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Reactive oxygen species (ROS) are reactive chemical species containing oxygen. They include peroxides, superoxide, hydroxyl radicals, singlet oxygen, and alpha-oxygen.
Due to their transient nature, they are easily measured in live cells using fluorescent dye-based assays, such as with DCFDA. They are harder to measure in tissue and biofluid samples.
DCFDA – cellular reactive oxygen species
Flow cytometry, plate reader
Microscope, flow cytometry
Cellular reactive oxygen species
Mitochondrial hydroxyl radical
Microscope, plate reader
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Oxidative stress can be measured indirectly by measuring the levels of DNA/RNA damage, lipid peroxidation, and protein oxidation/nitration, rather than a direct measurement of reactive oxygen species. These oxidative stress markers are more enduring than reactive oxygen species.
There are several types of DNA/RNA damage that can be measured as oxidative stress markers. 8-hydroxydeoxyguanosine (8-OHdG) is probably the most commonly used DNA damage marker for oxidative stress. Comet assays, assays for apurinic/apyrimidinic sites, and assays for aldehyde-induced damage can be used as less direct measures of DNA damage which is potentially related to oxidative stress.
Malondialdehyde (MDA) is the most commonly used lipid marker of oxidative stress. It is formed via peroxidation of polyunsaturated fatty acids and is typically quantified using the TBARS assay. The TBARS assay is not entirely specific for MDA, as other aldehydes also generate a signal with the assay, however, the TBARS assay is generally more convenient than using HPLC to measure MDA. Competitive ELISA assays for MDA are also available.
Other lipid peroxidation markers include 4-HNA, 8-isoprostane, lipid hydroperoxides, and oxidized LDL.
Protein oxidation / nitration
Oxidative damage to proteins can take the form of protein carbonylation and protein nitration (3-nitrotyrosines). Reactive oxygen species can also cause the formation of advanced glycation end products (AGE) and advanced oxidation protein Products (AOPP). All of these markers can be measured by standard assays.
Lipid hydroperoxide (LPO)
Lipid peroxidation (MDA)
Protein carbonyl content
DNA damage – apurinic/apyrimidinic sites
Antioxidant enzymes and other redox molecules counteract the ROS that cause oxidative damage. There are three classes of antioxidants used as oxidative stress markers: small molecules, enzymes, and proteins (such as albumin).
A number of assays exist to measure the total antioxidant capacity of a sample, including small molecule and protein antioxidant based capacity. One of the most common total antioxidant capacity assays is the Trolox equivalent antioxidant capacity assay (TEAC). The oxygen radical antioxidant capacity (ORAC) assay is another common oxidative stress assay that measures antioxidant capacity by measuring the ability of antioxidants to reduce the quenching of a fluorescent dye by ROS.
Antioxidant activity can also be measured at the level of specific analytes. For instance by looking at the relative levels of GSH and GSSG. Glutathione (GSH) is considered the most abundant molecule among endogenous antioxidants, forming GSSG in its oxidized form. It is recycled by glutathione reductase.
Otherwise, the levels of activity of antioxidant enzymes, such as GST and Superoxide dismutase can be measured in relation to the levels of oxidative stress.
Total antioxidant capacity: copper-based
Total antioxidant capacity - FRAP assay (Ferric Reducing Antioxidant Power Assay
Intracellular glutathione (GSH)
Flow cytometry, plate reader
Oxidative stress defense cocktail (catalase, SOD1, TRX, smActin)