidation.342 Some studies have shown
that plasma TBARS can predict cardiovascular events. For
example, it has been reported that elevated plasma TBARS
predict carotid atherosclerotic plaque progression over a
3-year period, as validated by carotid wall thickness measured with ultrasound.343 In the PREVENT trial, patients
with high TBARS levels at baseline were at increased risk
of vascular events.336 The vascular risk reflected by TBARS
was found to be independent of C-reactive protein, interleukin 6, and traditional risk factors.336 Of the oxidative
stress biomarkers used clinically, plasma TBARS is among
the most common; however, because of the nonspecificity
of the test, it is not clear which specific species are being
measured, and there is a high probability of increased artifactual generation of TBARS during sample processing.
Isoprostanes
Increased circulating and urine levels of F2-IsoPs have been
demonstrated in various cardiovascular diseases associated
with vascular injury, including hypertension, atherosclerosis,
ischemia-reperfusion injury, and heart failure.344,345 In addition, in healthy people with cardiovascular disease risk factors,
such as obesity, diabetes mellitus, and hyperlipidemia, plasma
concentrations of F2-IsoPs are elevated,346 which suggests
that indices of lipid peroxidation could be clinically relevant
biomarkers of cardiovascular risk. In support of this concept,
a recent prospective study that included 1002 patients with
atrial fibrillation who had undergone anticoagulation therapy
who were studied for 25 months demonstrated that the levels
of 8-iso-PGF2α and sNOX2-dp (a marker of Nox2 levels) correlated with cardiovascular events.347 Using various modeling
paradigms, it was shown that 8-iso-PGF2α predicted cardiovascular events and death. As such, it was suggested that F2IsoP might complement conventional risk factors in prediction
of cardiovascular events.
4-Hydroxy-Trans-2-Nonenal
Increasing experimental evidence indicates that HNE might
have a dual role as both a marker of systemic oxidative stress
and a contributor to the pathogenesis of cardiovascular disease.348,349 Despite experimental evidence linking HNE and
cardiovascular disease, there is a paucity of information in
humans. A few studies in dialyzed patients demonstrated that
the plasma levels of MDA and HNE correlate with severity of
cardiovascular disease.350,351
Nonenzymatic Total Antioxidant Capacity
Total antioxidant capacity (TAC) is a measure of the combined antioxidant effect of the nonenzymatic defenses in
biological fluids and does not take into account the enzymatic antioxidant systems such as superoxide dismutase,
catalase, and peroxidase. The assay measures low-molecular-weight antioxidants, both water soluble and lipid
soluble, and includes urate, bilirubin, vitamin C, thiols, flavonoids, carotenoids, and vitamin E.352 Experimental and
clinical studies have shown, for the most part, low levels
of TAC in various cardiovascular diseases.353–355TAC assays
have also been used extensively in human studies evaluating
the effects of dietary antioxidants. A recent comprehensive
study reviewed prospective cohort studies and clinical trials relating to associations between plasma/dietary antioxidants (TAC) and cardiovascular events.356,357 In long-term,
large-scale, population-based cohort studies, higher levels
of TAC were associated with a lower risk of cardiovascular
disease, which supports a protective effect of dietary antioxidant vitamins, carotenoids, and polyphenols. However,
results from large randomized, controlled trials fail to support long-term use of single-antioxidant supplements for
cardiovascular prevention because of their lack of benefit
or even adverse effects on major cardiovascular events or
e62 Circulation Research August 19, 2016
cancer. Although the use of antioxidant supplements has
been reported to have no benefit on cardiovascular events in
several large randomized, controlled trials, cohort studies
still support the protective effects of dietary antioxidants on
preventing cardiovascular disease. In particular, antioxidant
vitamins and polyphenols exhibit high antioxidant capacity in vitro and cardioprotective effects in vivo. Although
TAC assays could shed light on dietary antioxidant effects
in specific patient cohorts, there are technical concerns regarding specificity and sensitivity of these assays, and as
such, TAC of foods or populations should not yet be used
to make decisions affecting population health.358
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NO and Peroxynitrite as Biomarkers of Endothelial
Dysfunction
Biomarkers of impaired endothelial function reflect altered
NO bioavailability, increased oxidative stress, coagulation,
and endothelial inflammation. NO, produced by endothelial cells, is a major determinant of endothelium-dependent
vasodilation and is an inhibitor of coagulation, inflammation, and oxidative stress359,360 and consequently has been
considered as an important marker reflecting endothelial
status. Because NO has a short half-life, plasma levels of
oxidative degradation products of NO, including nitrite, nitrate, and nitrosothiols, have been used as surrogate indices
of NO generation.361 The most commonly used approach to
assess NO breakdown products is the Griess reaction assay, which can be used in various clinical samples such as
urine, plasma, serum, and cerebrospinal fluid. The Griess
assay, which is now available in commercial kits, can detect
nitrite levels in clinical samples as low as 0.5 mmol/L.362,363
NO products can also be measured with chemiluminescence
and fluorescence methodology,364,365 but the clinical utility
of these approaches is still unclear.
In addition to assessing levels of NO and its metabolites,
measurement of asymmetric dimethylarginine, a potent endogenous inhibitor of NOS-derived NO production, can
reflect NO bioavailability.366 Plasma asymmetric dimethylarginine levels are measured by LC/MS367 and have been
shown to correlate with endothelial NOS activity and to be
associated with endothelial dysfunction.368,369 Vascular injury is also associated with increased production of endothe.−
lial O2 , which readily reacts with NO to form peroxynitrite
(ONOO−), an injurious free radical that further contributes to endothelial dysfunction.370 Similarly, lipid peroxyl
radicals react with NO and could also be a source of NO
inactivation.371 Plasma measures of endothelial oxidative
stress include indices of lipid peroxidation (F2-IsoPs and
TBARS) and nitrotyrosine levels, reflecting peroxynitrite
generation, although these measurements are not specific
for endothelial ROS production.372 Lipid peroxidation is
increased in diseases associated with endothelial dysfunction, and there is a strong negative correlation between
oxidative stress and endothelial function as assessed by
forearm blood flow responses to acetylcholine in the presence of the antioxidant vitamin C.373,374
Recommendations for Measurement
Considering the important role of ROS and oxidative stress
in the pathophysiology of cardiovascular disease, there is an
increasing need to accurately measure oxidative status in the
clinical setting, as well as in translational studies. Current
approaches rely on the measurement of circulating biomarkers of oxidative stress (Figure 7). However, there are limitations to using such an approach, because (1) circulating
levels of ROS biomarkers reflect generalized oxidative status without any information about the tissue or enzymatic
source from which the ROS are generated; (2) biomarkers
are indirect indices of ROS; and (3) current methodologies
lack sensitivity and specificity. Development of new analytical methodologies should focus on direct measurement
of free radicals, using assays that are user-friendly, practical, and of clinical utility. Because the ideal biomarker of
oxidative stress has not yet been identified, it is prudent to
measure multiple independent biomarkers when evaluating
ROS status in humans. A practical guide to suggested biomarkers to evaluate ROS/RNS status in the clinic is summarized in Figure 8.
Measurements of ROS/RNS and oxidative stress are
often made in the context of antioxidant supplementation;
Figure 7. Approaches to assess redox
status in clinical and translational studies.
Schematic showing simplified practical
approaches to measuring oxidative
status in circulating cells, blood, urine,
and cerebral spinal fluid (CSF). These
approaches are used primarily in clinical
and translational research and are not used
as routine assays in clinical laboratories.
EPR indicates electron paramagnetic
resonance spectroscopy; FL, fluorescence;
GSH, reduced glutathione; GSSG, oxidized
glutathione; HPLC, high-performance
liquid chromatography; LC/MS, liquid
chromatography/mass spectrometry; RBC,
red blood cells; TAC, total antioxidant
capacity; and WBC, white blood cells.
Griendling et al Measurement of Reactive Oxygen Species e63
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however, antioxidant supplementation does not automatically have an antioxidant effect, an issue incompletely
considered in many previous clinical trials that failed to
show benefit. Failure to detect changes in oxidative stress
after antioxidant supplementation could have several possible causes: (1) Antioxidant pharmacokinetics did not
allow sufficient accumulation of antioxidant in the target
tissue. (2) Antioxidants did not have sufficient antioxidant
potency; for example, intracellular superoxide dismutase
.−
reacts 10 000 times faster with O2 than the frequently
used antioxidant supplement ascorbic acid. (3) Antioxidant
supplements were not targeted to specific subcellular sites
of ROS/RNS overproduction, such as mitochondria. It is
conceivable that the use of antioxidants targeted to compartments where ROS are generated, such as the mitochondria, might be more effective.375 Indeed, recent studies with
transgenic mice support the therapeutic potential of SOD
overexpression in mitochondria and cytoplasm. (4) Finally,
and relevant to this statement, many of these studies lack
validation of an antioxidant effect by measurement of
relevant biomarkers discussed herein. Hence, in assessing
changes in the oxidative stress, it is important not only to
measure changes in the ROS/RNS themselves but in their
secondary products as well.
Summary and Overall Recommendations
Reliable and reproducible measures of ROS/RNS are difficult
to achieve given the complexity of the biological systems with
which they interact, their short half-lives and high reactivity, the balance between production and antioxidant capacity,
and compartmentalization of reactivity and short diffusion
distances. However, these molecules have both physiological and pathological roles, and precise measurements are
not only desirable but also imperative for consistency across
studies. The principles and analyses in this statement provide
a basis for deciding which assays to use in specific experimental conditions, as well as recommendations for common
use in various settings.
As general guidance, we recommend that any method or
assay used for measuring ROS or RNS should be specific,
Figure 8. Decision tree for measuring oxidant status in clinical tissue. Clinical samples (blood, tissue, urine) can be assayed
for pro-oxidant and antioxidant biomarkers with a variety of bioassays. For measuring pro-oxidants, levels of lipid peroxidation
or DNA damage can be measured in biological fluids. Metabolites of lipid peroxidation products (aldehydes, isoprostanes, and
hydroperoxides) by gas chromatography (GC)/liquid chromatography (LC) mass spectroscopy (MS) provide the most sensitive
and specific assessment of oxidative stress and are shown as 1° assays. Proteins modified by 4-hydroxy-trans-2-nonenal and
malondialdehyde can also be measured by Western analysis or other immunoassays, and isoprostanes can be measured by ELISA,
but these measurements are generally less sensitive and less specific (2° assays). Lipid hydroperoxides (LOOH) can
also be measured by high-performance liquid chromatography (HPLC). The use of nonspecific and nonselective measurements
of lipid peroxidation products (thiobarbituric reactive substances [TBARS]) is not recommended (3° assay). DNA damage can be
measured by quantifying modified bases either by GC/LC/MS (1° assay), HPLC, or reverse transcription polymerase chain reaction
(RT-PCR; 2° assays) or by less specific immunoassays (3° assay). Assessment of antioxidant status could involve measurements
of the total antioxidant capacity (TAC) or measurement of reduced glutathione (GSH)/oxidized glutathione (GSSG) or reduced
cysteine (Cys)/oxidized cysteine (CySS) ratio either by HPLC (2° assays) or by colorimetric/fluorometric techniques, although these
measurements are relatively less sensitive and prone to artifacts (3° assays) than other techniques. ORAC indicates oxygen radical
absorbance capacity.
e64 Circulation Research August 19, 2016
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Figure 9. Recommended approaches for the detection of reactive oxygen species (ROS)/reactive nitrogen species (RNS) and
their effects: ROS and RNS can be quantified either by direct detection or by measuring secondary products or protein and
DNA modification induced by ROS/RNS-initiated reactions. For rigorous documentation of significant ROS/RNS production, it is
recommended that the ROS/RNS species be measured directly, along with the measurement of one of the consequences of their
production (protein/DNA modification, thiol depletion, or lipid oxidation). Assays that provide the most sensitive and selective
measurements of the species/product of interest are listed. Although the choice of the assay will depend on the specific research
question of interest, at a minimum, measurement of >1 outcome of ROS/RNS production is recommended. DAF-2 indicates
diaminofluorescein 2; EPR, electron paramagnetic resonance spectroscopy; Fe[DETC]2, Fe-diethyldithiocarbamate; GSH/GSSG,
reduced glutathione/oxidized glutathione; HNE, 4-hydroxy-trans-2-nonenal; LC, liquid chromatography; MDA, malondialdehyde; MS,
mass spectroscopy; SNO, S-nitrosylation; SOH, S-sulfenylation; and SSG, S-glutathionylation.
sensitive, and reproducible. Given the highly reactive nature
of these species, care should be taken to ensure the stability of the sample during isolation, preparation, storage, and
analysis, which is particularly challenging when measuring
oxidative modifications that can be influenced by exposure
to ambient oxygen. Because of the ephemeral nature of many
of these modifications, best practice is to confirm findings
with >1 assay and to use appropriate controls. In addition,
we recommend that investigators choose an array of assays
that directly and specifically measure the ROS of interest, as
well as its particular endpoints, rather than relying on global
measures of oxidative stress (Figure 9). We also suggest that
the values of antioxidants (such as glutathione) or products
of oxidation (such as lipid peroxidation products) should
be reported in absolute rather than relative values. Values
of these metabolites expressed in molar concentrations are
particularly useful for intertissue comparisons and for comparison of results obtained in different laboratories.
Acknowledgment
The authors thank Sofia Tsiropoulou for her help with the section on
protein oxidation.
Griendling et al Measurement of Reactive Oxygen Species e65
Disclosures
Writing Group Disclosures
Writing Group
Member
Other
Research
Support
Speakers’
Bureau/
Honoraria
Expert
Witness
Ownership
Interest
Consultant/
Advisory
Board
Other
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Employment
Research Grant
Kathy K.
Griendling
Emory University
NIH (HL038206,
HL095070)†
None
None
None
Ownership
interest in
hydrocyan dyes
(intellectual
property)*
None
None
Rhian M.
Touyz
University of
Glasgow Institute
of Cardiovascular &
Medical Sciences
British Heart
Foundation
(RG/13/7/30099,
RE/13/5/30177 and
CH/12/4/29762)†;
Canadian Institutes
of Health Research†
None
None
None
None
None
None
Aruni
Bhatnagar
University of Louisville
NIH (HL55477,
GM103492)†
None
None
None
None
None
None
Yeong-Renn
Chen
Northeast Ohio
Medical University
None
None
None
None
None
None
None
William Chilian
Northeast Ohio
Medical University
NIH (RO1HL115114-04)†
None
None
None
None
None
None
Sergey Dikalov
Vanderbilt University
Medical Center
AHA*; NIH (R01
HL124116-01A1)†
None
None
None
None
None
None
David G.
Harrison
Vanderbilt University
Medical Center
NIH†
None
None
Covington and
Burling†
None
None
None
Davis Heart and Lung
Research Institute, The
Ohio State University
None
None
None
None
None
None
None
Jay L. Zweier
This table represents the relationships of writing group members that may be perceived as actual or reasonably perceived conflicts of interest as reported on the
Disclosure Questionnaire, which all members of the writing group are required to complete and submit. A relationship is considered to be “significant” if (1) the person
receives $10 000 or more during any 12-month period, or 5% or more of the person’s gross income; or (2) the person owns 5% or more of the voting stock or share of the
entity, or owns $10 000 or more of the fair market value of the entity. A relationship is considered to be “modest” if it is less than “significant” under the preceding definition.
*Modest.
†Significant.
Reviewer Disclosures
Other
Research
Support
Speakers’
Bureau/
Honoraria
Expert
Witness
Ownership
Interest
Consultant/
Advisory
Board
Other
Reviewer
Employment
Research Grant
Ruhul Abid
Brown University
AHA (I have a Grant-in-Aid award
that involves roles of ROS in coronary
endothelium)*; NIH (I am a PI of
COBRE Project grant that involves
ROS measurement in vascular
endothelium in vivo and in vitro)*
None
None
None
None
None
None
CNR, Institute of
Clinical Physiology
Genetics Unit (Italy)
None
None
None
None
None
None
None
Northwestern
University
None
None
None
None
None
None
None
University Hospital
Jena (Germany)
None
None
None
None
None
None
None
Maria Grazia
Andreassi
Hossein
Ardehali
P. Christian
Schulze
This table represents the relationships of reviewers that may be perceived as actual or reasonably perceived conflicts of interest as reported on the Disclosure
Questionnaire, which all reviewers are required to complete and submit. A relationship is considered to be “significant” if (1) the person receives $10 000 or more during
any 12-month period, or 5% or more of the person’s gross income; or (2) the person owns 5% or more of the voting stock or share of the entity, or owns $10 000 or
more of the fair market value of the entity. A relationship is considered to be “modest” if it is less than “significant” under the preceding definition.
*Significant.
e66 Circulation Research August 19, 2016
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Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and
Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From
the American Heart Association
Kathy K. Griendling, Rhian M. Touyz, Jay L. Zweier, Sergey Dikalov, William Chilian,
Yeong-Renn Chen, David G. Harrison and Aruni Bhatnagar
on behalf of the American Heart Association Council on Basic Cardiovascular Sciences
Circ Res. 2016;119:e39-e75; originally published online July 14, 2016;
doi: 10.1161/RES.0000000000000110
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