Oxidative stress assays and oxidative stress markers

Learn about markers including ROS, MDA, GSH:GSSG, and more. Review assays for use with tissues, cells, and biofluids.

Oxidative stress reflects the toxic side of oxygen and metabolism. Oxidative stress has been defined as “An imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage” (Sies and Jones, 2007).

Oxidative stress markers fall into three main classes:
- Reactive oxygen species (ROS)
- DNA/RNA, lipids and proteins that have been damaged by oxidation
- Antioxidants

For these three classes:
- ROS represent the agents that transmit oxidative stress and damage components of the cell
- DNA/RNA damage, lipid peroxidation, and protein oxidation/nitration represent the damage caused by oxidation
- antioxidants represent the systems for managing oxidative stress

Reactive oxygen species (ROS)

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.

Assay	Readout	Assay kits
DCFDA – cellular reactive oxygen species	Flow cytometry, plate reader	ab113851
Cellular superoxides Cellular ROS/Superoxide	Microscope, flow cytometry	ab139477 ab139476
Cellular reactive oxygen species	Plate reader	ab186027, ab186028, ab186029
Cellular ROS/RNS	Microscope	ab139473
Mitochondrial hydroxyl radical Mitochondrial superoxide Hydrogen peroxide	Microscope, plate reader	ab219931 ab219943 ab138874, ab138886, ab102500

"We are currently using the product [ab113851] to measure microglial activation after 24 hours in response to activating stimuli. The product has been giving us very consistent results and is very easy to use.”
- Dr Neal Bennett

DNA/RNA damage, lipid peroxidation, and protein oxidation/nitration

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.

DNA/RNA damage

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.

Lipid peroxidation

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.

Assay	Readout	Assay kits
8-OHdG	Plate reader	ab201734
Lipid hydroperoxide (LPO) Lipid peroxidation (MDA) Protein carbonyl content DNA damage – apurinic/apyrimidinic sites	Plate reader	ab133085 ab118970 ab126287 ab211154
Oxidized proteins	Western blot	ab178020

Antioxidants

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.

Assay	Readout	Assay kits
Total antioxidant capacity: copper-based Total antioxidant capacity - FRAP assay (Ferric Reducing Antioxidant Power Assay	Plate reader	ab65329 ab234626
Ascorbic acid NAD/NADH NADP/NADPH GSH/GSSG ratio Thiol	Plate reader	ab65656 ab65348, ab176723 ab65349, ab176724 ab138881, ab205811 ab112158, ab219272
Intracellular glutathione (GSH)	Flow cytometry, plate reader	ab112132
GST Superoxide dismutase Glutathione reductase Xanthine oxidase Glutathione peroxidase Aconitase Catalase Thioredoxin reductase NQO1 Peroxidase	Plate reader	ab65325, ab65326 ab65354 ab83461 ab102522 ab102530 ab109712 ab118184, ab83464 ab190804 ab184867 ab155895
Oxidative stress defense cocktail (catalase, SOD1, TRX, smActin)	Western blot	ab179843