Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association

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.

Keywords: AHA Scientific Statements; free radicals; oxidants; peroxides; reactive nitrogen species; reactive oxygen species.

Figure 1

Choice of assays to measure ${\mathrm{O}}_2^{·-}$ and H₂O₂. When choosing an assay to measure ${\mathrm{O}}_2^{·-}$ 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.

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.

Figure 3

Oxidative posttranslational modifications on protein thiols (cysteine).^, 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.

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).^, 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.

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.

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.

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.

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.

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.